CA2112130C - Screening assay for the detection of dna-binding molecules - Google Patents

Abstract

The present invention defines an assay use-ful for screening libraries of synthetic or biological compounds for their ability to bind specific DNA
test sequences. The assay is also useful for deter-mining the sequence specificity and relative DNA-binding affinity of DNA-binding molecules for any particular DNA sequence. The assay is a competition assay in which binding of a test mole-cule to a DNA test sequence changes the binding characteristics of a DNA-binding protein to its binding sequence. When such a test molecule binds the test sequence the equilibrium of the DNA:protein complexes is disturbed, generating changes in the ratio between unbound DNA and DNA:protein complexes. The assay is versatile in that any test sequence can be tested by placing the test sequence adjacent to a defined protein binding DNA screening sequence.

Description

~ 21 lZ~30 S~~ i ASSAY FOR TXE DETECTION OF
DNA-BINDING MOLECULES
Field of the Invention The present i~vention relates to a method, a system, 5 and a kit useful for the identi~icatio~ o~ molecules that speci~ically bind to defined nucleic acid ~equences Ref erences Ambinde~, R. F ., et al ., J. Virol . 65 :1466-1478, 1991 .
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s B~IG1~U- ' OI~ the ~nvention several classes of small molecules that interact with double ..LL~ded DNA have been identified. Many of these small ~ c~ have l Lur~u~ iologic~l effects. For 10 example, many Aminn~rridines and polycyclic hy lLu-;alLu-bind DNA and are mutagenic, te:Lc~tog~l~ic~ or carcinogenic.
Other 6mall ---lec~ that bind DNA include biolo~i~Al metabolites, ~ome of which have applications as antibiotics and antitumor agents ;nrlllAln~ ac~ir y~in D, ~rh;- ~;in, 15 distamycin, and CJ'l irhr~m;c;n; planar dyes, such as ~h;~ -m and acridine orange; and --lec~ that contain heavy metals, such as cisplatin, a potent antitumor drug.
Most known DNA-binding molecules do not have a known ~ qll~nre binding preference. However, there are a few 20 ~mall DNA-binding lec~ that preferentially reco~n; ~e ~r~r;f{r nucleotide s~lu- -~, for example ~rhi- ~._in preferentially binds the s~ [(A/T)CGT]/tACG(A/T)]
(Gilbert et al. ); cisplatin covalently cross-links a platinum l~ar~l e between the N7 atoms of two adjacent 25 deUA,~"Al~S;r~fi (Sherman et al.); and CAl ;rhe~m;cir preferentially binds and cleaves the s~Tl~nre TCCT/AGGA
(Zein et al. ) .
The bi olo~ r~ D~u..~e elicited by ~ost therapeutic DNA-binding lec~ toxicity, speci~ic only in that 30 these l~r~ may preferentially ~ffect cells that are more actively replicating or LL~ ibing DNA than other cells. Targeting Erer;fir site~ may si~nific~ntly decrease toxicity simply by reducing the number of potential binding ~ite~ in the DNA. As sperifi~rity for longer seuual.~t,, is 35 acquired, the ~ ,æ~; f j c toxic effects due to DNA-binding WO 93/00446 PCr/US92/05476 6 2112t3 may decrease. Many therapeutic DNA-binding ~-~ec~lP~
initially identified based on their therapeutic activity in a h;o~o~;c~l screen have been later ~Pt~rm;n~l to bind DNA.
Therefore, there is a need for an in vitro assay 5 useful to screen for DNA-binding- lDr~1P~. There is also a need for an assay that allows the discrimination of sequence binding pref erences of such ~ Pc~ Q .
Additionally, there is a need for an assay that allows the ~ t~m~n~tion of the relative a~rinities of a DNA-binding 10 - l~Dr~l P for dirferent DNA s~lu~ 3 . Finally, there is a need for therapeutic ~ ~ec~lPs that bind to epDc';1~ic DNA
se-lu~
8umm~ry of the Invention The present invention provides a method for screening molecules or ' capable of binding to a selected test B ~ in a duplex DNA. The method involves adding a --le~lP to be ~ ,--ed, or a miYture containing the v-~Dr~lP-, to a test system. The test system tn~ D~ a DNA
20 binding protein that is effective to bind to a screening ~ ~ , i.e. the DNA binding protein's cognate binding bite, in a dupleY DNA with a binding affinity that i8 preferably ~u~L~n~ially ~ 3n~ of the 8~u~ 8 ad~acent the binding s~ e -- these adjacent ~eqllPnr~c:
25 are referred to a8 test s~u~nc~. But, the DNA binding protein is sensitiVe to binding of 1P~1 DC to such test ~;Dq~lPnr~, when the test s~ e is adjacent the screening o~ . The te8t 8y8tem further ~nr~ D~ a dupleY DNA
having the screening and test se~u~ es adjacent one 30 another. Also, the binding protein is present in an amount that ~ U-ate8 the 8creening 8P~l ~ e in the duplex DNA.
The test -ler~l P i8 ir.~;uLated in contact with the test system for a period ~-~ff~rjPnt to permit binding of the lPr~l P being te8ted to the test g~ ~DI~r~ ln the duplex 35 DNA. The amount of binding protein bound to the duplex DNA
_ _ _ _ _ . _ _ _ . _ _ _ _ _ _ _ 93/~0446 PCr/US92/05476 ~WO ._ .
7 2~ 17~3~
is compared before and after the addition of the test molecule or mixture.
Candidates for the screening sequence/binding protein may be selected from the following group: EBV origin of replication/EBNA, HSV origin of replication/UL9, VZV origin of replication/UL9-like, HPV origin of replication/E2, ~ terleukin 2 onh~nrr/NFAT--l, HIV-LTR/NFAT-l, HIV-LTR/NFkB, HBV nhAnr~r/HNP-1, fibrinogen promoter/HNF-l, lambda oL-o~/cro, and essentially any other DNA:protein interactions.
A pref erred ~ of the present invention utilizes the UL9 protein, or DNA-binding proteins derived therefrom, and its cognate binding sequence S~Q ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17, or SEQ ID NO :15 .
The test sequences can be any combination of sequences o:~ interest. The seyu~ es may be randomly generated ror shot-gun approach screening or specif ic 8~ may be chosen. Some specific sequences of medical interest include the following sequences involved in DNA:protein interactions: EBV origin of replication, HSV origin o~
replication, VZV origin of replication, HPV origin of replication, interleukin 2 PnhAnr~r~ HIV-LTR, HBV onhAnror~
and fibrinogen promoter. Furth~ ~, a set of assay test q comprised of all pnqqi hl P se~u~ s of a given length could be tested (eg., all four base pair sequences).
In t_e above method, comparison Or protein-bound to free DNA can be ~ qhod using any clPt~o~t; r~n assay, preferablY, a gel band-8hirt assay, a filter-binding assay, or a capture/~loto~ion assay.
In one i L of the DNA capture/~3ot~o~tjon assay, in which the DNA that is not bound to protein is ~ UL t d, the capture System involves the biotinylation of ~1 nucleotide within the qcreening s~lu-~ e (i) that does not eliminate t_e protein ' 8 ability to bind to the screening WO 93/00446 PCr/US92/05476 scuuCl~ce~ (ii) that is capable of binding :~LLc~avidin~ and (iii) where the biotin moiety i8 protected from interactions with ~ Lc~avldin when the protein is bound to the screening scUucl,ce. The capture/clet~ct; r n assay also 5 involves the d~tec~ 1rn of the ~a~uLcd DNA.
In another : ' i o~ the DNA capture/detection assay, th~ capture sy5tem in which the DNA:protein ,1r~YP~: are a~-uL~1, the capture system involve6 the use of nitroc~ 1 n~e f ilters under low salt conditions to 10 capture the protein-bound DNA while allowing the non-protein-bound DNA to pass through the filter.
The present invention also 1nr~ a screening system f or identi~ying le~ 1 r ~ that are capable of binding to a test gr. lu-~ c in a duplex DNA se~u~ c. The sy~tem 15 ~nrlllAr~: a DNA binding protein that is effective to bind to a screening ~ e in a duplex DNA with a binding affinity that i8 subst:~nt;~11y 1n~ rL of a test B ,.~ re ad~acent the screening 8c~u~ . The binding of the DNA protein i8, however, sensitive to binding of 20 ~ l~r~ c to the test _ ~u~ c when the test 6~ c is ad~acent the 8creening ~ . The 5ystem ;nnlt~A~s a duplex DNA having the ficreening and test s~ nrr~ adjacent one another. Typically, the binding protein i8 present in ~n amount that 8aturate8 the screening P,~l r - ~ in the 25 duplex DNA. The 8y8tem also inrl~A~ means for detecting the amount of binding protein bound to the DNA.
As A~-~rr; h-~A above the test 8~ can be any number of _, of interest.
The ~creening ~ /binding protein can ~e s~ c~rA
30 from known DNA:protein interactions using the criteria and guidance of the pre5ent A1~c~1rE-~re. It can also be applied to DNA: Protein interactions later discuvc~.d.
A ~cLcL.~d ' ~1 of the 6creening system of the pre6ent invention 1nrlllA~ the UL9 protein, or DNA-binding 35 protein deriv8d l.llcL~LL~ (e-g., the Lu--~ ~.ted ~L9 protein _ _ _ _ _ _ _ _ _ ~0 93/00446 Pcr/uss2/os476 21 1~130 designated UL9-COOH). In 1:his c '; the duplex DNA
has (i) a screening seyu~ e sPlPc~e~ from the group - consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:17 and SEQ ID NO:15, and (ii) a test se:~u~nce adjacent the 5 screening se ~ , where ~L9 is present in an amount that caturates the screening seyuence. The aystem further ;n~ dPc means for dPtec~inq the amount of UL9 bound to the DNA, ;nrl~ ;n~, band-shift assays, filter-binding assays, and capture/detection assays.
The present tl;~c~F~re describes the ~Lvc~duL-~ needed to test DNA:protein interactions for their suitability for use in the screening assay of the present invention.
The present invention further defines DNA capture systems and detection systems. Several methods are 15 described. A filter binding assay can be used to capture the DNA:protein 1PYP~: or, alternatively, the DNA not bound by protein can be LC~LUL~d by the following method.
In the first part of this Eystem, the cognate DNA binding site of the DNA binding protein is ; f i Pd with a 20 dPten~inn moiety, such as biotin or digQYigPn;n. The modification must be made to the site in such a manner that (i) it does ~ot eliminate the protein's ability to bind to the cognate binding s~lU- ~e, (ii) the moiety i5 a--cPQ~::ihle to the capturing agent (e. g., in the case of biotin the 25 ~gent is DL ~Lavidin) in DNA that i8 not bound to protein, and (iii) where the moiety is protected from interactions with the capture agent when the protein is bound to the screening sequPn~-e.
In the second part of this system, the target 30 oligonucleotide i5 lAhPllPd to allow dete~t;nn. T~hPll;ng of the target oligonucleotide can be a~ hPd by standard techniques such as r~iol~hPll;n~. Alternatively, ~ moiety such as dignY;~Pn;n can be ir~L~vL~ted in the target ol;~n~ ~lPntide and this moiety can then be detected 35 ~ftQrdcapture. . =
_ _ _ _ _ lo 2~ 12~30 Three embodiments of t~e capture/~l~trrt; r,n system described by the present disclosure are as follows:
(i) the target oligonucleotide (cnnt~;n;ngl for 5 example, the screening and test sequences) --modification of the cognate binding site with biotin and incorporation of digoxigenin Or radioactivity (eg, 33S or 32p);
capture of the target oligonucleotide using streptavidin attached to a solid support; and detection of the target 10 oligonucleotide using a tagged anti-digoxigenin antibody or radioactivity measurement (eg., autoradiography, counting in scintillation fluor, or using a phosphoimager) .
(ii) the target oligonucleotide -- modification of 15 the cognate binding site with digoxigenin and incorporation of biotin or radioactivity; capture of the target oligonucleotide using an anti-digoxigenin antibody attached to a solid support; and detection of the target oligonucleotide using tagged streptavidin or 20 radioactivity measurements.
(iii) separation of the target oligonucleotide which is bound to protein from the target oligonucleotide which is not bound to protein by passing the assay mixture through a nitrocellulose filter under conditions in which 25 the protein:DNA complexes are retained by the nitrocellulose while the non-protein bound DNA passes through the nitrocellulose; and detection of the target oligonucleotide using radioactivity, tagged anti-digoxigenin:digoxigenin interactions, or tagged 30 streptavidin:biotin interactions.

~' ~ lOa 2 ~ i 21 30 This invention provides a method of screening for 1 Pf'lll ~C capable of binding to a gelected test sequence in a duplex DNA, comprising:
( i ) adding a ~olecule to be 8~:L ~a-3ne(1 to a test system composed of (a) a DNA binding protein which is effective to bind to a screening sequence in a duplex DNA
with a binding affinity that is sub3tAntiAlly ;nrl~r~n~nt of said test 3equence adjacent the screening sequence, but where said protein binding is sensitive to binding of molecules to such test sequence, and (b) a duplex DNA
having said screening and test sequences adjacent one another, (ii) incubating the molecule in the test system for a period 8~- f f i ~ nt to permit binding of the ~ - ~
being tested to the test 3equence in the duplex DNA, and ( iii ) detecting the amount of binding protein bound to the duplex DNA before and after said adding.
This invention also provides a screening system for identifying molecules that are capable of binding to a test sequence in a target duplex DNA sequence, comprising:
a duplex DNA having screening and test sequences adjacent one another, a DNA binding protein that is ef f ective in binding to said screening sequence in the duplex DNA with a binding af f inity that is substantially i nrl~p-~nrlf.nt of said test sequence adjacent the screening sequence, but which is sensitive to binding of r 1 ~r~ to said test s~.q~nr e, and means for detecting the amount of binding protein bound to the DNA.
This invention also provides a method for inhibiting the binding of a DNA-binding protein to duplex DNA, comprising:
contacting a ~_ olln~l with a duplex DNA which contains a test sequence adjacent a screening sequence, . ~, lOb 21 1 Z 1 30 where the DNA binding protein is effective to bind to the screening sequence with a binding affinity that is 3ubstantially i n~f~p-~n~l~.nt of said test sequence, further where the binding of said ~< __ ' to the test sequence inhibits the binding of the protein to the screening 3equence .
~his invention also provides the aforementioned method for inhibiting the binding of a DNA-binding protein to duplex DNA wherein the a. ' is identified by the steps of preparing a series of duplex nucleic acid f ragments, each containing a test sequence ,- ~ ~8~d of one of the 4N
po~asible permutations of sequence3 in a sequence of base pairs having N-b~p~;rs, where said test sequence is adjacent the screening ~equence, measuring the binding af f inity of the DNA binding protein to each of the series of nucleic acid ~r~- 18 in the presence of the compound, and

2 0 selecting the compound if it lowers the binding affinity of the DNA binding protein for the ~creening sequence .
Bri~f D~3~criptioll of th~ Figur~s Figure lA illustrates a DNA-binding protein binding to a screening sequence. Figures 1~ and 1 C illustrate how a DNA-binding protein may be displaced or hindered in binding by a small molecule by two different r-~h~ni becau~e . ~

11 2t 1~3~
of = steric hinderance (1~3) or because of conformational (allosteric) changes induced in the DNA by a small molecule (lC) .
Figure 2 illustrates an assay for detecting inhibitory molecules based on their ability to preferentially hinder the binding of a DNA-binding protein to its binding site. Protein (0) iB displaced from DNA (/) in the presence of inhibitor (X). Two alternative capture/detection systems are lllustrated, the capture and detection of unbound DNA or the capture and detection of DNA:protein complexes.
Figure 3 shows a DNA-binding protein that is able to protect a biotin moiety, covalently attached to the oligonucleotide sequence, from being recognized by the streptavidin when the protein is bound to the DNA.
Figure 4 shows the incorporation of biotin and digoxigenin into a typical oligonucleotide molecule for use in the assay of the present invention. The 2 0 oligonucleotide contains the binding sequence ( i . e ., the screening sequence) of the UI,9 protein, which is underlined, and test sequences flanking the screening sequence. Figure 4 shows the preparation of double-stranded oligonucleotides end-labeled with either digoxigenin or 32p Figure 5 shows a series of sequences that have been tested in the assay of the present invention for the binding of sequence-specific small molecules.
Figure 6 outlines the cloning of a truncated form of the UL9 protein, which retains its sequence-specific DNA-binding ability (UI.9-COOH), into an exprecsion vector.
Figure 7 shows the pVL13 93 baculovirus vector containing the full length U~9 protein coding sequence.
Figure 8 is a photograph of a SDS-polyacrylamide gel showing (i) the purified U~9-COOH/glutathione-S-transferase fusion protein and (ii) the UIl9-COOH
polypeptide. In the figure the U~9-COO~ polypeptide is indicated by an arrow.

WO 93/00446 PCr/US92/05476 2~1213~ ~

Figure 9 shows the effect on UL9-COOH binding of alterations in the test seyu~ s that flank the Ul9 screening 8~ue ..ue. The d~ta are displayed on band shift gels.
Figure lOA shows the effect of the addition of several 6..".;~..LLc.Lions of Distamycin A to DNA:protein assay r~rt;nr- u~ ;n~ different tegt g~utsnces. Figure 10B
shows the erf ect Or the addition or AQt ~ - y~,ln D to DNA:protein assay reactions ut;l;7;n~ dirrerent test 10 5-~ ' Figure lOC show~ the ef rect Or the addition of Doxorubicin to DNA:protein nssay rP~Qtinn~ ut;l;s~;r~
dirrerent test 8~ ~ue...,. 3 .
Figure llA illustrates a DNA capture ~ystem o~ the present invention ut;l;5:1ng biotin and ~ Lavidin coated lS ; ~- beads . The presence of the DNA is detected using an /~lk~-l ;nr pl~o~hatase substrate that yields a ~.hpmil ;nP~Pnt product. Figure llB show~ ~ similar reaction using biotin coated agarose beads that are c;u..juyl,ted to 2-Lr~yLavidin, that in turn i8 .io~-Juy~Led to 20 the ~ yLu ad DNA.
Figure 12 d- L,cLes a test matrix ba~ed on DNA: protein-binding data .
Figure 13 lists the top strands (5'-3') of all the pnc:l:;hl~ rOur base pair ~ u~c~ that could be used as ~L
25 defined set of ordered te5t se~ in the assay (ror a ~creening s~ e having n bases, where n=4 ) .
Figure 14 list~; the top strands (5'-3') of all the po~ ;hl~ four base pair seguences that have the same base co:~position as the ~ -r~ S ~ -GATC-3 ' . This is another 30 example of A derined, ordered set Or s~ that could be test~d in the assay.
Figure 15 shows ~n example Or an ol ;~nml-3Pot;~
7q~nlP Cnnts~;n;n~ test sey,._,.. es f1 /~nl~in~ a screening - ~ . The 8~u_l~C.: of this - 1 P~ is ~l~D~.Le~ aE
35 SEQ ID NO: 18, where the "X" of Figure 15 i8 N in SEQ ID

WO 93/00446 Pcr/US92/05476 ~ ~ 7 ~

- NO :18 .
- D~t~ De~cription of th~ Inv~mtion Def inition6:
Ad~acent i6 used to describe the di6tance relaf;c~n~:hir between two nQiqhhnring DNA sites. Adjacent sites are 20 or les6 bp apart, or more preferably, 10 or less bp apart, or even more preferably, 5 or les6 bp apart, or most preferably, immediately abutting one another. "Flanking"
i6 ~ ~ynonym for adjacent.
Bo~n~ NA. a6 u6ed in thi~ alo~re~ refers to the DNA that is bound by the protein used in the a66ay (ie., in the le~ of thi6 ~i~r1ns~re, the UL9 protein).
Dissociation is the proces6 by which two molecules cease to interact: the process occurs at a f ixed average rate under specific phy6ical con~itions.
F~nrtional bin-l i nr- is the noncovalent a6sociation of protein or small lPr~ to the DNA - lPrll 1 ~ . In the assay of the pre6ent invention the fllrr~inn~l binding of the protein to the screening e_~ue~ = (i.e., it~ cognate DNA binding ~ite) has been evaluated u6ing filter binding or gel band-6hirt experiment6.
Heteromolecules are lP~ pc that are comprised of at lea6t two dif f erent type6 o~ - lea~ e: ~or example, the covalent rou~lin~J of at lea6t two 6mall organic DNA-binding le~ (eg., di6tamycin, a~t;r ~ in D, or acridine) to ~ach other or the covalent ~o~rl ~ n~ of such a DNA-binding e~lP(6) to ~ DNA-binding polymer (eg., deoxyol;~rn~rlPotide) .
On-rate i6 herein defined a6 the time required for two molecule6 to reach 6teady 6tate a660ciation: for example, the DNA:protein complex.
Off-rate i6 herein defined a6 the time required for one-half of the a6sociated lPYPe, e.g., DNA:protein 35 . lPYPe, to ~l~Ror;AtP.

WO 93/00446 PCr/US92/05476 Seau~ ;e L ~JP~ ic binding ref ers to DNA binding 1 PR which have a strong DNA se~ e binding preference. For e~ample, reætriction enzymes and t~te proteins listed in Table I P LLC~Le typical ~6~u.~". e ErPcific DNA-binding.
SeaU~ e .,- ~ferential binding refers to DNA binding r-~Prl~lPR that generally bind DNA but t~tat show preference for binding to some DNA seq tences over others. SPqllPnre-preferential binding i8 typified by several of the small -le~AlllPR tested in the present ~iR~ Rllre, a.g., aiStamyciit. S~l ~e },-e:ftL-,.Lial and ~ ,e~ific binding can be evaluated using a test matrix such as is pre_ented in Figure 12. For a given DNA-binding molecule, there are ~ ~6~.LL~ of differentiAl arfinities for di~ferent DNA se~UPnrPR ranging from r.o~ erific (no ~ AhlP preference) to æP l~.~ e pre~erential to Ahcol~ltP seTt~n~e fir~r;firity (ie., the recognition of only a single se~u~ e al~ong all pr~R~ hl P ~ f;, as i8 the case with many rest~riction ~ lP5~Rpc).
scrPPn;n~ uc~c is the DNA sc l -~ that defines the cognate binding site for the DNA binding protein: in the ca~e of UL9 the 8creening ~ e can, for example, be SEQ ID NO :1.
F--ll lprlll-~R ~re desirable as t~tAL- Lic~ for several rea50n8 related to drug delivery: (i) they are commonly less than 10 R le~ Ar weight; (ii) they are more likely to bc p~ hlP to cells; ( iii) unlike peptides or ol;g~n~lPotides, they are less susceptible to d~L laLion by man~r celllll~r - -n;r~ nd, (iv) they 30 are not as apt to elicit an immune ~ .,e. ~any rhAr-~r~P~ltical ~ ~; P~ have extensive libraries of rh~m;~A~l and/or b;ol-~q;~AAl mixtures, often fungal, bacterial, or Algal extracts, that would be de5ir~ble to ~creen with the ~I;say of the present invention. Sm~ll ~llPR may be l~ither bioloq;r~l or synthetic organic ~: = = == == =

93/00446 Pcr/uss2/o5476 ~0 - _ _ , or even inorganic ~- _ ' - ( i . e ., cisplatin) .
Test seauence is a DNA seuuc:l.ce adjacent the screening sequPnre. The as5ay of the present invention screens for -lec~lP~ that, when bound to the test sequence, affect the 5 interaction of the DNA-binding protein with its cognate binding site (i.e., the screening s~uue..~e). Test c can be placed adjacent either or both ends of the bcreening se~ue..~e. Typically, binding of --~Pcllllpc to the test ~PT~pnre interferes with the binding of the DNA-10 binding protein to the screening 6~lu~l - e. However, some le~ll P~ binding to these sequences may have the reverse ~ffect, causing an increased binding affinity of the DNA-binding protein to the screening sequence. Some molecules, Qven while binding in a seguence specif ic or sPql~nre 5 preferential manner, might have no effect in t_e assay.
mese -lPr~lP~ would not be detected in the assay.
Tlnh~und DNA. as used in this ~ ns~re, refers to the DNA that is not bound by the protein used in the assay (i~., in the 1P~ of this ~i~rlnsllre~ the I~L9 protein).
I. The Assay one f eature of the present invention is that it provides an assay to identify small lPr~lp~ that will bind in a s~ e ~ecific manner to --';r~lly 13iqn;f;rAnt 25 DNA target siteS. The assay facilitates the devPl~, L of new field of rh~rr--Pllt;r~l~ that operate by interfering ~it_ spec;fic DNA fl~nrt;nn~, such as crucial DNA:protein interactions. A sensitive, well-controlled assay to detect DNA-binding l~Pr~l P~ and to determine their sequence-30 ~rer;fi~ity and affinity has been developed. The assay canbe used to screen large biological and nhP~I;cs-l libraries;
for example, the assay will be used to detect s6.lu~ ;e srPr;fir- DNA-binding lerl~lPs in f L-tion broths or ~LL_CL:~ from various mi~ ,Ly~ isms. Fur~h~~ 6, another 35 spplication for the assay is to determine the 5''`l~-~" e WO 93/00446 PCr/US92/05476 - 2~2~30 fipe~if~r~ty and re]ative affinities of known DNA-binding drugs (and other DNA-binding -- -1 ec~ll D~) for dif$erent DNA
seguences. The drugs, which arQ primarily used in an~;t-AnrDr L,e~lt ~:, may have previously unidentified 5 activities that make them strong candidates f or therapeutics or therapeutic PL ~ UL D~L D in entirely different areas of - '~t-~nD.
The screening assay is b~ 1 Iy a competition assay that is ~lD~it3nD~ o test the ability of a molecule to lO compete with ~ DNA-binding protein for binding to a short, ~ynthetic, doubl~ ,L,t~ded ol i~o~DnYynucleotide that rnntAinc the reco~3nition seguence for the DNA-binding protein f lanked on either or both sides by a variable test site. me variable test site may contain any DNA seguence 15 that provides a rD~cnn~h~e recognition De~u~ ;e for a DNA-binding ~ ler~l D MnlPr~l D~ that bind to the test site nlter the binding cha, ~ L istic~ Or the protein in ~
m~nner that can be readily detected; the extent to which such -1DC~1 DC are able to alter the binding 20 char~.~L~listiCs of the protein i~ likely to be directly proportional to th~e a~finity of the test ~Dr~le rOr the DNA test 8ite. The relative affinity of n given molecule for different ol irJ~ r~Dotide Gr~ r~l at the test ite ~i.~., the te8t ~ Q) can be estAhl;RhD~l by ~yAm;nin~
25 its erfect on the DNA:protein interaction in each of the nl igonllrlentides. me rlDt~Drmin~tion of the high affinity DNA binding 8ites f or DNA-binding lDr~ l Pc will allow U8 to identify fire~i f i ~ target seguences for drug dev~l t ~ : .
A. General Cnnci~Drations.
The assay of the present invention has been ~3DQi~nDd for ~Dtt~rtinr3 test IDr--lDc or - that affect the rate o~ rDre~ Or a Erecifi~ DNA ~r4c~e from one protein ~lDr~lD to another identical protein in solution.
A miYtUre of DNA and protein iD ~LC}~t~LCd in ~oltltinn.
_ _ _ _ _ . _ _ _ _ _ _ _ _ _ _ 93~00446 Pcr/US92/0s476 ~0 17 21 12~3 - The co~lce ~l LL atiOn of protein is in excess to the concentration of the DNA so that virtually all of the DNA
is ~ound in DNA:protein complexes. The DNA is a double-stranded ol; gomlcl eotide that contains the recognition 5 sequence for a specific DNA-binding protein (i.e., the screening sequence). The protein used in the assay contains a DNA-binding domain that is specific for binding to the sequence within the c~ n~ ootide. The physical conditions of the 801uti~n (e.g., pH, salt cu~.ce..~L~ltion, ~ _ aL~Le) are adjusted such that the half-life of the complex i~ hle to performing the assay (optimally a half-life of 5-30 minutes), preferably in a range that is close to nor~al physiological conditions.
As one DNA:protein co~plex lliccori~Ates~ the released DNA rapidly ref orms a complex with another protein in ~oluti~n- Since the protein is in excess to the DNA, tl~ccon;~tions of one complex always result in the rapid r~A~Coc~Ation of the DNA into another DNA:protein complex.
At equilibrium, very few DNA - l~c~loc will be l~nho~ln~l The minimum ba~h~ of the assay is the amount of unbound DNA o~s~ v~d during any given measurable time period. The brevity of the observation period and the sensitivity of the detection system define the lower limit~
of ba-,h~ nd DNA.
Figure 1 illustrates how such a protein can be tl~crlr- ' from its cognate binding site or how a protein can be ~ ted from binding its coqnate binding site, or how the lr; no1-~ rc of the DNA:protein interaction can be altered. One - ~ni~~ i8 8teric hindt,c-~ce of protein binding by a small l~ e. Alternatively, a molecule may interfere with a DNA:protein binding interaction by 1n~ inq a conformational ~ hange in the DNA. In either event, if a test lo~le that binds the oligonucleotide hinders binding of the protein, the rate of transfer Or DNA
35 from one protein to another will be d~ ased. This will WO 93/00446 PCr/US92/05476 18 211213:
result in a net increa5e in the amount of unbound DNA. In other words, an increase in the amount of unbound DNA or a decrease in the amount of bound DNA indicates the pL t:sencc Or an inhibitor.
AlternatiVely, ~-lec~lP~ may be isolated that, when bound to the DNA, cause an increased affinity of the DNA-binding protein for its cognate binding site. In this case the amount of un}~ound DNA (v~ d during a given ~neasurable time period after the addition of the ~-lar~le) will decrease in the reaction mixture ~5 ~ L~1 by the capture/detection system described in Section II.
B. Other Methods There are sev~ral approaches that could be taken to look for small mo:Lecules that ~re~;1'it /~lly inhibit the interaction or a given DNA-binding protein with its binding E ~u~ (cognate ~ite). One c~-v- cl- would be to test biol~y;t~l or ~~haT~;cnl c ` for their ability to preferentially block the binding or o~ne spe~i1'; r~
DNA:protein interac:tion but not the others. Such an as~ay would depend on the devPl- L of at lea~t two, preferably three, DNA:protei~n interaction systems in order to establish control8 for distinguishing between general DNA-binding -l~r~lla~: (polycations like heparin or intercalating agentE. like e~h;l1;11Tn) and DNA-binding ac~ a~ having 8e~u~ binding pre~ e~ that would af~ect proteinlcognate binding æite interactions in one 6ystem but not the other(6).
one illustration of how this system could be used is as follows. Each cognate site could be placed 5' to ~
e~v,Lc. gene (such a6 genes anrr>~l;n~ l~-q~lArtoE;~a or luciferaE~o) sUCh that binding of the protein to the cognate ~ite would enhance tran8cription of the reporter gene. The ~E- IC~ of ~1 8~u~ c E,e-;f;c DNA-bi~nding drug that blocked the DNA:protein interaction would decrease the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ WO 93/00446 PCr/US92/05476 -21 12130 ~

pnh~n~~ ~ o~ the reporter gene expression. Several DNA

PnhAn~Prs could be coupled to reporter genes, then each construct ~d to one another in the presence or absence of s~all DNA-binding test molecules. In the case where multiple protein/cognate binding sites are used rOr screening, a competitive inhibitor that blocks one interaction but not the others could be ;~lPn~;~iD~l by the lack of transcription of a reporter gene in a transfected cell line or in an in vitro assay. Only one such DNA-binding ~equence, specific for the protein of interest, could be SL;L ael-ed with each assay system. This approach has ~ number of limitations i n~ A i n~ limited testing ,~ArAhility and the need to ~ aLLu~;~ the ~Lu~Liate reporter syste~ for each different proteintcognate site of interest.

C. ~'h-~osi ng and Testing an Appropriate DNA-Binding Protein.

Experiments perf ormed in support of the present invention have def ined a second approach ~or identi~ying le~l P~ having 8~lu- ~ L,L~ferential DNA-binding. In this approach small lec~lP~ binding to se~l~,P- ~c adjacent the cognate binding se~ual.c.. can inhibit the protein/cognate DNA interaction. This ~ssay has bee~

~1D~ ignPc~ to use a single DNA:protein interaction to scree~

5 ~or ~clu-~ e ~"e~fic or a6:~ut~ p.aferential DNA-binding lec~lP~ that rP~-o~n;7e virtually any sC~lu~ e.

While DNA-binding recognition sites are usually quite small (4-17 bp), the 5D~ that is ~-uL_-;L~d by the binding protein is larger (usually 5 bp or more on either 30 side of the recognition ae~u~ e -- aE; det~;Led by DNAase - I protection (Galas et al. ) or methylation interference (~iPhPnl ~ ~t et al. ) . Experiments performed in support o~

the present invention P L.~ted that a single protein and its cognate DNA-binding sequence can be used to assay 35 virtually any DNA sDTlDn~e by placing a ~eqllDnre of WO 93/004~6 PCr/US92/05476 .

interest adjacent t~ the cognate æite: a small molecule bound to the adjacent site can be detected by alterations in the binding characteristics of the protein to its cognat~ site. Such alterations might occur by either 5 steric hindrance, which would cause the dissociation of the protein, or induced conf ormational changes in the recognition gPfluA~ne for the protein, which may cause either ~~ 3 binding or more likely, decreased binding of the protein to it:s cognate site.
1) Criter;a ~or chnf~;n~ an ~ iate DNA-binding protein.
There are several ~ on~ Prations involved in choosing DNA:protein 1f~YP8 that can be employed in the assay of the present invention ; nf ~ 3 j n~
a) The ~Jff-rate (see "Definitions") should be ~ast enough to a~ h the assay in a r~A~nn~hle amount of time. The interactions of some proteins with cognate sites in DNA can bl~ ~d in days not minutes: s~ch tightly bound 1f~YP~ would inconveniently lengthen the 20 period of time it take~ to perform the assay.
b) The c~f f -rate ~hould be slow enough to allow the ~ ~ of unbound DNA in a L- ~ hlf" amount of time. For example, the level of free DNA is dictated by the rntio between t he time needed to measure free DNA and 25 the amount of free DNA that occurs naturally due to the of~-rate during th~ time period.
In view of tke above two ~n~ ~ations, practical use~ul DNA:protein off-rates fall in the range of appr~Y1r-t~ly two minutes to several days, although shorter 30 off-rates may be ~ - by faster eTl;. t and longer off-r~tes may be Al`~ ' ted by dest~h;l;~ln~ the binding conditions for the a6say.
c) A ~urther cnn~ ation is t~at the kinetic interaction8 of the DNA:protein complex i8 relatively 35 insensitive to the nucleotide 8~ ~tu~ ,eS f lanking the _ _ _ _ _ _ _ _ _ _ _ _ _ ~WO 93/00446 PCr/US92/05476 21 ~3~

- recognition ~equence . The arf inity of many DNA-binding proteins is affected by differences in the sequences ad; acent to the recognition sequence . The most obvious example of this rh~n -non is the preferential binding and cleavage of restriction enzymes given a choice of several ldentical recognition sequences with different flanking se~u-~nc~ (Polinsky et al.)O If the off-rates are affected by flAnl~in~ 5~T~n~ c the analysis of .,Live binding data between different flAnk;n~l olig~n~ otide sequ~
~ecomes dif f icult but is not i i hl~ .
2) Te5ting DNA:protein interactions for use in the assay.
Experiments perf ormed in ~upport of the present invention have identified a DNA:protein interaction that is particularly useful for the above described assay: the Herpes Simplex Virus (HSV) ULg protein that binds the HSV
origin of replication (oriS). The UL9 protein has fairly ~tringent 8~ e CrQcif;~ty. There appear to be three binding site8 f or UL9 in oxis ! SEQ ID NO :1, SEQ ID NO: 2, SEQ ID NO:17 (Elias, P. et al., Stow et ~1.). One ,_ (SEQ ID NO: 1) binds with ~t least 10-fold higher ~finity than the second 8~lu~ ~e (SEQ ID NO:2): the ~1 L~ ~escribed below use the higher affinity binding ~ite (SEQ ID NO:l).
DNA:protein a8sociation r~ t ionC are performed in solution. The DNA:protein complexes can be se},~ted from i~ree DNA by any of several methods. one particularly useful method for the initial study of DNA:protein interaCtiOns ha8 been vi ~ l i 7~tion of binding results using band shi~t gels (Example 3A). In this method DNA:protein binding rt~n~tiOnc are applied to polyacrylamide/TBE gels and the lAh~ lt~Yt~c and free labeled DNA are 8e~L~t~d electrophoreti~ally. These gels are fixed, dried, and exposed to X-ray ~ilm. The resulting autoradiogram8 are ~Y~mint~ for the amount of free probe WO 93/00446 PCr/US92/0~4~
- 22 Z11213~
that is migrating ~ a~at~ly rrom the DNA:protein c 1PY, These assays include (i) a lane containing only rree labeled probe, and (ii) a lane where the sample is labeled probe in the presenc~ of ~ large excess of binding protein.
5 The band shirt a86ay5 allow v; ~ 1; 7~tion Or the ratios between DNA:protein 1 oYo~ and rree probe. ~owever, they are le88 accuralte than filter binding a5says for rate-dotorm;n;n~ experiments due to the lag time between loading the gel and ele_LL~ ,L~tic separation or the - 9.
The filter binding method is particul~rly useful in d~to~;n;n~ the off-rates for protein:oligon~rleotide ~ lo~PIt (Example 3B). In the filter binding assay, DNA:protein l~oY~'~ are retained on a filter while free DNA passes through 1 he filter. This assay method is more 15 n6-;uLc-Le for orf-rate ~otorm;n~tions because the separation of DNA: protein ~ lo~o~ from rree probe is very rapid.
The di ~ ,allLa~e Of filter binding is that the nature o~
the DNA:protein complex cannot be directly vi~--5.1;- ~ 5O
if, for example, th~ ~ inq; -lor~lo was also a protein 20 - 'n~ for the binding of a site on the DNA lecl-lo, filter binding as8,ay8 cannot differentiate between the binding of the two proteins nor yield ; nf t tion about whether one or both proteins are binding.
There are many lcnown DNA:protein interactions that may 25 be useful in the practice of the present invention, ;nrll~;n~ (i) the D~YA protein interactions listed in Table I, (ii) bacterial, ~east, and phage systems such ~8 lambda oL-oR/cro, and (iii) modified restriction enzyme systems (e.g., protein binding in the ab~ence of divalent cation6).

3 0 Any protein that binds to a specif ic rQcognition ~ r~
may be usQful in the pre8ent invention. One constraining factor is the effect of the; A;AtQly ad~acent 8~u~ L
(the te~t ~ ) on the a~finity o~ the protein for its recognition ~ lon~ e DNA: protein interactions in which _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ ~WO 93/00446 Pcr/u592/05476 21 121~

there is little or no e~fect of the test_s~u~ es on the ~ffinity of the protein for its cognate site are preferable for use in the described assay; however, DNA:protein interactions that exhibit (test S~-{U~ API~ " L) differential binding may still be useful if algorithms are applied to the analysis Or data that _ -- Le for the differential affinity. In general, the effect of flanking sequence composition on the binding of the protein is likely to be correlated to the length of the recognition s~u~lce for the DNA-binding protein. In short, the k;n--t;rc of binding for proteins with shorter recognition u~nces are more likely to suffer from fl~nlr;ng se-lu~ .e effects, while the k;no~t;rc of binding for proteins with longer reco~nition sequences are more likely to not be a~fected by ~lanking s~T~nre composition. The present rl~lcllre proYides methods and gll;A~nre for testing the u~efulness of such DNA:protein interactions, i.e., other than the ~IL9 oriS binding site interaction, in the screening as6ay.
D. P ~y~LatiOn of Full Length IJL9 and UL9-COOH
Polypeptides.
IJL9 protein has been ~ yaL d by a number of le ' ;n~nt techni~aues (Example 2). The full length UL9 protein has l~een yL~al_~ from baculovirus infected insect cultures (Example 3A, B, and C). Further, a portion of the UL9 protein that cnnt~;n~ the DNA-binding domain (~L9-COOH) has been cloned into a bacterial expression vector and produced by bacterial cells (Example 3D and E). The DNA-- binding domain of ~L9 is ~nntA;n~ within the C-tDrm;n~l 317 amino acid~ of the protein (Weir et al. ) . The UL9-COOH polypeptide was inserted into the expression vector in-frame with the glut:~th;Qn~-S-transferase (g_t) protein.
The gst/UL9 fusion proteisl was purified using affinity ~,1 L~ t~yLaylly (Example 3E) . The vector also rnnt~;n~d a 35 Lh~ ;n cleavage site at the junction of the two 24 2~ 3~
polypeptides. Therefore, once the fusion protein was isolated (Figure 8, lane 2) it was treated with thrombin, cleaving the UL9-COOH/gst fusion protein from the gst 5 polypeptide (Figure 8, lane 3). The UL9-COOX-gst fusion polypeptide was obtained at a protein purity of greater than 9596 as determined using Coomaisie staining.
Other hybrid proteins can be utilized to prepare DNA-binding proteins of interest. For example, fusing a 10 DNA-binding protein coding sequence in-frame with a sequence encoding the thrombin site and also in-frame with the ~-galactoside coding sequence Such hybrid proteins can be isolated by af f inity or immunoaf f inity columns (Maniatis et al.; Pierce, Rockford IL) . Further, 15 DNA-binding proteins can be i~olated by affinity chromatography based on their ability to interact with their cognate DNA binding site. For example, the UL9 DNA-binding site (SEQ ~D NO :1) can be covalently linked to a solid support (e.g., Cnsr-activated Sepharose TM4B
20 beads, Pharmacia, Piscataway NJ), extracts passed over the support, the support washed, and the DNA-binding then isolated from the support with a salt gradient (Kadonaga) . Alternatively, other expression syst:ems in bacteria, yeast, insect cells or mammalian cells can be 25 used to express adequate levels of a DNA-binding protein for use in this assay.
The results presented below in regard to the DNA-binding ability of the truncated UL9 protein suggest that full length DNA-binding proteins are not required for the 30 DNA:protein assay of the present invention: only a portion of the protein c~-n~1 n1 ng the cognate site recognition function may be required The portion of a DNA-binding protein required for DNA-binding can be evaluated using a functional binding assay (Example 4A).
35 The rate of dissociation can be evaluated (Example 4B) and compared to that of the full length DNA-binding protein. However, any DNA-binding peptide, truncated or full length, may be used ~0 93/00446 Pcr/uS92/05476 ~1 ~?1~

in the ~s~ay if it meets the criteria outl; n~ in part I.C.1, "Criteria for rhnnc;n~ an a~lu~.late DNA-binding protein". T~i8 remainC tr~e whether or not the truncated form of the DNA-binding protein ha~ the same af`~inity a~
the full length DNA-binding protein.
E. El~nr~irnAl Binding and Rate of Diasoc~Ation.
The full length '~9 ~nd puri~ied '3L9-COOH proteins were tested for fl~nrtionAl activity in "band shi~'t" assays (see Example 4A). The bur~er conditions were optimized ~or DNA:protein-~7inding (Example 4C) using the '3L9-COOH
polypeptide. These DNA-binding conditions also worked well for the full-length '~L9 protein. Radiol Ah-~
r,l;qnmlrleotides (SEQ ID NO:14) that contained the ll bp 'JL9 DNA-binding recognitioll geq~l~nre (SEQ ID NO: l) were mixed with each 'lL9 protein in a~ L U~JL late binding buf~er.
The r~rt;nnC were incubated at room t~ LuLa for 10 minutes (binding occurs in less than 2 minute~) and the udu~ were separated electrophoretically on non-denaturing polyacrylamide gel_ (EYample 4A) . The degree of DNA:protein-binding could be ~ te~;n~ - from the ratio of labeled probe present in DNA:protein _ 1~Y--~ versus that pr~sent ~1; free probe. Thi~ ratio was typically f7~t~7;nD~ by optical ~c~nn;n~ of autoradiograms and comparison of band intensities. Other standard methods may be used as well for this ~'~t~;nAtir~-, such as scintillatio 1 r~ ollnt; n~ of eYcised bands. The 'ilL9-COOH
polypeptide and the full lengt_ '~L9 polypeptide, in their respective buffer conditions, bound the target nl ;~nnl~rl~ntide egually well.
The rate of ~ or;~tion was d~t~--m;n~d using co~petition assays. An excess of llnl Ah~ d 91 ;~nmlrl ~otide that contained the UL9 binding site was added to each re~ction. This lnl Ah~ d ol; ~nn~rlPntide act~ as ~ ~rer;f;r inhibitor, capturing the UL9 protein as 35 it d~oc;nt~8 from the lJ.h~ l nl ;~on~rlentide (Example WO 93~00446 Pcr/uS92/05~6 26 21 ~2130 4B). The ~ o~-;Ation rate, as detPrm~nP~l by ~ band-shift assay, l~or both full length UL9 and UL9-COOH was approximately 4 hours at 4C or approximately 10 minutes at room t~ Lu~ Neither nu-- -"P ~tfic oligonucleotides (a 5 lo~ooo-fold excess~ nor sheared herring sperm DNA ~a 100, 000-fold exce&6) ~ 1 for binding with the sl;~o~ lPntide cnnt~;nin~ the UL9 binding site.
F. or~s Flanking S~T~Pn~e Variation.
A~i - ; nn~l above, one feature o~ A DNA:proteirs-binding system for use in the assay of the present invention is that the DNA:protein interaction is not Affert~Pd by the nucleotide ~ e of the regions adjacent the DNA-binding sit:e. The sensitivity of any DNA:protein-15 binding reaction to the composition of the ~l~nlrin~
Q= ~ can be e~raluated by the fl~nrt;nn~l binding assay ~md ~ ci~tion a~isay described above.
To test the effect of flAnlrin~ n~ variation on UL9 binding to the or~s SEQ ID N0:1 ~ _ -20 ol~gnn~ lPntideri were c-,.._l_L..~;~ed with 20-30 different i.~., the test ~u_..c.2s) 1-lAnlrin~ the 5' and 3' sides of the UL9 ~inding site. Further, ol; ~nn~ ntides were .;o,._L~u- -ed with point t nn~l at severzl positions within the UI.9 birding site. Most point l~inn~ within 25 the binding site ~ u,_l recognition. Several changQti did not destroy r~o~n~tinr and these include variationEi at ~ites that dirfer ~etween the three ~L9 binding sites ~SEQ
ID N0:1, SEQ ID N0:2 and SEQ ID N0:17): the second llL9 binding site ~SEQ ID No:2) shows a ten-~old d~_~e&se in 30 ~L9:DNA binding a~finity ~Elias Qt al.) relative to the first ~SEQ ID N0:1). on the other hand, ~ ._..c., variation at the test site (also called the test B~ e), ad~cent to the screening Elite (Figure 5, Example 5), had virtually no ef~ect on bindLng or the rate of ~;- Ation.
3S The results ~ -ing that the n -- L~ot~P s~-l - e _ _ _ _ _ _ 93/00446 PCr/US9t/054~6 ~WO
, . . . .
27 ~ 3~
in the test site, which flanks the screening site, has no ef f ect on the kinetics of UL9 binding in any of the oligonucleotides tested is a striking result. This allows the direct comparison of the effect of a DNA-binding molecule on test oligonucleotides that contain different test sequences. Since the only difference between test oli~nmlrlPntides i8 the difference in nucleotide s~qll~nre at the test site(s), and since the nucleotide sequence at the test site has no effect on UL9 binding, any differential effect observed between the two test oligoml~leotides in response to a DNA-binding molecule must be due solely to the differential interaction of the DNA-binding molecule with the test sequence(s). In this manner, the insensitivity of UL9 to the test sequences flanking the UL9 binding site greatly facilitates the interpretation of results. Each test oligonucleotide acts as a control sample for all other test oligonucleotides.
This i8 particularly true when ordered sets of test sequences are tested (eg., testing all 256 four base pair sequences (Figure 13) for binding to a single drug).
Taken together the above experiments support that the UL9-COOH polypepticle binds the SEQ ID NO :1 sequence with (i) ~-~rV~Iiate ~LL-:IIYL}I, (ii) an acceptable d;~soriAtion time, and (iii) indifference to the nucleotide sequences flanking the assay (binding) site. These features suggested that the UL9/oriS system could provide a versatile assay for detection of small molecule/DNA-binding involving any number of specif ic nucleotide sequences .
The above-described experiment can be used to screen other DNA:protein interactions to determine their usefulness in the present assay.
G . Small Nolecules as Sequence-Specif ic Competitive Inhibitors .
To test the utility of the present assay system WO 93/00446 PCr/US92/05476 , ~
' 211~13 several small molecules that have sequence preferences (e.g., a preference for AT-rich versus GC-rich sequences) have been tested.
Distamycin A binds relatively weakly to DNA (KA 2 2 x 5 lO~ ~l) with a preference for non-alternating AT-rich sequences (Jain let al.; Sobell; Sobell et al. ) .
Actinomycin D binds DNA more strongly (K~ = 7 . 6 x 10 7 ~') than Distamycin A and has a relatively 6trong preference for the dinucleotide sequence dGdC (Luck et al.; Zimmer;
10 Wartel). Each of these molecules poses ~ stringent test for the assay. DiEtamycin A tests the sensitivity of the assay because of its relatively weak binding. Actinomycin D rhi~l 1 Pn~eS the ability to utilize flanking sequences since the rJL9 recognition sequence contains a dGdC
15 dinucleotide: therefore, it might be anticipated that all of the oligonucleotides, regardless of the test sequence flAnk;n~ the assay site, might be equally affected by ~ct i - ~ in D .
In addition, ]Doxorubicin, a known anti-cancer agent 2 0 that binds DNA in a S~UC~ c pref erential manner ( Chen, K-X, et al. ), has be~n tested for preferential DNA sequence binding using the assay of the present invention.
Act;- y in D, Distamycin A, and Doxorubicin have been tested for their ability to preferentially inhibit the 25 binding of UI9 to olig~.mlcleotides containing different r3~-lu~l~r~C flanking the UL9 binding site (Example 6, Figure 5). 8inding assayr were perforlDed a6 described in Example 5. These studies were completed under conditions in which UL9 is in excess of the DNA ( i . e ., most of the DNA is in 30 complex).
Distamycin A was tested with 5 different test r3equences ~lanking the UL9 screening sequence: SEQ ID NO:5 to SEQ ID NO: 9 . The re6ults shown in Figure lOA
d Lr c.te that distamycin A preferentially disrupts 93/00446 PCr/US92~05476 O~WO

binding to the test ses[uences UL9 polyT, UL9 polyA and, to a lesser extent, UL9 ATAT. Figure 10A also shows the concentration dPp~nrl~nre of the inhibitory effec~ of di6tamycin A: at 1 ~M distamycin A most of the DNA:protein S collplexes are intact (top band) with free probe appearing in the U~9 polyT and UL9 polyA lanes, and some free probe appearing in the UI9 ATAT lane; at 4 ~LM free probe can be 6een in the UL9 polyT and UL9 polyA lanes; at 16 ,lLM free probe can be seen in the UL9 polyT and UL9 polyA lanes; and 10 at 40 ~ the DNA:protein in the polyT, UL9 polyA and UI.9 ATAT lanes are near completely disrupted while some DNA:protein compleYes in the other lanes persist. These results are consistent with Distamycin A's known binding preference for non-alternating AT-rich sequences.
Actinomycin D was tested with 8 different test seSluences flanking the UI9 screening sequence: SEQ ID NO:5 to SEQ ID NO: 9, and SEQ ID NO :11 to SEQ ID NO :13 . The results 6hown in Figure 10B d-- ~Lc~te that acti~ ~.in D
preferentially disrupts the binding of UL9-COOH to the 20 oligon~rlPotides U~9 CCCG (SEQ ID NO:5) And UL9 GGGC (SEQ
ID NO:6). These oli~n~lrleotides contain, respectively, three or five dGdC dinllrleotides in addition to the dGdC
dlnllrleotide within the UL9 recognition sequence. This result is consistent with ACtinl ~-;in D's known binding 25 preference for the ~linl~rl~otide s~uc:ln_e dGdC. Apparently the presence of a potential target site within the screening se~uence (oriS, SEQ ID NO:1), a6 mentioned above, does not interfere with the function of the assay.
Doxorubicin was tested with 8 different test seguences 30 flanking the UL9 screening sequence: SEQ ID NO:5 to SEQ ID
NO:9, and SEQ ID NO:11 to SEQ ID NO:13. The result6 shown in Figure 10C ' ~; Ll-te ~hat Doxorubicin preferentially disrupts binding to oriEco3, the test SPqtlPnre of which differs from oriEco2 by only one base (compare SEQ ID NO: 12 35 and SEQ ID NO:13). Figure lOC also shows the c~ .e.,l_l~tion _ _ _ _ _ _ _ ~3erPn~Prl--e of the i3lhibitory erfect of Doxorubicin: at 15 ,ILM Doxorubicin, the UL9 binding to the screening sequence is strongly affected when oriEco3 is the test seguence, and more mildly affected when polyT, UL9 GGGC, or oriEco2 was 5 the test 8eq~lPnre; and at 35 ,lLM Doxorubicin most DNA:protein _ 1PY~5 are nearly completely disrupted, with UT 9 polyT and UL9ATAT showing 60me DNA still complexed with protein. Also, effects similar to those observed at 15 llM
were also o~séLved using Doxorubicin at 150 nM, but at a 10 later time point.
Further incubation with any of the drugs resulted in additional disruption of binding. Given that the one hour incubation time of the above assays is eguivalent to several half-lives of the DNA:protein complex, the 15 additional disruption of binding suggests that the on-rate for the drugs is comparatively slow.
T~e ability o~ the assay to distinguish se~luence binding preference Using weak DNA-binding molecules with poor se~uè~c~ _~ecificity tsuch as distamycin A) is a 20 stringent test. Accordingly, the present assay seems well-suited for the identification of le~ P~ having better se~ .ce specificity and/or higher seguence binding af f inity . Purther, the results ~ ~L ate s~ ~I Pl~ ~e preferential binding with the known anti-cancer ~rug 25 Doxorubicin. This result indicates the assay may be useful for screening mixture5 for molecules displaying similar charaCteri5tics that could be sllhseqllPntly tested for anti-cancer activities as well as seguence-specif ic binding.
Other c ' ~ that may be suitable f or testing the 30 present DNA:proteLn 6ystem or for defining alternate DNA:protein systems include the following: e~ h;n~ ~in, which preferentially binds to the seguence (A/T) CGT
(Quigley et ~1. ); 5mall inorganic molecules, such as cobalt hPY~m;nP, that are known to induce Z-DNA formation 35 in regions that contain repetitive GC seguences (Gessner ~t ~WO 93/00446 PCr/US92/05476 21 1 ? ~ ~

al. ~; and other DNA-binding proteins, such as EcoR1, a restriction Pn~lnn~lclease.
~ . Theoretical considerations on the concentration of 5 assay -n~nts.
There are two ,- __ -ntS in the assay, the test sequence (oligonucleotide) and the DNA-binding domain of UL9, which is described below. A number of theoretical considerations have been employed in establishing the assay 10 system of the present invention. In one ~ L of the invention, the assay is used as a mass-EcL~l.ing assay. In this capacity, small volumes and col~cel,~L~ltions were desirable. A typical assay uses about 0.1 ng DNA in a 15-20 ,~Ll reaction volume (approximately 0 . 3 nM) . The proteia 15 c-,..~t:.-L~ ~tion is in excess and can be varied to increase or decrease the sensitivity of the assay. In the simplest scenario, where the small molecule is acting as a competitive inhibitor via steric hindrance, the system k;n~tit c can be described by the following equations:
D + P D:P, where k,p/kbp = ~qp ~ [D:P]/rD] [P]
and D + X D:X, where k~/kb~ = tD:X]/[D] [X]
D = DNA, P = protein, X = DNA-binding molecule, k~p and kf~ are the rates of the forward reaction for the DNA:protein interaction and DNA:drug 3 0 interaction, respectively, and kbp and kb~ are the rates of the backwards reactions f or the respective interactions. Brackets, [ ], indicate molar concentration of the _ o Ls.

WO 93/0~446 PCr/US92/05476 In the assay, ~oth the protein, P, and the DNA-binding ler~1l e or drug, X, are competing for the DNA. If steric hindrance is the: ,f ` 5~ni F'n Of inhibition, the assumption can be made that the two molecules are ~ _~ inq for the 5 same site. When the c~ Lc-tion of DNA equals the cu~ Lration of the DNA:drug or DNA:protein complex, the eql~l;hrium bindi~g constant, K~q~ is equal to the reciprocal of the protein c ~ Gtion (l/[P]). For UL9, the calculated X4,~L9 ' 2 . 2 x 109 ~1. When all three 10 . _ - ts are miYed together, the relati~n~hir between the drug and the protei n can be described as:
Keqp = Z (K~
15 where "z" defines the difference in affinity for the DNA
between P and X. For example, if z =4, then the affinity of the drug i~ 4-fold lower than the affinity of the protein for the D]NA molecule. The c ~ ~r.L- ~tion of X, therefore, must be 4-fold greater than the cc..~c~ Leltion of 20 P, to compete equally for the DNA molecule. Thus, the equilibrium affinity constant of UL9 will define the minimum level of detection with respect to the co..c~ L~tion and/or affinity of the drug. Low affinity DNA-binding --1P~111P~ will be detected only at high 25 .;o..c;~,-LL~,tions; likewise, high affinity molecules can be detected at relatively low c.~,nce~.LLations.
With certain test sequences, complete inhibition of UL9 binding at markedly lower ao~c~ Lc.Lions than indicated by these analyses have been o~s~rved, probably indicating 30 that certain sites among those chosen for fPs~ih~ 1 ity studies have affinities higher than previously p--hli~hPrl.
Note that relatively high c .l~-- L~c.tions of known drugs can be utilized for testing sequence speci~icity. In addition, the binding .;~,I-sL~..L of ULg can be readily lowered by ~0 93/00446 PCr/US92/054~6 21 lZ130 nltering the pH or salt c~ -L~ ation in the assay if it is desirable to screen for molecules that are found at low CU..ct~.~Lation (eg., in a fermentation broth or extract).
Analyses such as presented above, become more complex 5 if the inhibition is allosteric (nc,~- _titive inhibition) rather than competition by steric hindrance.
Nonetheless, the probability that the relative effect of an inhibitor on different test sequences is due to its relative and differential ~ffinity to the different test 10 SeS~nrC~ is fairly high. This is particularly true in the assays in which all sequences within an ordered set (eg., possible se~lu~ s of a given length or all possible variations of a certain base composition and defined length) are tested. In brief, if the effect of inhibition 15 in the assay is particularly strong for a single sequence, then it is likely that the inhibitor binds that particular sequence with higher affinity than any of the other sequences. Fur~h~ ~, while it may be difficult to ~l~t~rmi n-~ the absolute af~inity of the inhibitor, the 20 relative affinities have a high probability of being r~nn~hly accurate. This information will be most useful in facilitating, for instance, the refinement of molecular - - ~^1 i n~ systems.
I. The use of the assay under conditions of high 25 protein col.ce..LLation.
When the screening protein is added to the assay system at very high C~ LatiOns, the protein binds to nol. _~ecific sites on the ol i~-~n~ 1eotide in addition to the screening sequence. This effect has been ~' ~L~ted 30 using band shift gels: in particular, when serial dilutions are made of the UI-9 protein and the dilutions are mixed with a fixed col.~-e..~Lation of c~ Qn~lc1eotide, no binding (as seen by a band shift) is observed at very low dilutions (e.g., 1:100,000), a single band shift is observed at 35 moderate dilutions (e.g., 1:100) and a smear, migrating _ _ _ _ _ _ _ _ _ _ _ _ _ WO 93/00446 PCr/US92/05~
21t213~ 34 higher than the single band Observed at moderated dilutions, is observed at high c-,.,ce--~La.tions of protein (e.g., l:lO). In the band shift assay, a smear is indicative of a mixed population of complexes, all of which 5 presumably have the screening protein binding to the screening sequence with high affinity (e.g., for UL9, K~ ~
1.1 X 10~ ) but in addition have a larger nu~ber of proteins bound with markedly lower af f inity .
Some of the low affinity binding proteins are bound to 10 the test sequence. In experiments performed in support of the present invention, using mixtures of UL9 and glutathione-S-transferase, the low affinity binding proteins are likely UL9 or, less likely, glutathione-S-transferase, since these are the only proteins in the assay 15 mixture . These low af f inity binding proteins are significantly more sensitive to interference by a molecule binding to the test sequence f or two reasons . First, the interference is li]cely to be by direct steric hinderance and does not rely on induced conformational changes in the 20 DNA; secondly, the protein binding to the test site i6 a low affinity binding protein because the te6t site Ls not a cognate-binding s~qU~n~-e. In the case of T~L9, the difference in affinity between the low affinity binding and the high affinity binding appears to be at least two orders 2 5 of magnitude .
Experiments performed in support of the present invention d LLc-te that the filter binding assays capture more DNA:protein complexes when more protein is bound to the DNA. The relative results are accurate, but 30 under moderate protein col.cellLLstions, not all of the bound DNA (as c' LLc~ted by band shift assays) will bind to the filter unless there i6 more than one DNA:protein complex per oligonucleotid~ (e.g., in the case of ULg, more than one UL9:DNA complex). This makes the assay exquisitely 93/00446 PCr/US92/05476 ~ 130 sensitive under conditions of high protein cc,l.~e"LLation.
For instance, when actinomycin binds DNA at a test site under condition5 where there i8 one DNA:UL9 complex per oligonucleotide, a differential-binding effect on GC-rich 5 oligonucleotides has been observed (see Example 6). Under conditions of high protein c ~ ation, where more than one DNA:UL9 complex is found per nl iqnn~ leotide~ the differential effect of actir- ~cin D is even more marked.
These results suggest that the effect of actinomycin D on 10 a test site that is weakly bound by protein may be more readily d~-tect~P~l than the erfect of actinomycin D on the adjacent screening sequence. Therefore, employing high protein ~u~ JILratiOns may increase the sensitivity of the assay .
II. Capture/Detection Systems.
As an alternative to the above described band shift gels and filter binding assays, the mea,u~ t of inhibitors can be monitored by measuring either the level 20 of unbound DNA in the y e:sel~ce of test molecules or mixtures or the level of DNA:protein complex ~ in;n~ in the ~L~sence of test molecules or mixtures. I~rsuL.
may be made either at equilibrium or in a llcinetic assay, prior to the time at which equilibrium is reached. The 25 type of mea:~UL ~ L is likely to be dictated by practical factors, such as the length of time to equilibrium, which will be detPrmined by both the kinetics of the DNA:protein interaction as well as the kinetics of the DNA: drug interaction. The results (ie., the detection of DNA-30 binding molecules and/or the determination of their6~ u--n~e preferences) should not vary with the type of mea,juL~ -- L taken (kinetic or equilibrium).
Figure 2 illustrates an assay for detPrtin~ inhibitory lec~llPu based on ~heir ability to preferentially hinder 35 the binding of a DNA-binding protein. In the ple se"ce of _ _ _ _ _ _ _ _ WO 93/00446 PCr/US92/05~

an inhibitory molecule (X) the equilibrium between the DNA-binding protein and it5 binding site (screening seguence) i8 di5rupted. The DNA-binding protein (0) is displaced from DNA (/) in the pLes~ e of inhibitor (X), the DNA free 5 of protein or, alternatively, the DNA:protein complexes, can then be captured and detected.
For maximum sensitivity, unbound DNA and DNA:protein ,1 ~YC~ should b~ sequestered f rom each other in an ef f icient and rapid manner ~ The method of DNA capture 10 should allow for the rapid removal of the unbound DNA from he protein-rich mixture cnntAin;ng the DNA:protein complexes .
Even if the l;est molecules are ~pecif ic in their interaction with DNP. they may have relatively low af f inity 15 and they may also l~e weak binder5 of no~ ecif ic DNA or have non-specific interactions with DNA at low tions. In either case, their binding to DNA ~ay only be transient, much like the tr~nsient binding of the protein in 5oll~ti nn . Accordingly, one feature of the assay 20 is to take a -ole~ A- r-..a~shv~ of the ~qn;l;hrium state of a ~;olution compri5ed of the target/assay DNA, the protein, and the inhibitory test l~ . In the pL~R~h~ of an inhibitor, the amount of DNA that is not bound to protein will be greater than in the absence of ~n inhibitor.
25 Likewise, in the pL.R~I.ve of an inhibitor, the amount of DNA that is bound to protein will be lesser than in the absence of an inhibitor. Any method used to separate the DNA:protein complexes from unbound DNA, 5hould be rapid, because when the ca~pture system is applied to the solution 30 (if the capture system is irreversible), the ratio of unbound DNA to DNA:protein complex will change at a prede~rm;ne-d rate, based purely on the off-rate of the DNA:protein comple~. Thi5 5tep, therefore, determines the limit5 of ba- hgLvu..d. Unlike the protein and inhibitor, 35 the capture 5ystem 5hould bind rapidly and tightly to the ~WO 93/00446 PCI/US92/OS476 21 7213a DNA or DNA:protein complex. The longer the capture system i8 left in contact with the entire mixture of unbound DN~
and DNA:protein complexes in solution, the higher the ba- k~L~u..d, regardless of the ~L~S~ e or absence of inhibitor.
Two ~ l~ry capture systems are described below for use in the present assay. One capture system has been devised to capture unbound DNA (part II.A). The other has been devised to capture DNA:protein complexes (part II.B).
Both systemC are ohl ~ to high th~ -,u~ u- screening assays. The same detection methods can be applied to molecules l a~LuLed using either capture system (part II.C~
A. Capture of unbound DNA.
one capture system that has been developed in the course of experiments perf ormed in support of the present invention utilizes a -LLe~L.lvidin/biotin interaction for the rapid capture of unbound DNA from the protein-rich mixture, which ;nr]llA~c unbound DNA, DNA:protein complexes, excess protein and the te5t ~ 1DCt11DC or te6t mixtures.
SLLe~-clvidin binds with exLr~ ly high affinity to biotin (Kd ~ 10 ~ (Chaiet et al.; Green~, thus two advantages of the "LLe~Lavidin/biotin system are that binding between the two l~o~llec can be rapid and the interaction is the ~LL~r.y~nL known nol. c;.,v.,lent interaction.
In this detection system a biotin molecule is covalently ~LL-scl.ed in the ol ;gon~ lDotide screening sequence (i.e., the DNA-binding protein's binding site).
This attA, L is accomplished in such a manner that the binding of the DNA-binding protein to the DNA is not de~LL~,yed. Further, when the protein is bound to the biotinylated sD~IU~n~~e, the protein prevents the binding of streptavidin to the biotin. In other words, the DNA-binding protein is able to protect the biotin from being reco~n; ~ed by the streptavidin. This DNA:protein . , _, . _,, . _,, , . . ,,, ,,, , , . ,, _ _ _ _ _ _ WO 93/00446 PCr/US92/05476 21 1213~

interaction is illustrated in Figure 3.
The capture sy~tem is described herein $or use with the UL9/oriS system ~escribed above. The following general testing principles can, however, be applied to analysis of 5 other DNA:protein interactions. The usefulness of this system depends on the biophysical characteristics of the particular DNA:protein interaction.
1) Modi f ication of the protein recognition S~S~uP~.aQ with biotiln.
The recognition sequence for the binding of the UL9 tKoff et al- ) protein is underlined in Figure 4.
01; gnnllr~ eotides w~re synth~ ; 7P~ that contain the UL9 binding site and site-specifically biotinylated a number of location~ throughout the binding sequence (SEQ ID NO: 14;
15 Example 1, Figure 4). These biotinylated olignn~lrl~otides were then used in band shift assays to determine the ability of the UL9 protein to bind to the olignn~rl~otide.
These experiments using the biotinylated probe and a non-biotinylated probe as a control d LLate that the 20 pLeSel.ce of a biotin at the #8-T (biotinylated deu~yuLidine) position of the bottom strand meets the requirement6 liste~ above: the l,esel-c~2 of a biotin moiety at the ~8 position of the bottom strand does not markedly affect the ~peGifirtty of UL9 for the recognition ~ite;
25 further, in the presence of bound UL9, ~,LLe~ vidin does not recogni~e the presence of the biotin moiety in the oligon~lrl~otide. ]Biotinylation at other A or T positions did not have the two n~C~ ry characteristicæ ( i . e ., UL9 binding and protection from b,L~elJL-vidin): biotinylation 30 at the adenosine in position #8, of the top strand, prevented the binding of UL9; biotinylation of either a~l~nnfii no~: or thymidines (top or bottom strand) at positions ~3, #4, #10, or ~11 all allowed binding of UL9, but in each case, streptavidin also was able to r~co~n;s:e 35 the ~L~Q..c-: of the biotin moiety and thereby bind the WO 93/00446 PCr/US92/05476 oligonucleotide in the presence of UI,9.
The above result (the ability of UL9 to bind to an oligonucleotide containing a biotin within the recognition ~;equence and to protect the biotin from streptavidin) was 5 unexpected in that methylation interference data (Koff et al. ~ 6uggest that methylation of the deoxyguz~nosi n-~residues at positions ~7 and ~9 of the recognition sequence (on either side of the biotinylated deoxyuridine) blocks UL9 binding . In these methylation interf erence lO experiments, gll~nos;n~C are methylated by dimethyl sulfate at the N7 position, which cuLL~ u"ds structurally to the 5-position of the pyrimidine ring at which the deoxyuridine i~ biotinylated. These moieties all protrude into the major groove of the DNA. The mcthylation interference data 15 ~uggest that the ~7 and #9 position deoxyg~l~nnsines are contact points f or ULg, it was theref ore unexpected that the ~L.s~l~ce of a biotin moiety between them would not interf ere with binding .
The binding of the full length protein was relatively 20 unaffected by the presence oi a biotin at position ~8 within the UL9 binding 6ite. The rate of dissociation was similar for full length ~JL9 with both biotinylated and un-biotinylated ol; g~r~ leotides . However, the rate of ~licSociAtion of the truncated UT9-CûO~ polypeptide waE
25 faster with the biotinylated ol ignn~ Potides than with non-biotinylated oligonucleotides, which is a rate comparable to that of the full length protein with either DNA .
The binding conditions were optimized for UL9-COOH so 30 that the off-rate of the truncated UL9 from the biotinylated ol ;gonll~leotide was 5-10 minutes (optimized conditions are given in Example 4), a rate compatible with a mass screening assay. The use of multi-well plates to conduct the DNA:protein assay of the present invention i8 WO 93/00446 PCr/US92/OS476 one approach to mass screening.
2) Capture of site-srec;fic biotinylated oligQn~ otides.
The streptavidin:biotin interaction can be employed in 5 several different ways to remove unbound DNA from the solution containing the DNA, protein, and test molecule or mixture. M~n~tiC poly~yL~ne or agarose beads, to which ~r._~Lavidin is covalently attached or attached through a covalently attached biotin, can be exposed to the solution 10 for a brief period, then removed by ufie, respectively, of magnets or a f ilter mesh . Magnetic streptavidinated beads are currently the method of choice. SLL~tavidin has been used in many of these experiments, but avidin is equally useful .
An example of a second method for the removal of unbound DNA is to attach ~.LLe~avidin to a filter by first linking biotin to the filter, binding ,L- e~L~Yidin, then blorl~;nq non~re~ific protein binding sites on the filter with a nnncpDc;fic protein such as albumin. The mixture is 20 then passed through the filter, unbound DNA is ~;alJLuLed and the bound DNA passes through the f ilter .
One convenient method to sequester ~c~Lu e:d DNA is the use of liLre ~ L~-vldin c~ uy~ted superpa~
polystyrene beads as described in Example 7. These beads 25 are added to the assay mixture to capture the unbound DNA.
After capture of DNA, the beads can be retrieved by placing the reaction tubes in a magnetic rack, which sequesters the beads on the reaction chamber wall while the assay mixture is removed and the beads are washed . The ~ LUL èd DNA is 30 then detected using one of several DNA ~ c~irn systems, as described below.
Alternatively, avidin coated agarose beads can be u~ed. Biotinylated agarose beads (; Ibi 1; 79d D-biotin, Pierce) are bound to avidin. Avidin, like s~L~e~JLavidin~
35 has four binding site8 for biotin. One of these binding WO 93/00446 PCr/US92/05476 .
21 ~3~

sites is used to bind the avidin to the biotin that is coupled to the agarose beads via a 16 atom spacer arm: the other biotin binding sites remain available. The beads are mixed with binding mixtures to capture biotinylated DNA
5 tExamPle 7) Alternative ~ethods (Harlow et al. ) to the bead capture methods just described include the following s~L~:~L~-vidinated or avidi~ated supports: 1~ ~ pLu~ein-binding filters or 96-well plates.
B) Capture of DNA:protein complexes.
The amount of DNA:protein complex r~ ;nin~ in the assay mixture in the pLcsel,Ce of an inhibitory molecule can also be rlP~PrminPd as a measure of the relative effect of the inhibitory molecule. A net decrease in the amount of DNA:protein complex in response to a test molecule is an indication of the presence of an inhibitor. DNA molecules that are bound to protein can be ~ayLuL~:d on nitrocellulose filters. Under low salt conditions, DNA that is not bound to protein freely passes through the filter. Thus, by passing the assay mixture rapidly through a nitrocPllulose filter, the DNA:protein complexes and unbound DNA molecules can be rapidly separated. This has been accomplished on nitrocPlll~loFe discs using a vacuum filter a~a~atu;- or on slot blot or dot blot apparatuses (all of which are available from Srhlpirhpr and Schuell, Reene, NH). The ~ssay mixture i8 applied to and rapidly passes through the wetted nitrocPl 11~l nse under vacuum conditions. Any apparatus employing nitrocellulose filters or other filters capable of ret~ining protein while allowing free DNA to pass through the filter are suitable for this system.
C) Detection systems.
For either of the above capture methods, the amount of DNA that has been ~ LuLed is quantitated. The method of quantitation depends on how the DNA has been IJL ~ared . If the DNA is r~lio~ctively labelled, beads can be counted in 1~ srintill;ltion counter, or autoradiographs can be taken of WO 93/00446 PCr/US92/05476 42 21 1~3~
dried gels or nitrccel 1ll1 ~rce ~ilter6. The amount of DNA
has been quantitated in the latter case by a densitometer (Nolecular Dynamic~, Sunnyvale, CA); alternatively, f ilters or gels cQnt~; n; ng radiolabeled samples can be 5 q;uantitated using a phosphoimager (Mnler~ul Ar Dynamics) .
The .;~Lu-~d DNA may be also be detected using a chPm;ll~minpccpnt or colorimetric aetection system.
p~ ;nlAhPlling and rhpmilllm;np~c~ e (i) are very sensitive, allowing the detection of sub-femtomole 10 quantitie6 of ol~ n~rlPotide, ana (ii) use well-ect~hl;chP~ techniques. In the case Or rhPm;1llminPCCPnt detection, protocols have beQn devised to ~ te the requirements of a mass-screening assay. Non-isotopic DNA
detection techniquels have pr;nr;rs~l Iy in~;u-~ ted alkal ;nP
15 phosphatase as the detectable label given the ability of the enzyme to give a high turnover of substrate to product and the avAilAhil;ty of nuLDL..ltes that yield chPm; lllm;nP~cPnt or colored products.
1) Radioactive 1 ~hPl i n~.
Nany of the experiments described above for UL9 DNA:protein-binding studies have made use of radio-lAhellPd ol i ~or ll(-lPotides . The techniques involved in radiolAhPllin~ of ol;~QnllrlPntides have been tq;C~llcspd above. A specific activity of 10~-109 dpm per ~g DNA is routinely achieved using standard methods (eg., end-lAhQl ;ng the oligonucleotide with ~r~Pn~ ;np ~y_[32p]-s~
tr; rh- crh~-te and T4 polynucleotide kinase) . This level of specific activity allows small amounts of DNA to be measured either by autoradiography of gels or rilters exposed to film IDr by direct co~lnt;n~ of samples in scintillation f luid .
2) rhPm; lllm;nP~cPnt detection.
For rhPmi 1 llm; nPCCP'lt detection, digoxigenin-l 71hel 1 Pd Ol ;~rnllrl ~Potides (Example 1) can be detected using the _ _ =

WO 93/00446 PCr~US92/05476 .

chemill~m~nPccPnt detection system "Su~Ln~ LIGHTS,"
developed by Tropix, Inc. The detection system is diagrammed in Figures llA and llB. The technique can be applied to detect DNA that has been u a~l_uLed on either 5 beads, filters, or in solution.
AlkAl ~nP phosphatase is coupled to the ~;a~LuL~d DNA
without interfering with t}le capture system. To do this several methods, derived from commonly used ELISA (Harlow et al.; Pierce, Rockford IL) techniques, can be employed.
10 For example, an antigenic moiety is in- uL~uLated into the DNA at sites that will not interfere with (i) the DNA:protein interaction, (ii) the DNA:drug interaction, or (iii) the capture system. In the UL9 DNA:protein/biotin system the DNA has been end-l~hPllecl with diqnyiqQnin-ll-15 dUTP (dig-dUTP) and tPrmin~l transferase (Example 1, Figure

4~. After the DNA was ~ap~uL~d and removed from the DNA:protein mixture, an anti-digoxigenin-Alk,-l inP
phosphatase conjugated antibody was then reacted (Boehringer MAnnhPim, Tn~iAnArolis IN) with the 0 digoxigenin-cnnt~ i n i n~ oligonucleotide . The antigenic qnYigPn~n moiety was rPco~ni7s~1 by the antibody-enzyme cc.~.juyc-te. The presence of dig-dUTP altered neither the ability of UL9-COOH protein to bind the oriS SEQ ID NO:1-cnnt~;nin~ DNA nor the ability of streptavidin to bind the5 incorporated biotin.
Captured DNA was cletectPcl using the ~lkAl inP
phosphatas~ ~u., j uy ated ant 1 hoA i PC to d i gnY i qPn i n as follows. One chemilllminPccPnt substrate for AlkAl inP
phosphatase is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-30 rhn~ ylOxy) phenyl-1,2-dioxetane disodium salt (ANPPD) (Example 7). D~ hn~ ylation of ANPPD results in an unstable _ ', which dP --~-, rPl--Acin~ a prolonged, steady Pmi csion of light at 477 nm. Light measu.~ ~r L is very sensitive and can detect minute quantities of DNA

WO 93/00446 PCr/US92/05476 .
44 21 121~
(e.g., 1O2-1O3 attomoles) (Example 7).
Colorimetric substrates f or the A 1 k;- ~ i n~ phosphatase system have also been tested and are useable in the present assay system.
An alternative to the above biotin capture system is to use digoxigenin in place of biotin to modify the ol ;g~n~cleotide at a site protected by the DNA-binding protein at the assay site: biotin is then used to replace the t~ Y;gPn;n moieties in the above described ~l~te~t;~n system. In this aLL_, the anti-~;gQv;~r~n;n antibody is used to capture the ol;grmlrleotide probe when it is free of bound protein. SLL~ La~,idin cG..juy,lted to ~lk~l ;nr, phosphatase is then used to detect the ~Lesal~ce of captured oligonucleotides .
D) Alternati~1~e methods for d~t~ct;n~J molecules that increase the affinity of the DNA-binding protein for its cognate site.
In addition to identifying molecules or - `- that cause a decreased af f inity of the DNA-binding protein f or 20 the screening s~r~nre, molecules may be identified that incrense the affil~ity of the protein for its cognate binding site. In this case, leaving the capture system for unbound DNA in contact with the assay for increasing amounts of time allows the establ; I ~ of a f ixed of f -25 rate for the DNA:protein interaction (for example SEQ IDNO:l/UL9) . In the presence of a st~hil ;~inrJ molecule, the off-rate, a5 detec~ed by the capture system time points, will be decreased.
Using the capture system for DNA:protein ~ Yr~I: to 30 detect --lec~ that increase the affinity of the DNA-binding protein for the screening seuu~=,.. e requires that an excess of unlAhr~lPd ol;~n~lrleotide containing the UL9 binding site (but not the test ~ -r~) is added to the assay mixture. '~his is, in effect, an off-rate experiment.

WO 93/00446 PCr/US92/05476 .
, 45 21 ~73~3 In this case, the control 5ample (no te5t molecules or mixtures added) will show a fixed off-rate (ie., 6amples would be taken at f ixed intervals after the addition o~ the llnl ~hPl e~ competition DNA molecule, applied to S nitrocellulose, and a decreasing amount of radiolabeled DNA:protein complex would be observed). In the presence of a DNA-binding test --lecllle that Pnh:-nred the binding of IJL9, the of f -rate would be decreased 1 ie ., the amount of radiola~eled DNA:protein complexes observed would not 10 decrease as rapidly at the f ixed time points as in the control sample).
III. Utility A. The Usefulness of Seyuence-Specific DNA-Binding 15 t- lec~
The present invention def ines a high thL uu~l. put in vitro screening assay to test large libraries of biological or -hPm;r~l mixtures for the p~e:sel~ce of DNA-binding molecules having sequence binding preference. The assay is 20 also capable of detP~m;n;n~r the 6PTl~ ..r,eciricity and relative affinity of known DNA-binding molecules or purified unknown DNA-binding molecules. S~yu~ e~.ific DNA-binding molecules are of particular interest for several reasons, which are listed here. These reasons, in 25 part, outline the rationale for determining the usefulness of DNA-binding ~ 1PC11 1 P~: as therapeutic agents:
1) Generally, for a given DNA:protein interaction, there are 6everal ~hnll~:~nrl~ fewer target DNA-binding seqllPnr~P~ per cell than protein molecule5 that bind to the 30 DNA. Accordingly, even fairly toxic molecules might be delivered in sufficiently low v v..v~..LLation to exert a biological effect by binding to the target DNA seqnPn~-P~:.
2) DNA has a relatively more well-defined structure compared to RNA or protein. Since the general ~LLUVLU'~ of 35 DNA has le55 tertiary 6tructural variation, identifying or WO 93/00446 PCr/US92/05476 .
~1 1?130 ~j~nin~ specific binding molecules should be easier for DNA than for either RNA or protein. Double-stranded DNA is a repeating ~LuuLuL3 of deoxyrih~n~ lP~tides that 6tack atop one another to form a linear helical structure. In s this manner, DNA has a regularly repeating " lattice"
~LU._Lu~-~ that makes it particularly -hl~ to molecular - '~1; n~ ref 1- and hence, drug design and development .
3) Many "single-copy" genes (of which there are only 1 or 2 copies in the cell) are transcribed into multiple, potentially ~ h^U-9n~lC, of RNA lec~ , each of which ~ay be translated into many proteins. Accordingly, targeting any DNA site, whether it is a regulatory sequence or ~
coding or nt~nnorl;n~ sequence, may require a much lower drug dose than targeting RNAs or proteins.
Proteins (e.g., enzymes, receptors, or structural proteins) are currently the targets of most theL.I~u-ic agents. Nore recently, RNA molecules have become the target~; for antise~se or ribozyme therapeutic molecules.
4) Blor~ng the function of a RNA, which encode~ a protein, or of a OU~L-~IJ ~lin~ protein, when that protein regulates several cell~llsr genes, may have detrimental ef f Qcts: particularly if some of the regulated genes are kll~t for the survival of the cell. However, hlorkin~
a DNA-binding sit~e that is specific to a single gene regulated by such a protein results in reduced toxicity.
An example situation (4) is HNF-1 binding to Hepatitis B virus (HBV): ~;IF-l binds an HBV ~nh.9nr~lr se~, e and stimulates transcription of HBV genes tChang et al. ) . In a normal cell HNF-~ is a nuclear protein that appears to be JUL ~ f or the regulation of many genes, particularly liv~ ,ecific genles (Courtois et al. ) . If molecules were isolated that speCif ically bound to the DNA-binding dor~ain o~ HNF-1, all of the genes regulated by HNF-l would be 35 down-regulated, ~r~ tn I both viral and c~l lul;lr genes.

Wo 93/00446 PCr~US92/OS476 .
-Such a drug could be lethal since many of the genesregulated by HNF-1 may be n~C~c~c;~ry for liver function.
However, the assay of the present invention presents the ability to screen for a molecule that could distinguish the

5 HNF-1 binding region of the Hepatitis B virus DNA from co~ 1 Ar HNF-1 sites by, for example, including divergent flanking se~u,~ es when screening for the l~ lo, Such a molecule would specifically block HBV expression without effecting ~ r gene expression.
B. General Applications of the Assay.
General applications of the assay include but are not limited to screening libraries o~ uncharacterized (e.g., bjQlo~;c~ ~h~mic~l or synthetic _ ) for se~uenc~ e~ecific DNA-binding lec~ c (part III.B.1);
15 de1 ~rm;n;n~ the sequel.ce ~yecificity or preference and/or relative affinities of DNA-binding ~ l~ c (part III . B . 2 ); and testing of - ' ; f i ecl derivatives o~ DNA-binding molecules for altered specificity or affinity (part III.B.3). In particular, since each test - ' is 20 screened against up to 4N sequences, where N is the number of bACPr~l;rS in the test se~u~ance, the method will y_.lcL~te up to 4N ,,LLu.;LuL~/activity data points for analysing the rela~; nn ch; r between ~ . LL uc LUL c and binding activity, as evidenced by protein binding to an adjacent 25 sequence.
1) Mas~-_Lcel~ing of libraries for the ~l~sel.~e of sequence-specif ic DNA-binding molecules .
Many organizations (eg., the National Institutes o~
Health, pharmaceutical and rh~m;C~l c ~,L~ulcLtions) have 30 large libraries of rh~m; ~ l or biological _ '~ from synthetic processes, fermentation broths or extracts that may contain as yet unidentified DNA-binding molecules. One utility of the assay of the present invention is to apply the assay system to the maf-~-E_L ~elling o~ these libraries _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ , . .

6 PCI /US92/05476 .
- ?1 ~?~3-~) -o~ dif f erent broth5, extracts, or mixtures to detect the specific samples that contain the DNA-binding molecules.
once the specif ic mixtures that contain the DNA-binding molecules have been identified, the assay has a further 5 usefulness in aidin~ in the purification of the DNA-binding le~ e from the cl~ude mixture.
As purificaiton schemes are applied to the mixture, the assay can be used to test the fractions for DNA-binding activity. The assay is: -~hle to high thL~u~l~uL (eg., 10 a 96-well plate for~at automated on robotics equipment such as a Beckman Biomelc ~.~Ll.~.~al ion tBeckman, Palo Alto, CA]
with detection using so~ nt~ ~ ted plate-rQading densitometers, 1~ Dr8~ or rh~srhoir-7Qrs).
2 ) The assay of the present invention is also 15 useful for screening molecules that are currently described in the literature as DNA-binding molecules but which. have uncertain DNA-bindi.ng sequence specif icity ( ie ., haviny either no well-defined preference for binding to ~r~c;fic DNA s~uuences or having certain higher af f inity binding 20 sites but without dlefining the relative preference for all pOc~h~R DNA binding se~u~l~c~s). The assay can be used to ~ Q~m~nD the specific binding sites for DNA-binding molecules, among all possible choices of seql~Qr^-e that bind with high, low, or moderate affinity to the DNA-binding 25 --lDC~1D. ACt;- r in D, Dist~mycin A, and Doxorubicin (Example 6) all provide lec of molecules with these modes of binding. ~any anti-cancer drugs, such as Doxorubicin tsee Example 6) show binding preference for certain identified DNA se:~luencts, although the absQlute 30 highest and lowest specificity se~ue---.es have yet to be dQ~Qrminpd~ because, until the invention described herein, thQ methods (Salas, X. and Portugal, J.; ~-11 in~nD, C. nnd Phillips, D.R.; Phillips, D.R.,; and Phillips, D.R. et al.) for detecting dirf~rential affinity DNA-binding sites for 35 any drug were limited. Doxorubicin is one of the most Wo 93/00446 PCr~US92/05476 21 ~2130 widely used anti-cancer drug6 currently available. As shown in Example 6, Doxorubicin is known to bind some sequences preferentially. Another example of such sequence binding preference is Daunorubicin (Chen et al. ) that 5 differs slightly in ~L~ u~LuL~ from Doxorubicin (Goodman et al. ) . Both Daunorubicin and Doxorubicin are members of the anthracycline antibiotic family: antibiotics in this family, and their derivatives, are important antitumor ~gent6 (Goodman et al. ) .
The ascay of the present invention allows the sequ~nre preferences or specificities of DNA-binding~ r~ to be det~rm;n~d. The DNA-binding - ler--lP~ for which 6equence preference or specificity can be determined may include small molecules such as Am;nn~rridines and polycyclic hydrocarbons, planar dyes, various DNA-binding antibiotics and anticancer drugs, as well as DNA-binding macromolecules E~uch as peptides and polymers that bind to nucleic acids (eg, DNA and the derivatized homologs of DNA that bind to the DNA helix).
The molecules that can be tested in the assay for s~ nre preference/specificity and relative affinity to different DNA sites include both major and minor groove binders as well as intercalating and non-intercalating DNA
binders .
3) The assay of the present invention facilitates the identification of different binding activities by moleculeq derived from known DNA-binding molecules. An example would be to identify derivatives and test these derivatives for DNA-binding activity using the assay of the present 3 0 invention . Derivatives having DNA-binding activity are then tested for anti-cancer activity through, for example, a battery of a6says performed by the National Cancer Institute (Bethesda MD). Further, the assay of the present invention can be used to test derivatives of known anti-cancer agents to examine the effect of the modif ications 1 3 (~

(6uch as methylation, ethylation and other derivatizations) on DNA-binding activity and specificity. The assay provide5 (i) an initial screen for the design of better therapeutic derivatives of known agents and (ii) a method 5 to provide a better understanding of the mode of action of such therapeutic derivatives.
4 ) The screening capacity of thi6 assay is much greater than screening each separate DNA seguence with an individual cognate DNA-binding protein. While direct 10 competition n66ays involving individual receptor: ligand ~ 1PYP~ (eg., a 6pecific DNA:protein complex) are most commonly used for mass screening efforts, each as~ay reguires the identification, isolation, purification, and pro~l~rtit~n of the assay _ Ls. Using the assay of the 15 present invention, libraries of synthetic rhP~nic~l~c or biological ~ 1 P~ can be screened for detecting ~ eclllP~ that have prefQrential binding to virtually any specified DNA 6Pg~lPnre using a single assay system.
6e~ ~ y screen6 involving the specific DNA:protein 20 interaction may not be ~Pc~sr-~y, since inhibitory rll 1~ detected in the assay may be tested directly on a binl~ l system (eg., the ability to disrupt viral replication in a tissue culture or animal model).
25 C. s~ n~ ~ Targeted by the Assay.
The DNA:protein assay of the present invention has been dP~cignPIl to screen for _ ' that bind a full range of DNA se~u~ es that vary in length as well as l~Y;ty. SPq~ ecific DNA-binding ~PCI71P~:
30 di6.w~.Led by the assay have potential usefulness as either -lprlll~r ~ag_..L~, therapeutics, or therapeutic precursors . Table I lists several potential specif ic test seuu~ ces. Seyue:~ce G~cifi~! DNA-binding molecules are potentially powerful therapeutics for P~6Pnt;Al ly any 35 di6ease or condition that in 60me way involves DNA.

Wo 93/J0446 ~ P~r/US92/0j476 .
~1 7~13~

r 1Pe: of test se~ue.lces for the assay include: a) binding sequences of factors involved in the maintenance or propagation of inf ectious agents, e~p~Pci~ 1 1 y viruses, bacteria, yea6t and other fungi, b) seq~l~nrpc causing the 5 i~ld~y~ Llate expression oP certain cPl l~ r genes, and c) sequences involved in the replication of rapidly growing cells .
Furth~ 'e~ gene expre6sion or replication does not n~ Pc~--rily need to be disrupted by blo~ ;n~J the binding of 10 specif ic proteins . Specif ic s~ within coding regions of genes (e.g., ~ .f f.~Je ,Pc) are equally valid test Pnf P~: since the binding of small le~lPQ to these sequences is likely to perturb the ~L~ns~Liption and/or replication of the region. Finally, any molecules that 15 bind DNA with some seuuence specificity, that is, not just to one particular test se~uence, may be still be useful as anti-cancer agents. Several 6mall -l~oc~lP~ with some s~J~el~ce preference are already in u~e ag ant;f~s~nf~pr therapeutics. M~leclllPc identified by the present assay ZO may be particularly valuable as lead _ _ '~ for the devPl~ ~ t of congeners (i.e., rhP-n;f ll derivatives of a - lec~1P having differenct specificities) with either different spo~if;city or different affinity.
One advantage of the present invention is that the 25 assay is capable of screening for binding activity directed against any DNA Spf~rlpnf~e. Such ce~uè.l.ies can be --';c~11y significant target seque~ces (see part 1, MPf1;C~11Y
Significant Target Sites, in this section), scrambled or randomly generated DNA sequences, or well-defined, ordered 30 sets of DNA 8Pf,~1nf'P~: (see part 2, ordered Sets of Test Se~ue~lces, in this section), which could be used for screening for molecules d~ LL&ting 8f ~Ue~l~ e prêferentiAl binding ( like Doxorubicin) to deter~ine the se~uellces with highest binding affinity and/or to lPtPrm;nP the relative 35 relative affinities between a large number of different _ _ _ _ _ _ _ _ _ , _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . . _ WO 93/00446 PCr/US92/05476 .
2~121~ 52 G.,. ~ . There i8 usefulnegs in taking either approach for d~tectin~ and/or rl~f;iqn;n~ new therapeutic agents.
Part 3 of this section, Theoretical ~ n~ ration6 for Ch~o~:in~ Target Seyuences~ outlines the theoretical 5 c~n~ Drations for t~hr^;n~ DNA target site6 in a biological system.
1) Mo~irAl ly significant target sequences.
Few effective viral therapeutics are currently ~vailable; yet several potential target ~yu~-ae6 for 10 antiviral DNA-binding drugs have been well _l-ar~cLerized.
Furth~ e, with the ~ lAtion of a~ e data on all h;olo~iAl gygtemc, inrIl~A;n~ viral genomeg, c~ llAr genomes, pathogen genomes (bacteria, fungi, eukaryotic parasites, etc. ), the nu~ber of target ~ites for DNA-15 binding drugs will increase greatly in the future.M~ 'A11Y ~ign;fi~-~nt target sites can be defined as short DNA 2;e~l n~ (approximately 4-30 base pairs) that are required for the expression replication of genetic material. For example, sequence6 that bind regulatory 20 ~actors, either t.Lc--ls-iLiptional or replicatory factor6, would be ideal ta~rget sites for altering gene or viral expression. Secorldly, coding fi~ ~s may be ad~y~.~te target site6 for disrupting gene function. Thirdly, Qven non-coding, non-regulatory seyu~ e6 may be of intere6t as 25 target sites (e.g., for disrupting replication ~ u~ 3eO or introducing an irlcreased mutational frequency. Some specific example6 of ';~Ally significant target site6 are shown in Table 1.
3 0 Tl~rl -F I. ~EDICALLY Slr-NllrlCANT DNA ~ IU~
EBV origin of replic~ EBNA infec~ous D~l ph~rynged cu~
HSV ongiD of Joplic~ion ULg o~l uld ge~ He~
__ _ _ . = ~ : : .

WO 93/00446 PCr/US92/05476 .
53 27 ~2~30 VZV origin of replication UL9~ e ghingleg IIPV ori~in of replication E2 ~enital wartg, cervic~l carcinor~
Interle~in 2 enhsncer NFAT-I .
HIV LTR NFAT-I AIDS, ARC
NF~B
5HBV er~ncer HNF-I hepatitig Fibrogen proDter HNF-I dige~ge Oncol~ene proter lmd ?? amcer coding gequulcog (Abbreviations: EBV, Epstein-Barr virus; EBNA, Rpstein-Barr virus ~uclear antigen; HSV, Herpes Simplex virus;
VZV, Vericella zoster virus; HPV, human papilloma virus;
HIV LTR, Human; -'^ficiency virus long t~-r~;n:~l repeat;
15 NFAT, nuclear factor of activated T cells; NFkB, nuclear factor kappaB; AIDS, acc~uired immune deficiency Dyl~dLI -;
ARC, AIDS related complex; HBV, hepatitis B virus; HNF, hepatic nuclear factor. ) The origin of replication binding proteins, Epstein 20 Barr virus nuclear antigen 1 (EBNA-l) (~ '-;n~ r, R.F., et al.; Reisman, D. et al.), E2 (which i5 encoded by the huma~
p,~; 1 lc virus) (Chin, ~.T., et al. ), UL9 (which i8 encoded by herpes simplex virus type 1) (McGeoch,D.J., et al.), and the ~ locJol~ protein in vericella zoster virus 25 (VZV) (stow, N.D. and Davison, A.J. ), have short, well-def ined binding sites within the viral genome and are tllerefore ~Yr~ nt target sites for a competitive DNA-binding drug. Similarly, recognition SeCr~-nr~ for DNA-binding proteins that act as transcriptional regulatory 30 factors are also good target sites for antiviral DNA-binding drugs. R~ pl~ include the binding site for hepatic nuclear ~actor (HNF-l), which is required for the expression of hu~an hepatitis B virus (HBV) (Chang, H.-K.), . . _ . _ . _ _ _ _ _ _ _ _ . .

WO 93/00446 PCr/US92/0~476 2-1~2130 and NFKB and NF'AT-l binding sites in the human - '~ficiency virus (HIV) long terminal repeat (LTR) ,f one or both of which may be involved in the expression of the virus (Greene, W.C.).
F _ leS of non-viral DNA targets for DNA-binding drugs are also 5hown in Table l to illustrate the wide range of potential ~pplications for se~u~l.c. L~ecific DNA-binding ~ - lec~ F . For exa~ple, nuclear factor of activated T cells (NFAT-l) is a regulatory factor that is lO crucial to the in~llrihle expression of the interleukin 2 (IL-2) gene in response to signals from the antigen receptor, which, in turn, iB required for the cascade of -~lec~l Ar event6 during ~ cell activation (for review, see Edwards, C.A. and Crabtree, G.R. ) . The ~ ni ~ of action 15 of two 1 - r/l~Callt~, cyclo~porin A and FK506, is thought to be to block the i n~ c i hl~ expression of NFAT-l (Schmidt,A. et al. ;~nd Banerji, S.S. et al.). However, the effects of these drugs are not specifi~ to NFAT-l;
therefore, a drug targeted specifically to the NFAT-l0 binding site in the IL-2 ~nhAnr~r would be desirable as an "._d i _, r ~ anL.
Targeting the DNA site with a DNA-binding drug rather than targeting with a drug that affects the DNA-binding protein (~ hly the target of the current 25 ; ~ s~allLs) is advantageous for at least two rea60ns: first, 1:here are many fewer target site8 for specific DNA seqUences than sperifi~ proteins (eg., in the case of glucocorticoid receptor, a handful of DNA-binding sites vs. about 50, 000 protein molecules in each cell) and 30 secondly, only the targeted gene need be a~fected by a DNA-binding drug, while a protein-binding drug would disable all the c~l 1 tll Ar functions of the protein.
An example of the latter point i8 the binding site for HNF-l in the human f ibrinogen promoter. Fibrinogen level 35 is one of the most highly correlated factor with Wo 93/00446 PCr/US92/05476 Z7 ~2~3~

cardiovascular disease. A drug targeted to either HNF-l or the HNF-1 binding site in the fibrinogen promoter might be used to decrease f ibrinogen expression in individuals at high risk for disease because of the uvele~ ssion of 5 fibrinogen. However, since HNF-l is required for the expression of a number of normal hepatic genes, hlorL-ing the HNF-l protein would be toxic to liver function. Ir contrast, by blocl~;ng a DNI~ seguence that is _ _-8fJ in part of the HNF-l binding site and in part by flanking 10 Sf ,ue~Ce5 for diveLge~ e, the fibrinogen gene can be t~Lrgeted with a high level of selectivity, without harm to normal cPllulAr HNF-1 functions.
The assay ha5 been designed to screen virtually any DNA seguence. As described above, test seguences of 15 medical significance include viral or microbial pathogen genomic seguences and seguences within or regulating the expression of onr~ogenPC or other intl~pLu~Liately e,.~Lfssed rPll~llAr genes. In addition to the detection of potential antiviral drugs, the assay of the present invention is also 20 applicable to the detection of potential drugs for (i) ~isrupting the - ' hol ;r~ of other infectious agents, (ii) hlnrlr;n~ or redlucing the LLG~-auLiption of ina~Lu~Li~tely e~ ssed cP11t~lAr genes (such as ~I tu~3~- ~F or genes associated with certain genetic disorders), and ( iii) the Pnhiln, ~ or alteration of expression of certain CP11171Flr genes .
2 ) Def ined sets of test seguences .
The approach described in the above section d;crllc~Pc screening large numbers of f~ tion broths, extracts, or other mixtures of lln~r- ~8 against specific --~;rAlly significant DNA target seguences. The assay can also be utilized to I creen a large number of DNA seguences against known DNA-binding drugs to detPrm;np the relative affinity of the single drug for every poCc;hle defined specific WO 93/00446 PCr/US92/05476 seguence. For example, there are 4~ ps~;hlP sey-uences, where n ~ the number of nucleotides in the sey--uence. Thus, there are 43 e 64 different three base pair 6Pq~lPnrP~ 44, 256 dif~erent four base pair sey-uences, 4~-- 1024 different 5 5 base pair sey-uences, etc. If these s~q~ P~ are placed in the test site, the site adjacent to the screening De r-Pnre (the exa~ple used in this invention is the ULg binding s$te), then each of the different test sequences can be 6~.Le ~l~ed against many different DNA-bin~ing ler~ . The te5t D~uences may be placed on either or both sides of the screening 6~yu~ce, and the se~U~"~cec flanking the other side of the test se~ue~.ces are fixed seyuences to stabilize the duplex and, on the 3' end of the top strand, to act as an AnnPAlin~ site for the primer (see 15 Example 1). For example, oligonucleotides sey-uencea could be ;o~ LLu~Led as shown in Figure 15 (SEQ ID NO: 18) . In Figure 15 the TEST and S~ De~ue~ces are indicated.
The preparation of such double-str~nded ol i q~n--rl P~tide8 i5 described in Example 1 and illustrated 20 in Figure 4A and 48. The test sey-uences, denoted in Figure 15 as X:Y (where X ~ A,C,G, or T and Y = the complementary ~e~ e, T,G,C, cr A), may be any of the 256 different 4 base pair seqn~nrP~ shown in Figure 13.
Once a set cf test ol i qr n--r~ Potides containing all 25 pr~ ;hlP four base pair S~yu~ S has been synthP~i7od (see Example 1), the set can be s~ L~ened with any DNA-binding drug. The relative effect of the drug on each oligon~r-leotide a~say system will ref lect the relative affinity of the drug for the test se~ e. The entire 30 D~e~LLulu of affinities for each particular DNA sequence can therefore be defirled for any particular DNA-binding drug.
The data generated using this approach can be used to facilitate lPrl~lAr 'e~;n~ yLo~L~h~3 and/or be used directly to design new DNA-binding ~ r~lP~ with increased WO 93/00446 PCr/US92/05476 ., .
57 211;''13~
af f inity and specif icity .
Another type of ordered set of ol ignm~rlPotides that may be useful for screening are sets comprised of scrambled sequences with fixed base composition. For example, if the 5 recognition sequence f or a protein is 5 ' -GATC-3 ' and libraries were to be screened f or DNA-binding molecules that reco~ni~ this seSIu~nre~ then it would be desirable to screen se5~u~ es of similar size and base composition as control sequences for the assay. The most precise 10 experiment is one in which all prceihl~ 4 bp s~quQnr~ are E;creened; this L~ ~L~Se~l~S 4~ = 256 different test seyu~nces, a number that may not be practical in every situation. However, there are many fewer pos6;hl~ 4 bp seyuences with the same base composition (using the bases 15 lG, lA, lT, lC; n! -- 24 different 4 bp se~lue~ es with this particular base composition), which provides ~xr~ nt controls without having to screen large numbers of sequences .
3) Theoretical con~ rations in rhr~osin~
20 biological target sites: Specificity and Toxicity.
One crn~irl~ation in rhoos;n~ s~ oc to screen using the assay of the present invention is test 6~qnonre arC~6ih~ 1 ity, that is, the potential ~AyO~UL~ of the sequence in vivo to binding molecules. r~lllllA~ DNA is 25 par~A~e-d in chromatin, rendering most se~tu~ ,~ es relatively ~n~rcc~ ihle. Sequences that are actively transcribed, particularly those sequences that are regulatory in nature, are less protected and more ~rce~sihle to b~th proteins and small molecule6. This observation is substantiated by a 30 large literature on DNAase I sensitivity, footprinting studie6 with nucleases and small molecules, and general studies on chromatin l~LUl_LUL~ ~Tullius). The relative accessibility of a regulatory sequence, as det~ mi ned by DNAase I lly~r~ensitivity~ is likely to be several orders WO 93/~0446 PCr/US92/05476 58 21~2~30 of magnitude greater than an inactive portion of the rP1 1t~1Ar genome. For this reason the regulatory sequences of cPl 1 ul Ar 9enes, as well as viral regulatory or replication sequences, are useful regions to choose for selecting specif ic inhibitory small 1PO1~ 1 P using the assay of the pre6ent invention.
Another rnncjtloration in rhnOF;n~ sel P r~R to be s~,L_el~ed using the assay of the present invéntion is the uniqueness of the potential test sequence. As d; RC""Eet above for the nuclear protein HNF-l, it is desirable that small inhibitory 1PI~I-1PC are gpecific to their target with minimal cross reactivity. Both se~ c composition and length ef f ect sequence uniqueness . Further, certain sequences are found less frequently in the human genome than in the genomes of other organisms, for example, n viruges. Because of base composition and codon utilization differences, viral 56:yu-~llCeS are distinctly different from - 1 i An sequences. As one example, the d;n-l- lPotide CG is found much less rL~uu~ltly in - 1 lAn cells than the rl;n~-lP~tide sequence GC: further, in SV40, a ~ n virus, the RPqllPn~-OR AÇT and ACGT are r.~L~e_l~Led 34 and 0 times, respectively. Specific viral regulatory s~yue:l~ces can be chosen as test seyuences keeping this bias in mind. Small inhibitory molecules identif ied which bind to such test se l - ~e6 will be less likely to interfere with colllllAr fllnrtj~nR.
There are approximately 3 x 109 base pairs (bp) in the human genome Of the known DNA-binding drugs f or which there is crystallographic data, most bind 2-5 bp soq~loncoR.
There ~re 44 ~ 256 different 4 base sPquon~-oR; therefore, on average, a single 4 bp site is found roughly 1.2 x 107 times in the human genome. An individual 8 base site would be found, on average, about 50,000 times in the genome. On the surface, it might appear that drugs targeted at even an WO 93/00446 PCr/US92/05476 .
59 2 f 1 2 1 3~
8 bp site might be deleterious to the cell because there are so many binding sites; however, several other ~r n~ PrationS must be rPco~ni 70Cl. First, most DNA is tightly wrapped in chr~ ~ ~ ~ 1 proteins and is relatively 5 i nF ~cPcsible to { n- i n~ DNA-binding molecules as a ~L-ted by the non~pecific Pn~ n~ oolytic digestion of chromatin in the nucleus (Edwards, C.A. and Firtel, R.A-) -Active LL~ns~Liption units are more ~c~slhle than 10 DNA bound in ~,IIL ~ ~ 1 proteins, but the most highlyexposed regions of DNA in cllromatin ~re the sites that bind regulatory factors. As d ~Lc,ted by DNAase hypersensitivity (Gross, D.S. and Garrard, W.T. ), regulatory sites may be 100-1000 times more sensitive to 15 Dn~mlclPolytic attack than the bulk of chromatin. This i8 one reason f or targeting regulatory sequences with DNA-binding drugs. Secondly, the ~ L that several ~nt~ cs~nrPr drugs that bind 2, 3, or 4 bp seqUnr~ have sufficiently low toxicity that they can be used as drugs 20 indicates that, i~ high affinity binding sites for known drugs can be matched with specif ic viral target se~lu~
it may be possi h~ P to use currently available drugs as antiviral agents at lower cu..~ LL~tions than they are currently used, with a concomitantly lower toxicity.
D. Using Test Matrices and Pattern Natching for the Analysis of Data.
The assay described herein has been d~ignPd to use a single DNA:protein interaction to screen for sPquPn--e-30 specific and sequence-preferential DNA-binding -~lec~
that can rPco~ni ~e virtually any specified sequence. By using se~,,ences flanking the recognition site for a single DNA:protein interaction, a very large number of different sequences can be tested. The analysis of data yielded by 35 such experiments displayed as matrices and analyzed by _ _ _ Wo 93/00446 PCr/US92/05476 O

pattern matching techniques should yield information about the relatedness of DNA sequences.
The basic principle behind the DNA:protein assay of the present inventlon is that when - - lecl7 l P~ bind DNA
5 sequences flanking the recognition sequence for a specific protein the binding of that protein is blocked.
Interference with protein binding likely occurs by either tor both) of two ~ ni l 1) directly by steric hindrance, or 2) indirectly by p~LLuLLations transmitted to 10 the recognition seqllence through the DNA lf-c~l 1 e, a type of allosteric ~r LuLLation.
Both of these -- -ni~ will presumably exhibit distance effects. For inhibition by direct steric hindrance direct data for very small ~olecules i8 available 15 from methylation and ethylation interference studies.
These data suggest that for methyl and ethyl moieties, the steric effect is limited by distance effects to 4-5 base pairs. Even still the number of different sequences that can theoretically be tested for these very small molecules 20 is still very large (i.e., 5 base pair combinations total 45 (=1024) different s~ut:.,-es).
In practice, the size of ~eyu~l~ce6 tested can be explored empirically for different sized test DNA-binding molecules. A wide array of s~"ut:r.~es with increasing 25 sequence complexity can be routinely investigated. This may be A~ hPd efficiently by synthesizing deyt~ L~te Ol; ~nnl~rlPotides an~d multiplexing oligonucleotides in the assay process (i.e., using a group of different ol igQnllrlPrtides in a single assay~ or by employing pooled 30 sequences in test m1trices.
In view of the above, assays employing a specific protein and ol ;~nllrlPotides containing the specific recognition site for that protein flanked by dii~ferent se~uences on either side of the recognition site can be WO 93/00446 PCr/US92/05476 - used to simultaneously screen for many different molecule~:, lnrl~ 1nq small molecules, that have binding preferences for individual sequences or families of related sequences.
Figure 12 ~ LL~tes how the analysis of a test matrix 5 yields information about the nature of competitor sequence specificity. As an example, to screen for - 1PC1~ that could preferentially r~ro~ni~e each o~ the Z56 po~h~e tetranucleotide se~ R (Figure 13 ), ol ~ nllr~ ~ntides could be uu-lDLLu- Led that contain these 256 8eqn~n~
10 immediately adjacent to a 11 bp recognition sequence of UL9 oris (SEQ ID NO:15), which is identical in each CU1~DLL~
In Figure 12 "+" indicates that the mixture retards or blocks the formation of DNA:protein complexes in solution and "-" indicates that the mixture had no marked 15 effect on DNA:protein interactions. A su~mary of the results of the test from Figure 12 are shown in Table .

2 o #1,4,7: oligos nolle detectod for the ~bove #2: for recognition site ei~er nonspecific or specific #3 AGCT
#5 CATT or ATT
K GCATTC, GCATT, CATTC, GCAT, or ATrC
25 #8 CTTT
These re5ults d LLclte how such a matrix provides data on the pre5ence of sequence specif ic binding activity 30 is a test mixture and also provides inherent controls for non-specific binding. For example, the effect of test mix ~8 on the dif f erent te5t a55ays reveals that the test mix preferentially affects the olig~n~l~lPotides that contain WO 93/00446 2 l 1 21 3 ~ PCI`/US92/0~476 the seTlPnre CCCT. Note that the sequence does not have to be within the test ~ite for test mix ~8 to exert an affect.
By displaying the data in a matrix, the analysis of the sequences affected by the different test mixtures is 5 facilitated.
E) Other Applications.
The potential pharmaceutical applications for sequence-specific DNA-binding molecules are broad, ;nr]l-A~n~ antiviral, antifungal, antibacterial, antitumor g~ 10 agents, ; , ef-~allLs~ and cardiovascular drugs.
S~ c~ L,er;fjr D]tlA-binding molecules can also be useful as molecular reagent~ as, for example, specific seqnQnre probes .
As more molecules are detected, information about the lS nature of DNA-binding molecules will be gathered, eventually facilitating the design and/or modification of nQw molecules with different or srQrjAl;7s~ activities.
Although the assay has been described in terms of the detection of 5Ql c _~,ecific DNA-binding ~ r~le~, the 20 reverse assay could be achieved by adding DNA in exces~ to protein to look for peptide sequence sr~r~;fic protein-binding inhibitors.
The following ~ illustrate, but in no way are 25 intended to limit the present invention.
~qaterials and rqethods Synthetic oligon~rleotides were prepared using commercially available automated ol; g~n~rl~Qotide synthe-30 sizers. Alternatively, custom designed synthetic oligo-, nucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA). Compl LaLy strands were Ann~AlP~3 to generate double ..LLal-d ol;~nn~r~eotides.
Restriction enzymes were obtained from Boehrinqer 35 MAnnhQ;m (Tntl;~nAr~ IN) or New England Biolabs (Beverly ~ 2~ ~2~3G

MA) and were used as per the manufacturer' 8 directions .
Distamycin A and Doxorubicin were obtained from Sigma (St. Louis, MO) . Actinomycin D was obtained from 5 Boehringer ~l~nnh~;m or Sigma.
Exam~ l ~ 1 Pre~aration g~ the O1 ;~onualeotide ~-.nt~n;n-T ~h~
Scre~n; n/~ ~ecn~e~ce This example describes the preFaration of (i) l0 biotinylated/digoxyginin/radiolabelled, and (ii) radio-labelled double-stranded oligonucleotides that contain the screening sequence and selected Test sequences.
A. Biotinylation.
The oligonucleotides were prepared as described 15 above. The wild-type control sequence for the UL9 binding site, as obtained from HSV, is shown in Figure 4.
The screenlng sequence, i.e. the Ul~ binding sequence, is CGTTCGCACTT (SEQ ID NO:l) and is underlined in Figure 4A.
Typically, sequences 5 ~ and/or 3 ' to the screening 20 sequence were replaced by a selected Test sequence ( Figure 5 ) .
One example of the preparation of a site-specifically biotinylated oligonucleotide iæ outlined in Figure 4. An oligonucleotide primer complementary to the 25 3~ sequences of the screening gequence-cnnt~;n1n~
oligonucleotide was synthesized. This oligonucleotide terminated at the residue corresponding to the C in position 9 of the screening sequence. The primer oligonucleotide was hybridized to the oligonucleotide 30 containing the screening sequence. Biotin-ll-dUTP
(Bethesda Research Laboratories (BRL), Gaithersburg MD) and Klenow enzyme were added to this complex (Figure 4) and the re~ulting partially double-stranded biotinylated complexes were separated f rom the unincorporated 35 nucleotides using either pre-prepared G-25 SephadexTM
spin columns (Pharmacia, Piscataway NJ) or "NENSORB"
columns (New England Nuclear) as per _ _ _ _ _ _ . . .

~WO 93/00446 PCr/US92/0~6 21~3~

. .
manufacturer's instructions. The l. ;nin~ single-strand region was converted to double-strands using DNA polymerase I Klenow rL _ and dNTPs resulting in a fully double-stranded nl ;~Jnm~rlPotide. A second G-25 SPrh~rlPY column S was used to purify the double-stranded oligonucleotide.
Ol ignnllrleotides were diluted or ~ lPcl in 10 mM Tris-~HCl, pH 7.5, 50 mM NaCl, and 1 mM EDTA and stored at -20C.
For rA~ir~lAhrllin~ the complexes, 3lP-alpha-dCTP tNew England Nuclear, Wilmington, DE) replaced dCTP for the double -LL~Ild completion step. Alternatively, the top strand, the primer, or the fully doublc .,Lr~i~ded olignnllrleotide have been rA~inlAh~lP~ with ~y-32P-ATP and polynucleotide kinase (NEB, Beverly, MA) . Pr~l iminAry studies have employed radiolabeled, double-stranded o1 i~TnnllrlPntides. The o1 i~nn~l~leotides are l.L~l.ared by radiol Ah~l i n~ the primer with T4 polynucleotide kinase and ,~,_32p_ATp, AnnPAl in~ the "top" strand full length O1 i~onllrlPotide, and ~filling-in~' with Xlenow fragment and deoxynucleotide trirht~qrhAtes. After rhosrhnrylation and second strand synthesi6, ol i~nmlrlPotides are separated from buffer and unir.c;uL~.L.-ted trirhosrhAtes using G-25 Sephadex preformed spin columns (IBI or Biorad). This process is outlined in Figure 4B. The reaction conditions for all of the above Klenow reactions were as follows: 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 ~M NaCl, 1 mM
dithioerythritol, 0.33-100 ~LM deoxytrirhosrhAtes, 2 units Klenow enzyme (Boehringer-MAnnhPim, Tn~;AnA~olis IN). The Klenow reactions were incubated at 25C for 15 minutes to 1 hour. The polynucleotide kinase reactions were incuhated at 37C for 30 minutes to 1 hour.
B) End-lAhPl in~ with digoxigenin. The biotinylated, radiolabelled oligonucleotides or radiolabeled t,l i~omlrlpntides were isolated as above and r-- ~L~ 9Pd in 0.2 M potassium cacodylate (pH=7.2), 4 ~M MgCl2, 1 ~M 2-~WO 93/00446 PCr/US92/0~476 2f t2130 mercaptoeth~nnl, and O . 5 mg/ml bovine serum albumin. To this reaction mixture llignyigpnin-ll-duTp (an analog of dTTP, 2'-deoxy-uridine-5'-trirhncrhAte, coupled to digoxigenin via an ll-atom spacer arm, Boehringer MAnnh~im, 5 Tnr~ nArolis IN) and tpnmin;~l deoxynucleotidyl transferase (GIBCO BRL, Gaithersburg, MD) were added. The number of Dig-ll-dUTP moieties inC~JLy~L~lted using this method ~ear~d to be less than 5 (probably only 1 or 2) as judged by electrophoretic mobility on polyacrylamide gels of the lO treated LL , L as compared to oligomlr] Potides of known length .
The biotinylated or non-biotinylated, digoxygenin-containing, radiolAhPlled olignnllrleotides were isolated as above and rPcllcppn~pd in lO mM Tris-HCl, 1 mM EDTA, 50 mM
15 NaCl, pH 7.5 for use in the binding assays.
The above ~LoceduL~ can also be used to biotinylate the other strand by using an oligomlr~Potide cr,~t~in;n~ the screening sequence compl; L-ly to the one shown in Fir~ure 4 and a primer compl ~ry to the 3 ' end of that 20 molecule. To accomplish the biotinylation Biotin-7-dATP
was substituted for Biotin-ll-dUTP. Biotinylation was also accomplished by chemical synthetic methods: for example, an activated nucleotide is in~iUL~ULo.ted into the nl i ~onllrl eotide and the active group is subsequently 25 reacted with NHS-LC-Biotin tPierce). Other biotin derivatives can also be used.
C . RadiolAhPl 1 i n~ the Oligonucleotides Generally, ol i gnnllrleotides were radiol ;Ihel 1 P~ with gamma-32P-ATP or alpha-32P-deoxynucleotide tri rhncrh~tes and 30 T4 polynucleotide kinase or the Klenow fL ~ of DNA
polymerase, respectively. TAhPllin~ reactions were performed in the buffers and by the methods re~ ~?d by the manufacturers (New England Biolabs, Beverly MA;
ge~hpcrl~ Research Laboratories, Gaithersburg ~D; or W093/00_ PCr,US92/05~
2 1 1 2 ~ 3 (~

Boehringer/MAnnh~;m, Tn~q;AnArolis IN). o1~ n~ tides were separated from buffer and unincuL~uLated trirhrlcrhAtes using G-25 S~rhA~Y preformed spin columns (IBI, New Haven, CT; or Biorad, Ri ~ , CA) or ''N~;NSûK~'I preformed columns (New England Nuclear, Wilmington, DE) as per the manuf acturers instructions .
There are several reasons to enzymatically synthesize the second strand. The two main reasons are that by using an excess of primer, second strand synthesis can be driven to near _ le~ i rn 50 that nearly all top strands are Annc~led to bottom strands, which ~v~.,Ls the top strand single strands fro~ folding back and creating additional and unrelated doubl~ Landed l.LLUULUL''S~ and secondly, since all of the ol; ~on~lrleotides are primed with a common primer, the primer can bear the end-label 80 that all of the o1; ~n~rleotides will be labeled to exactly the fiame specif ic activity .
~YAmnle 2 Preparation of the UL9 Pro~i n A. Cloning o~ the UL9 coding 6~ c into pAC373.
To express full length UL9 protein a baculovirus expression 5y5tem ]na5 been used. The se~uel,ce of the UL9 coding region of Herpes SimpleY. Virus has been rl;cclos~l by McGeoch et al. and is available as an EMBL nucleic acid se~ e. The ~ inAnt baculovirus AcNPV/UL9A, which contained the UL9 coding sequence, was obtained from Mark ohA11h~rg (National Institutes of Health, Bethesda MD).
The cu..--LL~ uLion of this vector has been previously described (Olivo et al. (1988, 1989)). Briefly, the NarI~-30 l~coRV fragment was derived from pMCl60 (Wu et al.). Blunt-ends were generated on this LL, by using all four dNTPs and the Klenow fragment of DNA polymerase I
(Boehringer MAnnhl~;m, Tn~liAnArolis IN) to fill in the terminal overhangs . The resulting LL , ~- L was blunt-end 35 ligated into the unique ~3am~I site of the baculoviral ~WO 93/~0446 PCr/US92/05476 21 12~30 vector pAC3T3 (Summers et al. ) .
B. Cloning of the UL9 coding 6eguence in pVL1393 The UL9 coding region was cloned into a second ~aculovirus vector, pVL1393 (Luckow et al. ) . The 3077 bp 5 NarI/Eco~V LL t_ containing the UL9 gene was excised from vector pEcoD (obtained from Dr. Bing Lan Rong, Eye Research Institute, Boston, MA): the plasmid pEcoD
c~nt~in~ a 16.2 kb EcoRI ~ t derived from HSV-I that bears the UL9 gene (Goldin et al. ) . Blunt-ends were 10 generated on the UL9-containing L as described above. ~coRI linkers (10 mer) were blunt-end ligated (Ausubel et al.; ~ ~.ok et al. ) to the blunt-ended NarI/-EcoRV l L ~
The vector pVL1393 (Luckow et al. ) was digested with 15 EcoBI and the linearized vector isolated. This vector coTIt~;n~ 35 nucleotides of the 5' end of the coding region of the polyhedron gene upstream of the polylinker cloning site. The polyhedron gene ATG has been mutated to ATT to prevent translational initiation in recombinant clones that 20 do not contain a coding sequence with a functional ATG.
The EcoRI/UL9 fragment was ligated into the linearized vector, the ligation mixture transformed into E. coli and ampicillin resistant clones 5~lected. Plasmids LeCuvc:Led from the clones were analy~ed by restriction digestion and 25 plasmids carrying the insert with the amino t~rm;n~l ULg coding seguences oriented to the 5 ' end of the polyhedron gene were selected. This plasmid was designated pVL1393/UI,9 (Figure 7).
pVL1393/UL9 was cotransfected with wild-type 30 baculoviral DNA (AcMNPV; Summers et al. ) into SF9 (spodoptera frugiperda) cells (Summers et al. ) .
R~c ' in~nt baculovirus-infected Sf9 cells were identified and clonally purified (Summers et al. ) .
C. Expression of the UL9 Protein.
35 Clonal isolates of ~,:c ' ;~nt baculovirus infected WO 93~00446 PCr/US92/0~6 :; 21~2~30 Sf9 cells were grown in Grace's medium as described by Summers et al. The cells were scraped from tissue culture plates and collected by centrifugation (2,000 rpm, for 5 minutes, 4C). The cells were then washed once with 5 phosphate buffered saline (PBS) (Maniatis et al. ) . Cell pellets were fro~en at -70C. For lysis the cell~ were L~ d in 1.5 volumes 20 mM HEPES, pH 7.5, 10%
glycerol, 1.7 M NaCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF).
10 Cell ly~ates were cleared by ultracentrifugation (Beckman table top ultracentrifuge, TLS 55 rotor, 34 krpm, 1 hr, 4C). The supernatant was dialyzed overnight at 4C
against 2 liters dialysi6 buffer (20 mM HEPES, pH 7.5, 10%
glycerol, 50 mM NaCl, 0.5 ~M EDTA, 1 mM dtt, and 0.1 mM
15 PMSF).
These partial] y purif ied extracts were E~L ~al ed and used in DNA:protein binding experiments. If n~r~cSA~y extracts were cv..~ rated using a "CENTRICON 30"
f iltration device (Amicon, Danvers MA) .
D . Cloning the Truncated UI 9 Protein .
The sequence ~nro~l;n~ the C-t~nminAl third of ULg and the 3' flanking seT~nr~C, an approximately 1.2 kb LL, -nt, was sllh~lonPd into the bacterial expression vector, pGEX-2T (Figure 6). The pGEX-2T is a modification of the pGEX-l vector of Smith et al. which involved ~he insertion of a thro~bin cleavage sequence in-frame with the glut~thione-S-transferase protein (gst).
A 1,194 bp Bam~I/EcoRV fragment of pEcoD was isolated that contained a 951 bp region Pnro~ling the C-t~n;nAl 317 amino acids of UL9 and 243 bp of the 3 ' untranslated region .
This Bam~I/EcoRV UL9 carboxy-~rm;nAl (UL9-COOH) containing L , ~ was blunt-ended and EcoRI linkers added as described above. The EcoRI linkers were d~cign~d to _ _ _ _ . _ _ _ . _ .. . _ . . . _ . . . . _ _ _ . . _ ~WO 93/00446 PCr/US92/05476 allow in-frame fusion of the UL9 coding ~e~uellce to the gst-thrombin coding soqn~nr~. The linkered fragment was isolated and digested with EcoRI. The pGEX-2T vector was digested with Eco~I, treated with Calf Integtinal Alk~lin~
5 Phosphatase (CIP) and the linear vector isolated. The EcoRI linkered UL9-COOH rL _ was ligated to the linear vector (Figure 6). The ligation mixture was transformed into E. coli and ampicillin resistant colonies were E~elected. Pla6mids were isolated from the ampicillin 10 resistant rolon; ~ and analyzed by restriction enzyme digestion. A plasmid which generated a gst/thrombin/UL9-COOH in frame fusion was identified (Figure 6) and designated pGEX-2T/UL9-COOH.
A. Expression of the Truncated UL9 Protein.
E. coli strain J~I109 was transformed with pGEX-2T/C-UL9-COOH and was grown at 37C to saturation density overnight. The overnight culture was diluted 1:10 with LB
medium containing ampicillin and grown f rom one hour at 30C. IPTG (isopropyllthio.-,B-galactoside) (GIBCO-BRL) was added to a final ul-ut~ L-Lion of 0.1 mM and the incuoation was continued for 2-5 hours. Bacterial cells containing the plasmid were subjected to the temperature shift and IPTG conditions, which induced transcription from the tac promoter .
Cells were harvested by centrifugation and r~Fll~p~n~ d in 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM
Na2HPO" 4 m~ NaH2PO4). Cells were lysed by sonication and lysates cleared of c~ r debris by centrifugation.
The fusion protein was purified over a glutathione agarose affinity column as described in detail by Smith et al. The fusion protein was eluted from the affinity column with reduced glutathione, dialyzed against UL9 dialysis buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 1 mM
DTT, 0.1 n~ PMSF) and cleaved with tll~;, hin (2 ng/ug of WO 93/00446 PCr/US92/ 6 0~7 fusion protein).
An aliquot of the z-u~e:L~ lL obtained from IPTG-induced cultures of pGEX-2T~C-UL9-COOH-rnntAin;n~ cells and an aliquot of the affinity-purified, t~lL- ' in-cleaved 5 protein were analyzed by SDS-polyacrylamide gel ele~_LLuuhoLesis. The result of thi6 analysis is shown in Figure 8. The 63 kilodalton GST/C-UL9 fusion protein is the largest band in the lane marked GST-UL9 ( lane 2 ) . The first lane rnnt~inc protein size 2.~ d~-~1s. The UL9-COOH
10 protein band (lane GST-UL9 + ~IIL ` in, Figure 8, lane 3) is the band located between 3 0 and 4 6 kD: the glutathione transferase protein is located just below the 30 kD size standard. In a separate eYperiment a similar analysis was per~ormed using t]le lln;nrlllred culture: it showed no 15 protein cVLL~ ng in size to the fusion protein.
Extracts are dialyzed before use. Also, if ne~ cy~
the extracts can ~e cu..~-enLL~ted tYpically by filtration using a "CENTRICON 30" filter.
R~:-mnle 3 B~ n-l; n-l AssaYs A. Band shift gels.
DNA:protein binding reactions containing both l:~hPlle~
~ l PY~ and free DNA were separated electrophoretically on 4-10% polyacrylamide/Tris-Borate-EDTA (TBE) gels (Freid et al.; Garner et al. ) . The gels were then fixed, dried, and exposed to X-ray f ilm . The autoradiograms of the gels were eY~minp~ for band shift patterns.
B. Filter Binding Assays A second method used particularly in detPrmin;nrJ the off-rates for protein:oligonucleotide 1PYP~: is filter binding (Woodbury et al. ) . Nitrocellulose disks (SrhleirhPr and Schuell, BA85 filters~ that have been soaked in binding buffer (see below) were placed on a 35 vacuum filter a~ ~L-Lus. DNA:protein binding reactions ~10 93/00446 PCr/US92/05476 (see below; typically 15-30 ~Ll) are diluted to 0.5 ml with binding buffer (this dilutes the c~,..cel.~,ation of ~- ^ntS without ~ sor; Ating complexes) and applied to the discs with vacuum applied. Under low salt conditions S the DNA:protein complex sticks to the filter while free DNA
passes through. The discs are placed in scintillation counting fluid (New England Nuclear), and the cpm detDrminP~l using a scintillation counter.
This technique has been adapted to 96-well and 72-slot nitrocP~ ln~e filtration plates (SrhleinhPr and Schuell) using the above protocol except (i) the reaction dilution and wash volumes are reduced and (ii) the flow rate through the filter is controlled by adjusting the vacuum ~L~S2~ULe:~
This method greatly facilitates the number of assay samples that can be analyzed. Using radioactive nl ;gnn~rleotide8, the samples are applied to nitrocPlllllnse filters, the filters are exposed to x-ray film, then analyzed using a Molecular Dynamics scAnnin~ densitometer. This system can transfer data directly into analytical software ~L~r (e.g., Excel) for analysis and graphic display.
~yAmnle 4 Fllnrtional UL9 Bindinq Assay A. Functional DNA-binding Activity Assay Purified protein was tested for functional activity using band-shift assays. Radiol AhP7 1 P~l oligonucleotides (prepared as in Example lB) that contain the 11 bp recognition seSr~Pnre were mixed with the ULg protein in binding buffer (optimized reaction conditions: O.1 ng 32p_ DNA, 1 ul UL9 extract, 20 mM HEPES, pH 7.2, 50 mM KCl, and 1 mM DTT). The reactions were incubated at room tl aLUL~ for 10 minutes (binding occurs in less than 2 minutes), then separated ele~.LL~l~hoLetically on 4-10% non-denaturing polyacrylamide gels. UL9-specific binding to WO 93/00446 ~ PCr/US9~/0~6 - 27 7?~3~

the ol;~on~ Potide is indicated by a shift in mobility of the ol i~nn~lrl~Pntide on the gel in the presence of the UL9 protein but not in its absence. Bacterial extracts ~ nn~A~nin~ t+) or 1~ithout ~-) UL9 protein ana affinity purified UL9 protein were tested in the assay. Only bacterial extracts cnnt~in;n~ UL9 or affinity purified UL9 protein generate the gel band-shift indicating protein binding .
The degree of ~xtract that needed to be added to the reaction mix, in order to obtain UL9 protein excess relative to the ol;~nnllrlpotide~ was empirically detPrm;nP~
for each protein preparation/extract. Aliquots of the preparation were ad~ed to the reaction mix and treated as above. The guantity of extract at which the majority of the lAhPllP~ ol;gnl~llrlPntide appears in the DNA:protein complex was evaluated by band-shift or filter binding assays. The assay is most sensitive under conditions in which the minimum amount of protein is added to bind most of the DNA. Excess protein can decrease the sensitivity of the assay.
B. Rate of Dissociation The rate of ~ sociAtion is detPrm;nPd using ~
competition assay. An oligonucleotide having the seguence presented in Figure 4, which contained the binding site for UL9 (SEQ ID NO:14), wa5 radiolAhPlle~ with 32P-ATP and polynucleotide kinase t~ethpq~lA Research Laboratories).
The competitor DNA was a 17 base pair oligonucleotide (SEQ
, ID NO:16) containin,g the binding site for UL9.
In the competition assays, the binding reactions (Example 4A) were assembled with each of the olignmlrlPotides and placed on ice. T~nlAhPllP~
ol;~nnllrlPotide (1 ILg) was added 1, Z, 4, 6, or 21 hours before loading the reaction on an 8% polyacrylamide gel (run in TBE buffer (Naniatis et al. ) ) to separate the ~/0 93/00446 PCr/US92/05476 21 12t30 reaction ~ ts. The tl;R~oriAtion rates, under these conditions, for the truncated UL9 (UL9-COOH) and the full length UL9 is approximately 4 hours at 4C. In addition, random oligonucleotides (a 10,000-fold excess) that did not 5 contain the UL9 binding se~uence and sheared herring sper~
DNA (a lOO, OOO-fold excess) were tested: neither of these control DNA6 _LQ~A~ for binding with the oligonucleotide containing the UL9 binding site.
C. Optimization of the UL9 Binding Assay (i) Truncated UL9 from the bacterial expression system.
The effects of the following _ -nts on the binding and ~ oci~tion rates of UL9-COOH with its cognate binding site have been tested and optimized: buffering conditions 15 (;nrlt-A;n~ the pH, type of buffer, and ~u..-~..LL-tion of buffer); the type and uUII-~llLL~lt.iOn of monovalent cation;
the presence of divalent cations and heavy metals;
temperature; various polyvalent cations at different cu~ lLLc.tions; and different redox reagents at different 20 rvl,o-~ l ations. The effect of a given _ - L was evaluated starting with the reaction conditions given above and based on the A i ~:so~; Ation reactions described in Example 4B.
The optimized conditions used for the binding of UL9-25 COOH contained in bacterial extracts (Example 2E~ toOl i qrnllrleotides containing the HSV ori sequence (SEQ ID
NO:1) were as follows: 20 mM HEPES, pH 7.2, 50 mM KCl, 1 mM DTT, 0.005 -- 0.1 ng rA~linlAhPle~A~ (specific activity, approximately 10~ cpm/~g) or rli~oYigin~ted~ biotinylated 30 nl ;~rnllrl~ntide probe, and 5-10 ~g crude UL9-COOH protein preparation (1 mM EDTA is optional in the reaction mix).
Under optimized conditions, UL9-COOH binds very rapidly and has a fl;~ori~A~tion rate of about 4 hours at 4C with non-biotinylated oligonucleotide and 5-10 minutes with WO 93/00446 PCr/US92/0 ~6 biotinylated nl;~ clPotides. The AiCcori~tion rate of UL9-COOH changes markedly under different physical conditions. Typically, the activity of a UI9 protein ~L~La~ion was ARsPcc~d using the gel band-shift assay and related to the total protein content of the extract as a method of standardization. The addition of herring sperm DNA Apron~lP~ on the purity of UL9 used in the experiment Binding assays were incubated at 25~C for 5-30 minutes.
( ii) Full length UL9 protein from the baculovirus system.
The binding reaction conditions for the full length baculoviL US-pL uduced UL9 polypeptide have also been optimized . The optimal conditions f or the current assay were detPrm;nP~A to be as follows: 20 mM Hepes; 100 mM
NaCl; 0.5 DIM dithiothreitol; 1 nM EDTA; 5% glycerol; from 0 to 104-fold excess of sheared herring sperm DNA; 0 . 005 -0.1 ng radiolabeled (specific activity, approximately 10~
cpm/~g) or digoxiginated, biotinylated n~; gr,n~lr.l eotide probe, and 5-10 ~g crude UL9 protein preparation. The full length protein also binds well under the optimized conditions estAhl ;chP~A~ for the truncated UL9-COOH protein.
r le 5 The ~ffect of Test Seauençe Variation on the Off-Rate of the TTT.9 Protein The ol i ~on~nlentides shown in Figure 5 were r~A; olAhPl 1 Pd as described above. The competition assays were perf ormed as described in Example 4B using UL9-COOH .
p~ nlAh~lpcl oligomlrleotides were mixed with the UL9-COOH
protein in binding buffer (typical reaction: 0.1 ng ol ;grml~rlf~tide 3~P-DNA, 1 ,ul UL9-COOH extract, 20 mM HEPES, pH 7.2, 50 mM KCl, 1 ~I EDTA, and 1 mM DTT). The reactions were incubated at room temperature f or 10 minutes . A zero time point sample was then taken and loaded onto an 8%
_ _ _ _ _ _ _ _ _ _ . _ _ . . . _ . _ ~VO 93/00446 PCr/US92/05476 polyacrylamide gel (run use TBE). One ~g of the l]nli~hQll~cl 17 bp competitive DNA rl i~on~l~leotide (SEQ ID NO:16) (Example 4B) was added at 5, 10, 15, 20, or 60 minutes before loading the reaction sample on the gel. The results 5 of this analysis are shown in Figure 9: the ficreening s~-~ual.~es that ~lank the UL9 binding site (SEQ ID NO:5-SEQ
ID NO:13) are very ~ 2imil~r but have little effect on the off-rate of ~L9. Accordingly, these results show that the UL9 DNA binding protein i8 effective to bind to a screening 10 ~;eqnQnre in duplex DNA with a binding affinity that is subst~nti~lly in~r~n~nt of test 8c~ placed adjacent the screening se~uence. Filter binding experiments gave the same result.
Example 6 The Effect of ActinomYcin D. Dist~mvcin A and Doxorllhicin on UL9 Bindinq to the screeninq Se~uence is DePendent on the S~ecif ic Test Sec~uence Different oligon~rleotides, each of which c~nt:~in~cl 20 the 6creening s~ nre (SEQ ID NO:1) flanked on the 5' and 3' sides by a test s~Tl~nre (SEQ ID NO:5 to SEQ ID NO:13), were evaluated for the effects of distamycin A, actinomycin D, and doxorubicin on UL9-COOH binding.
Binding assays were performed as described in Example 25 5 . The ol i ~n~ tides used in the assays are shown in Figure 5. The assay mixture was allowed to pre-eSr~ilihrate for 15 minutes at room t~ ~ILUL~ prior to the addition of drug .
A c, ~ -"LraLed solution of Distamycin A was ~L~aLe:d 3 0 in dH20 and was added to the binding reactions at the ~ollowing cv--~ ~..L.cltions: O, 1 ~M, 4 ,uM, 16 ,~LM, and 40 ~N.
The drug was added and incubated at room t~ ~LuLe for 1 hour. The reaction mixtures were then loaded on an 8%
polyacrylamide gel (Example 5) and the ~ Ls separated WO 93~00446 PCI /US92/0 j~6 21 1213~

ele~LLu~huL~ Lically. Autoradiographs of these gels are shown in Figure lOP.... The test sec~uences tested were as follows: UL9 polyT, SEQ ID NO:9; UL9 CCCG, SEQ ID NO:5;
UL9 GGGC, SEQ ID NO:6; UL9 polyA, SEQ ID NO:8; and UL9 5 ATAT, SEQ ID NO: 7 . These results ' - LL ate that Distamycin A preferentially disrupts binding to UL9 polyT, ~lL9 polyA and UL9 ATAT.
A c~ t-c.tecl solution of Actinomycin D was pL~aLOd in dH20 and was added to the binding reactions at the 10 following c ~ l r a~ions: O ~ and 50 ~M. The drug was added and incubatecl at room ~ ALul~ for 1 hour. Equal volumes of dH20 were added to the control samples. The reaction mixtures ~ere then loaded on an 8% polyacrylamide gel (Example 5) and the ~ ^-ts separated 15 ele.:~Lu~l.uL~ Lically. Autoradiographs of these gels are shown in Figure lOB. In addition to the test sec~uences tested above with Distamycin A, the following test secluences were also tested with ~rt;r ~uin D: AToril, SEQ
ID NO:ll; oriEco2, SEQ ID NO:12, and oriEco3, SEQ ID NO:13.
20 These results d L,ute that ~ct;r- ~.in D preferentially disrupts the binding of UL9 to the ol; gnm~ leotide5 UL9 CCCG and UL9 GGGC.
A u~ lLLat~d solution of Doxorubicin was prepared in dH20 and was ~dded to the binding reactions at the following 25 uullu~l~LLations: O I~M, 15 IL~ and 35 ~M. The drug was added and incubated at room temperature f or 1 hour . Eclual volumes of dH20 were added to the control samples. The reaction mixtures were then loaded on an 8% polyacryla~ide gel (Example 5) and the ~ _ Ls separated 30 ele~.LLu~ horetically. Autoradiographs of these gels are shown in Figure lOC. The same test s~ tu~.ces were tQsted at, f or Actinomyci n D . ThesQ rQsults ~ LL c~ LQ that Doxorubicin preferentially disrupts the binding of UL9 to the ol;gnn~c~Pntides UL9polyT, UL9 GGGC, oriEco2, and oriEco3. Doxorubicin appears to particularly disrupt the UL9: screening sequence interaction when the test sequence oriEco3 is used. The sequences of the test sequences for 5 oriEco2 and oriEco3 differ by only one base: an additional T residue lnserted at position 12, compare SEQ
ID NO :12 and SEQ ID NO :13 .
ExamPle 7 U~e of tke 3io~;n/S~rePtaYidin RePorter SY~tem A. The Capture of Protein-Free DNA.
Several methods have been employed to sequester unbound DNA from DNA:protein complexes.
(i) Magnetic beads Streptavidin-con~ugated superparamagnetic 15 polystyrene beads (Dynabead3TM M-2aO Streptavidin, Dynal AS, 6-7X108 beads/ml) are washed in binding buffer then used to capture biotinylated oligonucleotides (Example 1). The beads are added to a 1~ ul binding reaction mixture c~)n~n;ng binding buffer and biotinylated 20 oligonucleotide. The beads/oligonucleotide mixture is incubated for varying lengths of time with the binding mixture to determine the incubation period to maximize capture of protein-free biotinylated oligonucleotides.
After capture of the biotinylated oligonucleotide, the 25 - beads can be retrieved by placing the reaction tubes in a magnetic rack (96-well plate magnets are available from Dynal ) . The beads are then waæhed .
(ii) Agarose beads Biotinylated agarose beads (immobilized D-biotin, 30 Pierce, Rockford, IL) are bound to avidin by treating the beads with 50 ~g/lll avidin in binding buffer overnight at 40C. The beadg are waghed in binding buffer and used to capture biotinylated DNA. The beads are mixed with binding mixtures to capture biotinylated DNA. The beads 3 5 are ~WO 93/00446 PCr/US92/0~6 ? l 1~

removed by centrifugation or by Collp~rt~ on a non-binding f ilter di6c .
For either of the above methods, quantification of the presence of the ~ nn~rleotide depends on the method of 5 lRhPll;n~ the ol;~onl-rl~otide. If the ol;~onl~rl~otide is r~dina~rt;vely l~hPlled: (i) the beads and supernatant can be loaded onto polyacrylamide gels to separate protein:DNA
complexes from the bead:DN~ complexes by elecL~ hul~sis, and autoradiography performed; (ii) the beads can be placed 10 in Srint~ tion fluid a~d counted in a sc;ntill~tion counter. Alternatively, presence of the oligonucleotide c~n be dPt-~m;nP~ using a t~hF~m;lllm;nPF~c~nt or colorimetric detection system.
B. Detection of Protein-Free DNA.
The DNA is end-l~h~ d with d;~oy;~rn;n-ll-duTp (Example 1). The antigenic digoxigenin moiety is rec'o~n; 7Pt9 by an antibody-enzyme conjugate, anti-digoxigenin-;~lk~l;np phosphatase (Boehringer MlnnhP;m Tn~ n~rolis IN). The DNA/antibody-enzyme co~ y~lte is then exposed to the substrate of choice. The presence of dig-dUTP does not alter the ability of protein to bind the DNA or the ability of ~.LLc:~Ldvidin to bind biotin.
(i) rhPm; lllm;npcrpnt Detection.
Digoxigenin-l ~hPl 1 ed o~ligonucleotides are detected using the rhPmilllm;nPccpnt detection system '~SOU~A~
LIGATS" developed by Tropix, Inc. (Bedford, NA). Use of this detection system is illustrated in Figures llA and llB. The technique can be applied to detect DNA that has been c~L-lL~d on either beads or filters.
Biotinylated ol ;~nn~ otides, which have terminal digoxygenin-containing residues (Example 1), are -~Lu- e:d on magnetic (Figure llA) or agarose beads (Figure llB) as described above. The beads are isolated and treated to block non-specif ic binding by incubation with I-Light .
-~0 93/00446 PCr/US92/0~476 -21 1213~

h]n-kin-~ buffer (Tropix) for 30 minutes at room UL-::. The ~Le:8e~ e of oli jnm--7P^tides i6 ~PfP~-tP~
using AlkAl inP phosphatas~ _u~Ju~lt.ed Antiho~li~G to digoxygenin. Anti-dignYi~pn~n-AlkAlinD phoD~haLase (anti-dig-AP, 1:5000 dilution of 0.75 units/ul, Boehringer M~nnhoim) i5 incubated with the sample for 30 minutes, dPcAnt~pd~ and the sample washed with 100 mN Tri~-HCl, pH

7.5, 150 mM NaCl. The sample is pre-P~-,ui 1 ~h-ated with 2 washes of 50 mM sodium bi~-L~u..ate, pH 9.5, 1 M MgCl2, then incubated in the same buffer containing 0.25 mM 3-(2'-6pi~ P)-4 Y~4~(3' 1~ D1~ yloxy) phenyl-1,2-~i nY~-tAnP ~i cor4i~1m _alt (AMPPD) for 5 minutes at room t~ _ _LUL~ PPD was developed (Tropix Inc. ) as a -hDmilllminPG~-Pnt substrate for _lksllin~ phosphatase. Upon 15 d~rhnGrhnrylation of AMPPD the resulting ~e- _ ~-, rPlp~cin~ a prolonged, steady PmiGSinn of light at 477 nm.
Excess liyuid is removed fro~ filters and the Pmicci-n of light occurring as a result of the tlPrhnsrhnrylation of 20 AMPPD by _lk_l inP phosphatase can be measured by e~O~-ULe:
to x-ray film or by ~Ptectinn in a ll-mi- ter.
In solution, the bead-DNA-anti-dig-AP is r-G ~ Pd in 'ISUU~ ;~N LIGHT" assay buffer and AMPPD and measured directly in a 1 i- Ler. Large scale screening assays 25 are performed using a 96-well plate-reading lumi- t~r (Dynatech Labcl-toLies, Chantilly, VA~. Suhpi~so~ram yuantities o~ DNA (102 to 103 aL~ -1PC (an attomole is 10 l~
moles) ) can be detected using the Tropix system in cùl~ju.,~ Lion with the plate-reading ll-mi-(ii) Colorimetric Detection.
Standard Alk-l in~ phosphatase colorimetric sub~LL-te~:
are A lso suitable f or the above detection reactions .
Typically substrates include 4-niLLu~h~ l phosphate WO 93/00446 PCr/US92/0~

(Boehringer M:~lnnh~;n), Results of colorimetric assays can be evaluated in multiwell plates (as above) using a plate-reading 6pectrophotometer (Nolecular Devices, Menlo Park CA). The use of the light emission system is more 5 sensitive than the colorimetric systems.
While the invelltion ha~ been described with re~erence to specific methods and: ' ~ 'i ~5, it will be appreciated that various modif ication~ and changes may be made without o departing from the invention.

~0 93/00446 PCI/US92/0~476 SEQUENOE LISTING
~1 ) GENERAL lNr ~ ~ :
~1) ADPLICANT: Edwarda, CynthLa A.
Cantor, Charle~ R.
Andrews, Beth M.
~li) TITLE OF INVENTION: screenLng Assay for the r ir~n of DNA-Blndlng ~ c~ .o (lil~ =ER OF SEQUENCES: 18 ( iv ) ~.~n~r~ ADDRESS:
(A~ DnnD~CCF~ LaW OFFICES OF PETER J. n~T~r Tr~rD
(B) STREET: P.O. Box 60850 (C) CITY: Palo Alto ( D ) STATE: ca (E) COUNTRY: USA
(F) ZIP: 94306 ( v) CONDUTER DEADABLE FORtl:
(A) ~SEDIUM TYPE: Floppy dLsk (B) COMPUTER: IBM PC ihl~.
tc) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWAD~E: Patent~n Release tl.0, Version tl.25 (vi) CURRENT APPLICPTION DATA:
(A) APPLICATION NUMBER:
( B ) FILING DATE:
(C) CLaSSIFICaTION:
(vii) PREVIOUS APPLICaTION DATA:
(A) APPLICATION =ER: 07/723,618 (B) FILING DATE: 27-JUN-1991 (C) CLAS:,lr~ UN:
(vLLL) ATTORNEY/AGENT lNrl ~ :
NAI~E: FahLan, Gary R.
(B) D~ Tc~TTnN NUMBER 33,875 (C) h~rr,r~~ /DOCXET NUMBER: 4600--0075.41 WO 93/00446 Pcl/US92/0~ 6 21 1213~

~iX) ~FT.- ION lNr~ :
(A) TELEPE~ONE: (415) 323-8302 (B) TBLEFAX: (415) 323-8306 ~2) LN~ ru FOR SEQ ID NO:l:
(i) SEQTlENOE rRho~.. r.. i".
(A) LENGT13: 11 balle pair~
(B) TYPE: nucleic ~cid (C) STP~ : double ( D ) TOPOLOGY: linear ( ii ) ~OLECULE TYPE: DNA ( genomLc ) (iii) n~r~ L: L~o (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOUROE:
(C) INDIVIDUAL ISOLATE: UL9 BINDING SITE, HSV oriS, higher af f inity (xi) SEQUENOE uL.~w~Lr..lur~: SEQ ID NO:l:

(2) ~N~I l'TrN FOR SE~ ID No:2:
(i) SEQUENOE rR~T~rTT~pTcTIcs (A) LENGTH: 1.1 base pair~
(B) TYPE: nucleic ~cid (C) C~P~ : double (D) TOPOLOGY: linear (ii) L50LECULE TYPE: DNA (genor~ic) (iii) n~r J . ~., J~ hT.. NO
(iv) ANTI-SENSE: NO

93/00446 PCl tUS92/0~476 ~0 _ 2 1 1 2 l 30 ~vL~ ORIGINAL SOUROE:
(C) INDIVIDUAL ISOLATE: UL9 BINDING SITE, EISV orlS, lower f f inity (xi~ SEQTlENOE L~ lUI!~: SEQ ID NO:2:
$GCq'CGCACT T 11 t2) l~r~ rOR SEQ ID NO:3:
(i) SEQuENOE ro7~Op~
(A) LENGT.: 30 bl~se pairs (B) TYPE: nucleic ~cid (c) ~:Tr-~ : double (D) TOPOLWY: llneAr ( ii ) MOLEC~LE TYPE: DNA ( g--nomic ) CAL: NO
( iv ) ANTI -SENSE: NO
(vi) ORIGINAL SOUROE:
(C) INDIVIDUAL ISOLATE: UL9Zl TEST SEQ. / TJL9 ASSAY SEQ.
(xi) SEQUEI;IOE Llc.;~ lUII: SEQ ID NO:3:
C GTTCGCACTT ~ 30 (2) lr~r~ ~ FOR SEQ ID NO:4:
(i) SEQUENOE r~-.o. ., ~_ "~
(A) LENG~I: 30 b~e pAir~
(B) TYPE: nucleic ilcid ( C ) 5~ : double (D) TOPOLWY: linear (ii) MOLECULE TYPE: DNA (genomic) ..

WO 93/00446 PCl`/US92/0~j6 (iLi) n~r~ T.- NO
~iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: UL9Z2 TEST SEQ. / UL9 ASSAY SEQ.
(xl) SEQUENOE L)c~ Klr~ N: SEQ ID NO:4:
;w~ ~ ~ GTTCGQCTT ~ . 30 ~2) INFORMATION FOR SEQ ID NO:5:
~i) SEQUENOE rl~T~
~A~ LENGT~: 30 base pairs ~B) TYPE: nuc~eic ~cid (C) srP~ ~: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) ~.lw L: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
~C) INDIVIDUAL ISOLATE: UL9 CCCG TEST SEQ. / UL9 ASSAY SEQ.
(xi) SEQUENCE U~ ;Kl~luN: SEQ ID NO:5:
C ' ~ GTTCGCACTT ~ 30 (2) INFORMATION FOR SEQ ID NO:6:
~i~ SEQUENOE r~ rFT T~TTrC:
~A) LENGT~: 30 b~e pair~
(B) TYPE: nucleic acid ~C) ~:TT ~`~T ~r~ : double (D) TOPOLO~Y: linear ~VO 93~00446 PCI`/US92/05476 2 1 1 2 ~ 30 ( Li ) NOLECULE TYPE: DNA 1 genomlc ) (Lli) r.~rul r~T. NO
( lv ) ANTI -SENSE: NO
(vi) ORIGINAL SOUROE:
(C) INDIVIDIIAL ISOLATE: UL9 GGGC TEST SEQ. / UL9 ASSAY SEQ.
~xL) SEQUENOE L~,.~l~ : SEQ ID NO:6:
GT~ Gr~ 30 (2~ lhl~. FOR SEQ ID NO:7:
~i) SEQUENOE rr~ r~ TCTTrc (A) LENGTII: 30 ba~u p~ir~
(B) TYPE: nucleic acid ( C) ~ ouble (D) TOPOLOGY: line~r (ii) NOLECULE TYPE: DNA (gonomi~) (iii) h~ru-~lw~L: NO
(iv) ANTI--SENSE: NO
( vi ) ORIGINAL SOUROE:
(C) INDIVIDUAL ISOLATE: UL9 ATAT TEST SEQ. / UL9 ASSAY SEQ.
(xi) SEQUENOE IJ~ u~ lUN: SEQ ID NO:7:
~"'`'I'ATaT~'r' GTTCGCACTT TAATTATTGG 30 (2) lN-!~ -Tt7~ FOR SEQ ID NO:8:
(i) SEQUENOE r~ L1~;~
(A) LENGT~: 30 b~e p~sir~
(B) TYPE: nucleic acid WO 93/00446 PCT/US92/0~6 21 1~130 (C) STP~ : double (D) TOPOLOGY: linear ( ii ) MOLEC~JLE TYPE: DNA ( genomic ) ru~ T- NO
( iv ) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: UL9 polyA TEST SEQ. / IIL9 ASSAY SEQ.
(xi) SEQUENCE ~ K~ un: SEQ ID NO:B:
C~~r.r7~ GTTCGQCTT ~ r ~ 30 (2) INFORMATION FOR SEQ ID NO:9:
i ) SEQUENCE rTTr D r ~A) LENGT~I: 30 ba~ie p2~ir~
~B) TYPE: nucleic ~cid ~C) sTDr~ nu~cc double (D) TOPOLOGY: line~r ( ii ) MOLECULE TYPE: DNA ( genomic ) (iii) }lypnT~n3TIrrr~ NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) IND~VIDUAL ISOLATE: UL9 polyT TEST SEQ. / UL9 ASSAY
SEQ .
~xi) SEQllENCE ll~ Kll-~lUN: SEQ ID NO:9:
W~L~L1U GTTCGQCTT ~ I ~ ) L~ 30 ~2) ln~l ~ FOR SEQ ID NO:10:

~10 93/00446 PCI /US92/05476 (i) SEQUENOE ~-~D
~A) LENGT~: 30 bA~e p~ir~
~8) TYPE: nucleLc acid ~C) STT ' : double ~D) TOPOLOGY: line~r ~ii) NOLECVLE ~CYPE: DNA ~genomic) ~iii) ~,,~,,. ~ " AT ~ NO
iv ) ANTI--SENSE: NO
~vi) ORIGINAL SOVROE:
~C) VlL~UAL ISOLATE: VL9 GCAC TEST SEQ. / UL9 ASSAY SEQ.
~xi) SEQUENOE L.c~aw~l~LlUi~: SEQ ID NO:10:
t---l~rrArrC GTTCGQCTT CrA~r~rr~C 30 ~2) lN~ FOR SEQ ID NO:ll:
~i) 8EQVENOE ~O-A~DT~TICS:
~A) LENGT~I: 30 b~e p~ir~
~B) TYPE: nucleic acid ~C) SrDA : double ~D) TOPOLOGY: linear ~ii) NOLECVLE TYPE: DNA ~gen~mic~
~iii) n~ru. Il ..T., NO
iv ) ANTI -SENSE: NO
~vi) ORIGINAL SOUROE:
~C) INDIVIDUAL ISOLATE: VL9 ATori-1 TEST SEQUENOE / UL9 A8SAY SEQ.
~xi) SEQUENCE D~l,r~ Ua: SEQ ID NO:11:
.

WO 93/00446 ~ PCr/US92/0~6 ccr.TAT~T~T CGTTCGCACT TrGTccrD~T 3b (2) le~u~ATlun FOR SEQ ID NO:12:
~i) SEQUENOE rlT3P~ ..Ih~
(A) LENGTH: 31 bal~e pz~irs (B) TYPE: nucleio acid (C) .CT~r : double ~D) TOPOLOGY: line~r (ii) MOLECULE TYPE: DNA (geno:llic) (iii) rYru~ L: NO
(iv) AMTI-SENSE: NO
(vl) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oriEC02 TEST SEQ. / UL9 ASSAY SEQ.
(xi) SEQUENCE IJ}~I,r~lrLlul~: SEQ ID NO:12:
GGCGAATTCG ACGTTCGCAC T1`CGTCCCAA T 31 (2) le~ ~Tl-M FOR SEQ ID NO:13:
(L~ SEQUENCE rTT~D~rTF~TcTTcs (A) LENGTH: 32 b~l~e pairs (B) TYPE: nucleic ~cld (C) CTP~ : double (D) TOPOLOGY: lino~r (ii) MOLECULE TYPE: DNA (geno~ic) ~iii) rYru.A~lCAL: ~iO
( iv ) AMTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVI~UA]. ISOLATE: oriEC03 TEST SEQ. / UL9 ASSAY SEQ.

~WO 93/00446 PCI/US92/05476 ~xl) SEQUENCE LL.;u,l~lr.luN: SEQ I3 NO:13:
GGCGAATTCG A~ , L ~ .L l./_W~ AT 32 (2) lhr~ lVW FOR SEQ ID NO:14:
(i) SEQUENOE ~ 'DII~ "~
(A) LENGTH: 36 bAse palr~
(B) TYPE: nucleic ~cld (C) .STPT~ : double (D) TOPOLOGY: llnear (Ll) MOLECULE TYPE: DNA (g~nomlc) (lll) nrr~ T-- NO
( lv ) ANTI-SENSE: NO
( vl ) ORIGINAL SOUROE:
(C) INDIVIDUAL ISOLATE: WILD TYPE
(xl) SEQUENCE D~ .lUI~: SEQ ID NO:14:
PT"'T~'T""~"T TCGAAGCGTT CGCACTTCGT CCCAAT 36 (2) LNr~ TTtl~T POR SEQ ID NO:lS:
(1) SEQUENOE rP-lDT~,.,_~,~" , (A) LENGTH: 9 bllse p~lr~
(B) TYPE: nucleic acid (C) sTP~'~"~T~n'~cs: double (D) TOPOLOGY: line~r (ll) MOLECULE TYPE: DNA (genomic) (lLi) n~r~ lr_HL: NO
(Lv) AIITI-SENSE: NO
(vL) ORIGINAL SOURCE:

WO 93/00446 PCI`/US92/0~j6 --- 2t ~213() (C) INDIVIDUAL ISOLATE: ~ UL9 BINDING SITE, COMPARE
SEQ ID 1!10 :1 (xi) SEQUENCE D~a~:Kl~r~lurl: SEQ ID NO:15:
TTcaQCTT 9 (2) lr~r~ ~ FOR SEQ ID NO:16:
(L) SEQUENOE rY~DD, ,~ " ~
(A) LENGT}~: 17 base pairD
(B) TYPE: nucl~Lc acid (C~ .CTPD : double (D) TOPOLOGY: line~r (li~ HOLECULE TYPE: :DNA (genomLc) (iil~ h~ru~ AL: N~
( iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: ~ISVB1/4, SEQUENCE OF t.vr~G~l~v~ DNA
MOLECULE
(xl) SEQUENCE l~a~.nl~lON: SEQ ID NO:16:
~C ACTTCGC 17 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE ru~vDrTFvTcTIcs (A) LENGT~}: ll bl~se pairs (B) TYPE: nuclelc acLd (C) CTPD : double (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: DNA (genomic~

~/0 93/00446 PCl'/us92/05476 ~iii) n~r ~ ~ . IL'~ NO
~Lv) ANTI--SENSE: NO
tVl) ORIGINAL SOUROE:
~C) INDIVIDUAL ISOLATE: UL9 BINDING SITE, HSV orlS
(xi) SEQUENOE ~ L;I~ _LJn SEQ ID NO:17:

~2) l;lr~ FOP~ SEQ ID NO:18:
i ) SEQUENCE C~ a ~A) L15NGTEI: 37 base pairs ~B) TYPE: nueleic acid ~C) Sl'P'` : do~ble ~D) TOPO~OGY: lin-ar ~ii) lSOLE= TVPE: DNA ~genomic) ~iil) n~r~ YES
~iv) ANTI-SENSE: NO
~vi) ORIGINAL SOURCE:
~C) INDIVIDUAL ISOLATE: UL9 ASSAY SEQUENCE, FIGURE lS
(xi) SEQUENOE Llc.i~Lir~lr.lU..: SEQ ID NO:18:
C~nTP~ AAT 37 . .

Claims (24)

IT IS CLAIMED:

1. A method of screening for molecules capable of binding to a selected test sequence in a duplex DNA, comprising (i) adding a molecule to be screened to a test system composed of (a) a DNA binding protein which is effective to bind to a screening sequence in a duplex DNA with a binding affinity that is substantially independent of said test sequence adjacent the screening sequence, but where said protein binding is sensitive to binding of molecules to such test sequence, and (b) a duplex DNA having said screening and test sequences adjacent one another, (ii) incubating the molecule in the test system for a period sufficient to permit binding of the compound being tested to the test sequence in the duplex DNA, and (iii) detecting the amount of binding protein bound to the duplex DNA before and after said adding.

2. The method of claim 1, wherein the screening sequence/binding protein is selected from the group consisting of EBV origin of replication/EBNA, HSV
origin of replication/UL9, VZV origin of replication/UL9-like, and HPV origin of replication/E2, and lambda oL-oR/cro.

3. The method of claim 2, wherein the DNA
screening sequence is from the HSV origin of replication and the binding protein is UL9.

4. The method of claim 3, wherein the DNA
screening sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, and SEQ ID NO:17.

5. The method of claim 1, wherein said detecting is accomplished using either a gel band-shift assay or a filter-binding assay.

6. The method of claim 1, wherein the test sequences are selected from the group consisting of EBV origin of replication, HSV origin of replication, VZV origin of replication, HPV origin of replication, interleukin 2 enhancer, HIV-LTR, HBV enhancer, and fibrinogen promoter.

7. The method of claim 1, wherein the test sequences are selected from a defined set of DNA
sequences has [XN]N combinations, where XN is sequence of deoxyribonucleotides and the number of deoxyribonucleotides in each sequence is N, N is greater than or equal to three.

8. The method of claim 7, wherein N is 3-20.

9. The method of claim 8, wherein N is 4-10.

10. The method of claim 9, wherein N is 4 and the number of combinations is 256.

11. The method of claim 10, where said deoxyribonucleotides are selected from the group consisting of deoxyriboadenosine, deoxyriboguanosine, deoxyribocytidine, and deoxyribothymidine.

12. The method of claim 1, wherein said detecting includes the use of a capture system that traps DNA free of bound protein.

13. The method of claim 12, wherein the capture system involves the biotinylation of a nucleotide within the screening sequence (i) that does not eliminate the protein's ability to bind to the screening sequence, (ii) that is capable of binding streptavidin, and (iii) wherein the biotin moiety is protected from interactions with streptavidin when the protein is bound to the screening sequence.

14. The method of claim 1, wherein said binding protein is present in a molar concentration less than or equal to the molar concentration of the screening sequence present in the duplex DNA.

15. The method of claim 1, wherein said binding protein is present in molar excess over the screening sequence present in the duplex DNA.

16. A screening system for identifying molecules that are capable of binding to a test sequence in a target duplex DNA sequence, comprising a duplex DNA having screening and test sequences adjacent one another, a DNA binding protein that is effective in binding to said screening sequence in the duplex DNA
with a binding affinity that is substantially independent of said test sequence adjacent the screening sequence, but which is sensitive to binding of molecules to said test sequence, and means for detecting the amount of binding protein bound to the DNA.

17. A screening system for identifying molecules that are capable of binding to a test sequence in a target duplex DNA sequence, comprising a duplex DNA having screening and test sequences adjacent one another, a DNA binding protein that is effective in binding to said screening sequence in the duplex DNA with a binding affinity that is substantially independent of said test sequence adjacent the screening sequence, but which is sensitive to binding of molecules to said test sequence, and means for detecting the amount of binding protein bound to the DNA, wherein the test sequence is as defined in claim 6, 7, 8, 9, 10, or 11.

18. A screening system for identifying molecules that are capable of binding to a test sequence in a target duplex DNA sequence, comprising a duplex DNA having screening and test sequences adjacent one another, a DNA binding protein that is effective in binding to said screening sequence in the duplex DNA with a binding affinity that is substantially independent of said test sequence adjacent the screening sequence, but which is sensitive to binding of molecules to said test sequence, and means for detecting the amount of binding protein bound to the DNA, wherein the screening sequence/binding protein is as defined in claim 2, 3, or 4.

19. The system of claim 18, where the DNA
screening sequence is SEQ ID NO:1.

20. The system of claim 19, where the U
residue in position 8 is biotinylated.

21. The system of claim 20, where said detection means includes streptavidin, and the streptavidin is bound to a solid support.

22. The system of claim 21, where streptavidin is used to capture the duplex DNA
when it is free of bound protein.

23. A method for inhibiting the binding of a DNA-binding protein to duplex DNA, comprising contacting a compound with a duplex DNA which contains a test sequence adjacent a screening sequence, where the DNA binding protein is effective to bind to the screening sequence with a binding affinity that is substantially independent of said test sequence, further where the binding of said compound to the test sequence inhibits the binding of the protein to the screening sequence.

24. The method of claim 23, wherein the compound is identified by the steps of preparing a series of duplex nucleic acid fragments, each containing a test sequence composed of one of the 4N possible permutations of sequences in a sequence of base pairs having N-basepairs, where said test sequence is adjacent the screening sequence, measuring the binding affinity of the DNA
binding protein to each of the series of nucleic acid fragments in the presence of the and selecting the compound if it lowers the binding affinity of the DNA binding protein for the screening sequence.