CA2044173A1 - End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids - Google Patents

End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids

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Publication number
CA2044173A1
CA2044173A1 CA002044173A CA2044173A CA2044173A1 CA 2044173 A1 CA2044173 A1 CA 2044173A1 CA 002044173 A CA002044173 A CA 002044173A CA 2044173 A CA2044173 A CA 2044173A CA 2044173 A1 CA2044173 A1 CA 2044173A1
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Prior art keywords
groups
oligonucleotides
supports
process according
thiol
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CA002044173A
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French (fr)
Inventor
Soumitra S. Ghosh
Eoin D. Fahy
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Akzo Nobel NV
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

This invention concerns methods and means for covalent attachment of oligonucleotides to solid supports substantially at their 5'-ends. According to the invention thio-oligonucleotides are attached to bromoacetyl-derivatized polyacrylamide supports, or conversely, bromoacetyl-oligonucleotides are immobilized on thiol-polyacrylamide supports. In a further aspect, this invention relates to bromoacetyl-oligonucleotides that may be immobilized on thiol-polyacrylamide solid supports, thiol-oligonucleotides immobilized on bromoacetyl-derivatized polyacrylamide supports as well as to methods for capture of nucleic acids by oligonucleotides attached to polyacrylamide solid supports, either by direct capture or in sandwich hybridization formats.

Description

W090/07582 ~ `'PC~/US90/00089 END-ATTACHMENT OF OLIGONUC~EOTIDES TO PO~YA~RYL~MI3E
50LID SUPP~RTS FOR ~APTURE
AND DETECTION OF NUCLEIC ACIDS

~ield ~f the Invention The present invention generally relates to certain developments in the chemistry of solid supports for the attachment of oligonucleotides.
More particularly, the pr~sent ~nvent~on ~s directed to solid supp~rts ~ontaining oligonucleotides in end-attachment, for c~pture and detection of nucleic acids, including ~ingle- and d~uble-stranded DNA and RN~ -targets.
1~ This inve~t~on further c~ncerns m~thod~ nnd means or covalent attachment of olig~nucleotldes to solid supports substant~ally at thelr 5'-end~. According t~ the ~nvention thiol-oligonucleotides are attached to bromoacetyl-derivatized polyacrylamide 6upport6, or conversely! bromoacetyl-ol~gonucleot~d~s are ~mmobilized on th~ol-polyacryla~ide supports.
In a further aspeck, th~ in~ent~ on relat~ to ~romoacetyl oligonucleotide~ t~at may be i~mobili~ed on thiol-polyacrylamide 601~d 8Upport~ thiol-~0 oli~onucleot~des immobilized on bromoacetyl derivatized polyacrylamide supp~rts as well as to method~ for ~aptur~
of nuclei~ acids by ol~gonuoleotldee attached to `
polyacrylamide solid Cupport~, e~ther by direct capture or in ~andw~ch hybrid~2ation ~orma~s~
~ackq~ound o ~he Ir~n~1Q~
It ~ often des~rable to d~tect Yery ~mall amounts of extracted or in vitro amplified nucle~c ~cid~, for example ~n ~iological 6amplè~O Aecordlng to the most common appr~æeh, th@ taryet nu~leio aa~d ~g hybr~d~2ed to ' - ' : i ., ` ! - ' ' '- .

an oligonucleotide. In order to obtain a detectable 6ignal, proportionate to the amount of the targ~t, either the target nucleic acid or the oligonuclaotide needs to be associated with a signal generating reporter element, such as a radioactive atom or a chromogenic molecule, or, an enzyme such as alkaline phosphatase. The signal generated by a pr~perly hybridized nucleic acid can be detected and measured by methods known in the art. Many of the commonly used techniques of molecular biology require the immobilization of the target~ on solid - supports, to enable fractionation and identification of specific seq~ences. The target nucleic acid may be captursd by aligonucleotides immobilized on solid supports, or more frequently, so-cialled "sandwich"
hy~ridization systems are employed, using a capture oligonucleotide covalently attacheld to a ~olid support for capturing detection oligonucle~tide-tàrget nucleic acid adducts formed ~n solution. Typical solid 6upports are, for example, nitrocellulose or nylon membranes, activated agaro~e supports or dia~otized c~llulose supports. However, the bonds ~etween these supports and the oligonucleot~des are oither not covalent~ th~reby allowins a certain release o~ the oligonucleotideR ~ro~
the 5upport ~ or the supports have other shortc~mings.
For example, N-hydroxysuccinimide or cyanogen bromide activated polysaccharid~ affin~ty supports have a seriou~
drawback in the leakage of ligand~. This not on-y leads to mi leading r~sults ~ut, even more i~portantly, po es he~lth hazards whsn im~unoa~finity-puri~ied products produc~d ~y reco~blnant DNA ~ynthesi~ are complexed wi~
~ouse ~onoclonal antibodi~s ~s~e e.g. Wilchek et ~
~iochemi5try ~, 2155 (1987) and Wilchek ek al. PNAS 7~, 10S5 (1975)~o ~ea~age fro~ ~olid support obviously inter~er~s with af~inity puri~ication: if the free ligand that leaks fro~ the support is more effective as a ,: :

W090/07582 2 ~ PCT/US90/00089 binder than the insolubilized l~gand, the free ~igand will bind the target macromolecule essentially irreversibly, and preYent affinity adsorptlon to the column. Further, cyanogen bromide activati~n of polysaccharide supports leads to the formation of N
substituted isoureas on the sur~ace of the matr~x. These confer undesirable ion exchange properties to the support, which become problematic in affintty chromatography, when analy~es (such as nucleic acids~ are present in v2ry minute concentrat~ons.
The attachment of oligonucleotides containing -~
an aldehyde or carboxlylic acid group at the 5'-terminus t~ non-porous polystyrene latex solid microspheres is disclosed in Kremsky et al., ~ucleic Ac~ds Research 15, 2891 (1987~. Although this method prov~des good end-attachment results, it is disadvantageous in that at the end of the coupling reaction, non-lcovalently bound oligonucleotide requir~ removal ~y a tedious gel electrophoresis step.
Th~refore, ~olid supports with cross-linked, porouæ polymer~c matrix ~tructures that are able to capture and covalently ~ind oli~onucleotides are preferred. For example Sephacryl beads are wldely used due to their excellen~ hybrid~zation properties~
A so called "bead-bas~d sandwich hybridization syst~m" (BBSHS) ~ or exa~ple, descr~bed in th~
following publ~at~on : EP ~7~ 302.
~ccording to thi~ method, ~n ~ ~ir6t ~tep, ~ target nucleic acid and an oligonucleotids probe used for its detect$on, which 1~ ~omplementary to at least a region of the target~ are hybr~dized. ~h~
obtained adduct ls then captured by ~ second ol~gonucleotide, th~t iB csmplementary to a d~fferent reg~on of the target, and is end-attached to a Golld support. The a~ount of the detect~on ol~gonucleot~d~
2 PCTtVS90/00089 associ~t2d with the solid support is directly related to the amount of the target captured. In this way/ the BBSHS ~an be used to determine the amount of a single-stranded nucleic acid in a sample. In this and simil~r assays most commonly radioactively (e.g., 3ZP) labeled cloned DNAs or synthetic oligonucleotides are employed.
32P-labeled oligonucleotide probes used in conjunction with Sephacryl beads in BBSHS experiments provide about 10:1 or better signal to noise ratios wi~h target sequances present in about 0.5 fmole amountsO
~~ In practice, non-radioisotopic reporter systems are often preferred, pr~marily due to the inconveniences associated with handlinq, storage and disposal of radioisotopes. Successful applici~tion of a non-radioisotopi~ reporter system re~ires a detection system which exhibits high sensitivities and low background properties when used in conjunction with the reporter system on a given solid support. ~he Sephacryl beads suppor~s show serious limitations when used in conjunction w~th non-radioisotopic, e.g. colorimetric, detectio~ systems. For exa~pl~, the colorimetric signal from enzy~e-~iigonucle~tide con~ugates in sandwi~h for~ats and direct capture experi~ents on Sephacryl beads was compro~ised by undesir2ble background, thereby giving low ~ignal to noi~e ratios. In the presence of targe~, the non-~peci~ic background can be ~ result o~:
1) hybridizat~on o~ the deteot~on and capture oligonuclaotida to non-exact sequence~ o~ the target nucle~c ac~d;
2) hybridization o~ ~he detec~ion olîgo~ucleotide to the capture oligonucleot~de;
3) non-specific attachment of the det~ction oligonucleotide to the besd ~upport or walls o~ the reaction vessel.

.
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W090/0~582 PCT/US90/00089 While the first two of these possi~le cause.
can be min mized by su~ficiently stringent s~lution hybridization, capture and wash conditions, the reasons for non-speeific binding properties are poorly understood.
It would be desirable to find solid supports t~at have better binding properties, e.g. on which the non-specific attachment of the oligonucleotides used for detection of the target nucleic acids is lower and which show a greater capture potential of the immobilized probe, especially when used in conjunction with non-radioisotopic detection systems.
The properties ~ost sought for in solid supports used for detection of nucleic acids are:
- hydrophilicity - ease of handling, 6uch as compatibility with centri~ugation techniqu~s - t~e presence of suitable ~unctional yroups - low non-6pecific bindin~ of the detec~ion oligonucl20t~des.
In search for new solid supports, attention focused on polyacrylamide-based matriceC. These ~upports are co~mercially av~ilable in a wid~ range of pore sizes, and are u~ed r~utinely, for exampls, in affinity c~romatography. Their hydrophilicity, lack of ch~rged re6idu~s on thei~ surface, and ease o~ derivatization axe some of the properties which make them potentially attractive as supports for th~ attach~nt of ~ligonucleotides~ Chem~c~l derivatization of these cross-linked p~lyacrylamide beads for use in ~ffinity chromatography provided ~paced out functional groups ~or att~ching spec~fic ligand~ in ori~n~ations ~a~ora~le for speci~ic ~indin~ with ~rious ~acromolec~les, ~hereby en~bling the selecti~e re~ention o~ these ~acro~ol~cules, ~.y., protein~ tInman, J~X., Meth. Enzy~l 34:30 : . .... .
- .. : . , ~ , ~ ..
,, .. ; ., : . : ,.
. :. . . -.:, W090/07582 P~T/US90/00089 .,;, ~
~ 6 (1974)~. Inman presented a ~onvenient method for the preparation Qf hydrazide derivatives of cross-linked polyacrylamides by reactin~ their primary amide groups with hydrazide. Depending on the reaction conditions, hydrazide derivatives with different levels of hydrazide fu~ctionality were obtained. The hydrazide derivatives are suitable starting materials for the preparation of other derivatives. For example, bromoacetyl-derivatized polyacrylamide matrices can be obtained by reacting the hydrazide derivatives with N-hydroxysuccinimidle ester of - bromoacetic acid, on-th~~analogy of the reaction described by Bernatowicz et al., Anal. Bioohem~ 155, 95 (1986).
our further goal was to Idevelop a methodology ~or the attachment of oligonucleotides to solid supports by which the nucleic acids are tethered to the ~olid supports by their 5'-ends. In case of the conventionally used solid ~upports, a.g. Sephacryl beads the end-attachment is relatively low (about 50-55%; Ghosh et al., Nucl. ~cids Res. 15:5353; ~1987)). ~ higher degree of end-attachment would be manifested in greater capture poten~ial of the i~mobilized oligonucleotide probe~
As an object o~ the present anvention, it h~s been found that thiol a~d bromoace~yl groups of suitably derivatized oligonucleotides and polyacrylamide ~ol~d supports reaa~ w~th raasonable yields and their reaction, ~urprisingly, results in an almost 100% end attachment of the oligonucleotides. Accordingly, thiol-derivatized oligonuclectides were used in ~ombination wit~
bromoacetyl-deriv~tized polyacrylamide 601id supports.
Thiol-oligonucleotides can, ~or example, be prepared as desçribed by Peng Li e~- al., Nucl~ ~cids ~ , 5275 ~1987) or Orgel et al., ~ucl._ ~cids Res. 14, 6513 ~1986 . , -: . . ~.
:, . : . -' ~' , ''' ' , W090/07582 PCT/U~90/000~9 ~ lternatively, bromoacetyl-oligonucleotides were a~tached to thiol-derivatized polyacrylamide supports. Bromoacetyl-derivatized oligonucleotides are new compounds. The thiol-derivatized polyacrylamide supports ~re known in the art, and ars, for example disclosed in ~eth. Enzymol. 34:30 ~1974)~ The known thi~l-affinity supports were prepared by the coupling of carboxyl groups with cystamine, f~llowed by reduction with exces~ DTT and not via hydrazide derivatives as in the process of the present invention.

Summary o~ the_Invention The present invention relates to oligonucleotides immobilized on solid supports and to new methods for the attachment of oli.gonucleotides to solid supports.
~ ore particularly, thi~; invention relates to oligonucleotid~s immobilized on polyacrylam~de solid suppor~s and processe~ ~or coupling said oligonucleotides to ~aid polyacrylamide 6upports. By developing a new methodology for th~ immobllization of oligonucleotides on polyacryla~id~ matrice , our primary goals were to ~nv~stigat~ the influence o~ pore s~zes on the hybridization properties of immobilized oligonucleotides, and to ~ncrease the coupling e~fic~ency, particularly, tc increase the percent~ge of oligonucleotides that are attached to the ~olid support by their ~'-termi~0 As hereina~ove describad, the experiments were perfomled with two essential system~
3~ E~ther khiol-derivatized oligonucleotides were at~ached to bro~oacetyl-derivat~ed solid supports or bro~oacetyl oligonucleotides were coupled with thiol-derivati~ed polyacryla~ide ~olid supports. Although coupli~g efficiencies varied considerably depe~ding on the ~ctual format used, we have ~urpri~in~ly ~ound that in both - , ;.
- " ~ : ~

.~ :

W090/0758~ PCT/US90/00089 ~ c~, formats essentially ali (more than 95%) oligonucleotides were end-attached to the polyacrylamide solid supports.
This was reflected in the superior direct capture ability of the oligonucleotides immobilized according to th~
present invention for complementary oligonucleotides and double stranded DNAs. Polyacrylamide supports with very high (about 2 x 107 daltons) exclusion limits performed particularly well.
In anoth~r aspect of the invention, we have found that non-specific adsorption of the ~egatively charged nucleic acids can be considerably~reduced by converting the residual functionalities of solid supports (that do not participate in the coupling reaction) into ~ther functionalities having anionic properties, e.g.
carboxylic or trinitrophenyl groups. The obtained supports with mixed functionaliti.es are particularly preferred for practicing the pres;ent invention.
Accordingly, the present invention relates to polyacrylamide supports having covalently attached thereto oligonucleotides substant:ially at their 5'-ends.
In anoth~r aspect, the present inv~ntion is directed to a process for coupling thiol-derivatized oligonucleotides to polya~rylamide supports substantially at tAeir 5'-ends, the primary amide groups o~ ~aid supports being at least partially converted into bromoacatyl groups prior to end-attach~nentO
According to another aspect, the invention relates to a process for coupling bromoacetyl-derivatized oligonucleotides to polyacrylamide suppoxts substantially at their 5'-ends, the primary amide groups of said supports being at least partially converted into thiol groups prior to ~nd~attachment.
Following eith~r coupling strategy, the i~vention includes ~urther derivatization of the polyacrylamide supports by converting th~ir . .
: . ~ .: .

WO90/07582 PCTtl~SgO/00089 t''~) :

functionalities not involved in coupling into anionic functional groups to provide better non-specific ~dsorption results.
According to a ~urther aspect, there are provided oligonucleotides derivatized at their 5'-ter~ini with bromoacetyl groups.
According to a still further aspect, the i~vention concerns bromoacetyl-derivatized oligonucleotidec immobilized on thiol-derivatized polyacryla~ide solid supports, or conversely, thiol-derivatized oligonucleotides immo~ilized on bromoacetyl-derivatized polyacrylamide solid supports.
The present invention is directed to the above aspects and all associated methods and means for accomplishing such. For example, methods for preparation and purification of the detection and capture oligonucleotides, including synthesis or isolation from a natural source via restriction cleavage and subsequent purification;
preparation of oligonucleotide-signal element (e.g., radioactive atom, enzyme, fluorescent or chemiluminescent probes, mercury-based detectors, etc.) adducts for use in hybridization with the target nucleic acids:
hybridization techniques for hybridizing the target nucleic acid to the detection (and capture) oligonucleotide;
and so forth, are within the scope of this invention.
Brief Description of the prawinqs Fi~ure 1 illustrates the synthesis of bromoacetyl- (Reaction A) and sulfhydryl-containing (Reaction B) Biogel polyacrylamide supports.

. . .
... . . .;

::'` : , ' ,:, ` : : ', . '' WO90/07~2 PCT/US90/00~89 .~ .~

Figure 2 ~hows the synthetic route of thiol-oli~onucleotides and their coupling t~ br~moacetyl-derivatized 8iogel polyacrylamide supports. (The residual hydrazide groups are convert~d to carboxyl groups to lower non-specific binding.) Figure 3 is a reaction chart of the synthesis of bromoacetyl oligonucleotides.
Figure 4 shows the acylation of thiol-derivatized Biogel polyacrylamide supports and the use of the polyacrylamide matrices with mixed functionalities in ~ coupling with bromoacetyl oligonucl~otides.
Figure 5 is a synthetic scheme for the preparation of Trisacryl-SH supports.
Figure 6 shows the acylation of thiol-derivatized Trisacryl supports and the use o~ theproducts with mixed functionalit:Les in capturing bromoacetyl oligonucleotides.

Detailed DesçriE~tion_of the Invention 1. Definitions and aeneral methods The kerm "oligonucleotide" as used throughout the speci~ication and the claims re~rs to nucleic acids including bDth single and double stranded RNA and DNA
molecules that m~y be isolated from a natural source, may be synthesized or pxoduced by restriction digest.
By the term "detection oligonucleotide" or grammatical variations thereof is meant a nucleic acid (~NA or DNA) sequence (isolated ~rom a natural source, synthetically produced or a product of restrictiQn digest carrying a reporter labal) that has su~ficient homology with a target nucleic acid ~equence such that under ~uitable conditions it is capable o~ hybridizing with said target se~uence.

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' ~ . ., - ........ .

WO90/075$2 PCT/US90/00089 .. . '. i t ~

The term "capture oligonucleotide'i specifically refers to a nucleic acid (RNA or DNA) sequence (isolated from a natural source or synthetically produced or a product of restriction digest) that is attached to a solid support, preferably substantially at its 5'-end, and that has suf~icient homology with a target nucl~ic acid sequence (different from the sequence hybridized to the detection oligonucleotide) suc~ that under suitable conditions lt is capable of hybridizing with said target sequence.
- Typical~detection and capture oligonucleotides are about 12 to 200 nucleotides, preferably about 15 to 40 nucleotides in length, and usually share at least about 12 bp, preferably about 25 bp complementarity with the target nucleic acid sequenoe.
"Polyacrylamide supports" are cross-linked polyacrylamide matrices that are commercially available in a wide range of pore sizes. 'rypical representatives of such matrices are "Biogel" be,ads, manufactured by Blo-Rad (USA) that are further cat2glDrized according to theirexclusion volu~es. The molecula:r weight exclusion limits 3f Biogel P-2, Biogel P-10, Biogel P-60 and ~iogel P-200 are 2000, 10000, 60000 and 200000 daltons, respectively.
~lthough our experiments ware predominantly carried out with Biogel beads, other polyacrylamide suppor~s, including thos~ in which certain groups, e.g. the amide groups are substituted, ~ay also be used for practicing the present invantion. A typical repres~ntative of such supports is Tri8acryl GF-200 (TBF Biotechnics, USA) that is produced by copolymerization of N-acryloyl 2-amino-2-hydroxymethyl-1,3-propane diol and has an exclusion li~it of 2x107 daltons. In this r~sin the secondary a~ides contain ~-hydroxymethyl-1,3 propane diol ~bstituents, aach of it~ repeating units containin~ thr~e hydroxymethyl groups and one secondary amide group.

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WO 90/07~82 P~/llS90tO0089 e. ~
~9'' 12 Consequently, this polymer is more hydrophilic in character than the Biogel resins.
The primary amide yroups of the polyacrylamide supports used according to the present invention are at least partially converted into bromoacetyl groups or thiol groups prior to the attachment to the oligonucleotides. The hromoacetyl and thiol derivatives were generated from hydrazide-derivatized supports.
Further details of the respective procedures are to be found in the description of preferred em~odiment and in the examples. - - ~~~~~
Since the conversion of hydrazide supports into bromoaoetyl or thiol derivatives is not guantitative, the ~nconvertecl hydrazide functionalities are available for 15 further derivatization; thus, if desired, they can be further convertéd into other groups, such as carboxyl, trinitrophenol, etc., groups. All of ~uch derivatives are within the scope of the present inventio~. It should be noted that, although the bromoacetyl and thiol-derivatized polyacrylamide supports were prepared fromthe respective hydrazide compounds, other synthetic routes may also be available and are encompassed by the invention, provided that they are suitable for the production of polyacrylamide supports the pximary amide groups of which are at least partially converted into bromoacetyl or thiol groups (see e.g. Inman, ~eth.
Enzy~ol. 34, 30 ~1974)).
Oligonucleotides can be synthetized and purified by any method known in the art, for example using t~ ~olid-phase cyanoethyl pho~phoramidite method and HPLC purification ~Ghosh, et al., Nucl. Acids Re~.
15, 5353 (1~87)]. AlternatiYely, they can be isolated from natural sources or produced synthetically or by restriction enzyme cleavage and, if desired, tailored so as to be suitable for the intended use.

.: .
.:, .- . , :
:, Thiol- and bromoacetyl-derivatized oligonucleotides can be prepared, as hereinbefore described, by literature-kn~wn processes~ Further details of their preparation are given in the description of preferred embodiments of the invention and in the examples.
In the specification and claims, when describing the process of hybridization between oligonucleotides and solid supports, the terms "attachment", "coupling", "tether", "binding" and "immobilization" are used interchangeably and refer tG
covalent linkage of oligonucleotides to the solid supports~
The term "TASI' i5 used to refer to the transcription amplification system disclosed in PCT International Publication No. W088/10315, This method involves using oligonucleotides to prime the synthesis of a ~ouble-stranded DNA copy (cDNA) of the target DNA or RNA
sequence.
In an e~bodiment o~ TAS, one of the oligonucleotides, primer A contains, within it sequenc , the T7 RNA polymerase promoter bind:Lng sequence ~P~S) attached to ~equen~es complementary to the target sequence ~TCS). ~lsngation fr~m this primer by rever~e transcriptase results ~n the generation of a ~ingle-stranded cDNA conta~ning the T7 promoter at its 5' end.
A ~econd primer oligonucleotide, primer B, ~5 . comple~entary to the first cDNA strand at B~me dist~nce (100-300 bases) downstream of primer ~. Pri~er ~ i~ used to initiate synthesis of the second cDNA 6trand, producing a double-stranded cDNA with the T7 ~NA
polymerase promot~r attached. Incubat~on of the double~
~tranded cDNA with T7 RNA polymerass and ri~onucleotide ~riphosphates will result ~n the ~ynthe~is of ~NA

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transcripts from the cDNA. Additional amplification can be achieved by repeating 'l'AS on the newly synthesized RNAD
As to cther aspects of the invention, including preparation and purification of oligonucleotides, preparation of oligonucleotide-target nucleic acid adducts, methods for attachment of oligonucleotides to solid supports, hybridization methodologies, detection and measurement of signals generated by properly hybridized nucleic acids, etc., reference is made to standard textbooks of molecular biology.
See, for example, Maniatis, et al., Molecular Cloning: A Laboratory Manual, Colcl Spring Harbor Laboratory, New York 1982, and the various references lS cited therein; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York (1986) and Hames, et al., "Nucleiç Acid Hybridization", IRL Press, (1985~.

2. DescriptiQn of preferred embodiment Before further derivatization, the polyacrylamid~ matrices, e.g., Biogel beads sr~ trea~ed with hydrazin~, essentially following the procedure of Inman, Supra. (See Reference Example 2.~ Depending on the hydrazine concentrations, reaction temperatures and reaction times hydrazide sllpports with various substitution levels of the hydrazide functionality may be obtained. The reaction can ~e carried out at room temperatuxe o~ at higher temperatures (see Inman Supra). . .
Hydrazide gr~up densities may be measured by the method given in the Analytical Procedures section of the In~an re~erence referred to above or Reference Example ld. In the~e experiments polyacrylamide resins with dif~erent . exclusion li~its can be e~ployed, the higher exclusion limits b~ing preferred. According to a particularly ..;.

, : , . .. " , ~ ;

W~90/07582 PCT/US90/~00~9 .7~, ~5 pr~ferred embodiment, polya~rylamide supports having large pore sizes, in particular those with exclu~ion , limits over 400000 daltons, mo~t preferably about 2x107 daltons, such as Trisacryl GF 2000 (IBF Biotechnics, USA) are used.
. Although the non-specific binding of [~2p~_ labeled oligonucleotides was found to be low and not ~ependent on the level of substitution of the hydrazide functionalities on hydrazide-derivatized Biogel resins, hydrazide-derivatized supports did not perform well ln binding experiments with enzyme-oligonucleotide conjugates primarily due to increased non-specific binding, principally attributed to the enzyme c~mponent of the conjugate. This problem was thought to be eliminated by further derivatization of the supports.
According to an embodiment of the invention, thiol-oligonucleotides are coupled to bromoacetyl-derivatized polyacrylamide supports.
The bromoace~yl derivatives of polyacrylamide supports are preferably obtained by treatment of the hydrazide functionaliz2d support~ wit~
excess of bromoacetic acid-N-hydroxyguccinimide est~r, essentially following the procedure described by ~ernatowit~ et al., SuDra. The extent of derivatization ~an be determined by ~ two-~tep procedureO In the ~ir~t step, the ~upports are exposed to a large excess of dithio~hreitol (DTT) t~ effect a quantitative bromoa~etyl-to-thiol fun~tional~ty conversion. The thiol groups are then titrated with Ellma~'g reagent 15.S'-dithio-bis(2-nitr~benzoic ac~d)~. Under normal reaction conditions, the conYersions u~ually range between 16-S~, there~ore, the res~dual hydrazide grcup~ ~re still available ko further derivati~ation. Carboxyl derivatives can be obtained by tr~a~ing the bromoacetyl : .
,-~, , . . :

woso/o75~ PCT/US90/00089 derivatizecl supports with excess glutaric anhydride, to effect a hydrazide-to-carboxyl transformation In non-specific binding experiments with oligonucleotide-alkaline phosphatase conjugates the S bromoacetyl-derivatized supports perform considerably better than the hydrazide-derivatives. Non-specific binding is further reduced by the hydrazide-to-carboxyl transformation. It was found to be advantageous to silanize the eppendorf tubes and have 1% BSA in the hybridization solutions to prev~nt adhesion of the oligonucleotide-enzyme conjugates to the walls of the tubes.
~ o effect coupling, the oligonucleotides are also derivatized. Since thiol co~pounds, due to their greater nucleophilicity over amin~s, show enh~nced reactivities towards alpha-halo calrbonyl substituted compounds, according to the invent:ion, the bromoacetyl-derivatized polyacrylamide support:s are coupled with thiol-oligonucleotides. Preferably, thiol-derivatized oligonucleotides are prepared using a [32p] -labeled 5'- 1 phosphoxylated oligonucl~otide in two steps: (1) reaction of the phosphate group with imidazole in the presence of a diimide and displacement of the imida~ole leaving group with cystamine in one reaction step: and reduction o~ the disulfide bond of the cystamine linker with DTT. A
simil~r procedure was described by Orgel et al~, Supra.
The 5'-phosphorylated starting oligonucleotides are prepared as described by ~aniatis et al., Supra, p. 122.
D~tails of the synthesis of their thiol-derivativas are set forth in Example lc. The overall phosphate-to-thiol transformation obtained by this process was estimated to be 60 75% by polyacrylamide qel analysis of ~32P]-labeled products, ~nd susceptibility of clearanc~ of 5~_32po~
label of unreacted phosphorylated oligonucleo~ide by , . . :: , , .......................... . , :

: . . . .
: i ", ~, .. . ;: :

WOsO/n75s2 PCT/US90/00089 alkaline phosphatase treatment, as described in Example ld.
The thiol-derivatized oligonucleotides are coupled with the bromoacetyl-derivatized polyacrylamide solid supports under an atmosphere of nitrogen (see also Example le~. The attachment of thiol-oligonucleotides to the supports can be monitored using radiolabeled nucleic acids. The coupling efficiency of thiol-functionalized oligonucleotides to the bromoacetyl-derivatizad solid supports is dependent on the pH of the reaction, with pH
of about 9.O being preferr~d. We have found a considerable variability in the attachment efficiencies `~
in thes2 experiments, probably due to the susceptibility o~ the reactive thiol group to air oxidation in storage and duFing the course of the reaction. The overall attachment efficiencies were found to be between about 3.5% and 48%. However, since the yields of the coupling ~xperiments are calculated as percentages of cpms o~ the total radioactivity introduced in the system, due to the incomplete conversion o~ the 5'-plhosphorylated oligonucleotides to their thiol-dlerivatives, the actual yields are considerably hiqher. Nor~ovex, following this method, ~ore than 99% of the olig~nucleotides are attached to the solid support through their 5'-termini.
This is a striking advantage over other systems known in the art, for example Sephacryl beads, where merely about 50-55% of the Oligonucleotides are end-attach~d.
Duriny the course of thi~ work, we found lt beneficial to: (1) use ~resh thiol oligonucleotides for the coupling reaction; (2) degas all reaotion soluticns prior to use; and (3~ car~y out the coupling under an inert atmosphere.
The hybridization potential of thiol-oligo~ucleotides immobilizPd on bromoacetyl polyacrylamide supports wa5 evaluat~d using either ~ 32p_ ~` :` ` :; . , , `, ` - "~ ;, :: ,::: :: ~., :,:: : : :: ::.. ,. :.: .. . . .
: .. ~ ~. ::: .. :: .

.. . , ,, : ,. ~;: . . ~ . :
~. : :, , ,:

WO90/07582 PCT/~S90/00089 '~ '';i ~J
1~
labeled oligonucleotide in a direct capture experiment, or a single-stranded 7Kb plasmid DNA target in a sandwich format. The oligonucleotides immobilized according to the in~ention generally gave better results than Sephacryl beads for direct capture of target oligonucleotides in this construction. The results w~re somewhat worse in case of sandwich capture of long target DNA.
Using the reverse format, bromoacetyl-derivati2ed oligonucleotides are coupled with thiol-derivatized~polyacrylamide solid supports. The introduction of thiol functionalities on polyacrylamide matrices can be achieved by reacting hydrazide~
polyacrylamide supports with N-acetylhomocysteine thiolac~one, essentially as described in Example 2a hereinafter. The extent of derivatization can-be determined, by titration of the thiol group with Ellman's reagent, as hereinbefore describ~d. The conversion usually is between about 4% and about 15%, and is strongly dependent on the amount of hydrazide groups in the solid supports: supports with higher hydrazide substitution levels being preferred. Since the conversion of hy~razide groups into thiol groups i~ low, there are always unreacted hydrazide groups left that are available for further derivatization. Natrices wit~
mixed functionalities, in which the residual hydrazide groups are converted, for example, into carboxyl or ``
trinitrophenyl groups, without modi~ying the thiol-- functionalities, are easy to prepare by literature-known processes and may have advantages in certain hybr~dization reactions, by reducing non-specific absorption of the negatively charged nucle$c acids, due to the anionic properties of carboxyl and trini rophenyl groups. Treatment o~f the polyacrylamide ~upports with glutaric a~hydride and/or iodoacetic ac~d converts the ,. . ..
....
, .. . : : , . ~

WO90/075~2 PCT/US90/00089 ~ ~ 'J

hydrazide and sulfhydryl functionalities to carboxylic groups. These supports were found to provide better non-specific adsorption results and are, therefore, preferred for use in direot capture experiments.
Oligonucleotides derivatized at their 5'-termini with bromoacetyl groups can, for example, be prepared by reacting 5'~aminohexyl-phosphoramidate oligonuclPotides with bromoacetic acid-N-hydroxysuccinimide ester. The performance of the reaction i5 further illustrated in the examples. The ~ phosphate-t:o-bromoacetamide transformation is about 60-70% determined by polyacrylamide gel analysis of th~
~32P]-labeled products, and susceptibility of cleavage of 5~_32po~ label of unreacted phosphorylated oligonucleotides to alkaline phosphatase treatment.
The covalent attachment of the bromoace~yl-oligonucleotides to thiol-derivatized polyacrylamide ~olid supports is illustrated in the examples. The coupling efficiencies are between about 3% and about 30%, depending on ~he pore size (molecular weight exclusion limit) of the solid support employed. Ac not~d earlier, the actual coupling efficiencies are hi~her, since these figures do not reflect the incomplete conversion of the 5'-phosphorylated oligonu~leotides to their bro~oacetyl derivatives. Surprisingly, more porous ~atrices with high exclusion li~its provide substantial~y better roupling results than thP highly cross-linked variants.
Tha hybridization potential o~ bromoacetyl oligonucleotides immobilized on thiol-derivatized solid supports was tested by direct capture of oligonucleotide targets and in candwich format, using TAS ~NA
trans~ripts. The results were similar to those obtained with the reverse fo~mat. While the oligonucleotides immobilized on thiol polya~rylamide supports were clearly batter in direct capture o~ oligonucleo~ides than .~ . . "., . . . ",...,; :
, . " :, ` .' q~

Sephacryl beads, their capture pot2ntial was less express~d in sandwich capture experiments, especially when long targets were to be identified.
The direct capture and background properties of bromoacetyl ester derivatized oligonucleotides immobilized on thiol-polyacrylamide solid supports were also tested with oligonucleotide-alkaline phosphatase conjugates. Although the background from non-specific binding was found to be low, the greater capture potential of polyacrylamid~ supports was lost in the case of oligonucleotide-enzyme conjugates.
According to a particularly preferred embodiment of the invention, polyacrylamide supports with exclusion limits of about 2x107 daltons are employed. A
typical support from this group is ~risacryl GF-2000 ~IBF
Biotechnics, USA). Since, as hereinabove described, the amide hydrogens of this polyacrylamide resin are ~ubstituted with 2-hydroxymethyl-1,3-propane diol groups, be~ore further derivatization, reactive amine groups need t~ be introduced, for example by transamidation with Pthylene di~mine. This reaction is carried out at slightly elevated temperatures, preferably at about 90C. -~
Thiol-derivatives of these supports are prep~xed in an analogous manner to the preparation of the thiol; r derivatized Biogel products. To reduce electrostatic interactions between the positively charged amine groups and the negatively charged oligonucleotide backbone, the residual unr~acted amine groups on the Trisacryl supports are pre~erably-acylated either with glutaric anhydride or succinic anhydride. The substitution lev~l o~ the sulfhydryl groups on the Trisacryl-SH supportsj determined by titration of the sulfhydryl functionalities with DTNB, is in the range of about 10 to about 16 ~moles/gr. With these supports, the coupling efficiencies to brom~acetylated oligonucleotides is about .- ; . . , ,- , . . .
.. . . ..

WO90/075~2 PCT/US90/00089 20%, and the level of end-attachment is extremely high (about 97%). The oligonucleotide substitution level of these supports is about 60 pmoles oligonucleotide/gram support.
In hybridization studies involving direct capture of radioactively labeled oligonucleotides, Trisacryl-SH supports containing immobilized oligonucleotides, especially supports in which the unreacted sulfhydryl groups were alkylated with iodoacetate, p~rformed particularly well. The oligonucleotides immobilized on Trisacryl-SH supports showed excellent hybridization potential also in sandwich hybridization ~xperiments, essentially carried out as hereinabove described. The signal-to-noise ratio was about 4-times higher in sandwich hybridization experiments performed with Trisacryl supports than on Sephacryl beads.
The bromoacetylated oligonucleotides couple to thiol-derivatized polyacrylamide supports with reasonable yields, and the reproduci~ility o~ the reaction is very good. In case of the reverse ~or~at, usiny thiol-oligonucleotides and bromoacetyl derivatized supports, the col~pling a~ficiencies show greater variability, probably du~ to the higher susceptibility of thiol-oligonucleotides to oxidation. ~owever, both ~pproaches provide extremely good end-attachment results.
Essentially all of the oligonucleotides (95% or more) are attached to th~ solid supports by their 5'-termini, by using eiSher couplin~ ~ethodology of the present invention.
The hybridization potential of oligonucleotides i~mobilized on polyacryla~ide solid supporSs following any o~ the processes disclosed in t~e present invention, is v~y good in direct capture experiments, primarily due to the superior direct capture ability of end-attached :: :.: .. . : ; : , - : .- - . . .. ... .

W~90/07582 PCT/US9~/00089 oligonucleotides. In sandwich hybridiza~ions with TAS
RNA products or long, single-stranded DNA fragments, the polyacrylamide supports having large pore sizes perform especially well in contrast to their highly cross-linked counterparts. The results can be fur~her improved by functionalization of these porous supports, e.g., with carboxyl groups. The best results were clearly obtained with polyacrylamide supports having an exclusion limit of about 2x107 daltons (Trisacryl GF 2000).

- 3. Examples ~ ~~~~
eference Example 1 Te~t methods a. ~irect c~pture of 32P-labeled targets on oligonucleotida-deriv~t~zed suppcrt3 ~ he bead sa~ples (50 mg) were aliquoted into 1 ml eppendorf tubes, and the supernatant was removed after centrifugation. Prehybridization in 250 ~l o~ 5x SSPE
~0.75 M NaC1, 50 mM NaH2P0~, pH 7.4, and 5 mM ethylene diamine ~etracetate~4 Na (EDT~)], 10% dextran sulfate, 0.1~ sodiu~ dodecyl sul~ate (SDS) was carried out at 37^C
~or 30 minute~, after which tha supernatant was drawn off. Sampl~s of complementary and non-complementary 32p_ :
labeled oligonucleotides (approximately 30 bases) were preincu~ated at 65-C for 5 minutes and then added to the bead samples ~typically 3.75 fmoles oligonucleotide/lO0 ~l hybridi2ation bu~fer~. The beads were incubated at 37-C for l hour with occasional shaking. After washing with 5 x l ml o~ 2x SSC (0.3 M NaCl, 0.03 ~ Na citrate, p~ 7.0) at 3~-C, the amount o~ label bound to the supports was deter~ined by Cerenkov counting.

nd~ hybri~izstio~ Or ~ ge~er~t~
~N~ tra~cxipts o~ ~oll~ support3 , , WO~0/07582 P~T/US90/0~89 Samples of Biogel (Bio-Rad, USA) or Trisacryl (IBF Biotechnios, USA) (50 mg) were prehybridized as outlined a~ove.
The target, DNA or ~NA, was denatured at 65-C
for 5 minutes immediately prior to hybridization.
Solution hybridization of the target RNA (O.5 f~oles) with a complementary, 32P-labeled detection oligonucleotide (5 fmoles) was performed in a total volume of 20 ~1 in 5x SSP~, 10% dextran sulfate, 0.1%
SDS for 2 hours at 42'C. The sample was then diluted to - . 100 ~1 with hybridization-buffer and added to the solid support. Sandwich hybridization was performed at 37 C
for 1 hour with occasional shaking. Finally, the beads were washed with 5 X 1 ml 2x SSC at 37-C. Non-complementary RNA target was usecl as a control to determine the level of non-specii.ic binding in the assay.

c. Direct captura o~ ~lkaline phosph~tase-oligo~ucl~otide con~ugates o~ so$~ ~uppor~s Oligonucleotide-containing Biogel or Trisacryl supports ~50 mg) were prehybridized with 250 ~1 of 5X
SSC, 10~ dextran ~ulfate, 0.1% SDS, 1% bovine serum albumin fraction V bovine seru~ ~lbumin fraction V tBSA) in 1 ml eppendorf tubes for 30 minutes at 37-C.
~omplementary and r~on-complemer~tary oligonucleotide-alkaline phosphatase conjugates were dilul:ed with 0.1 M
Tris, O.1 M NaCl, 1% BSA, pH 7 . 5, and preincubated t 55 C for 5 minutes. ~fter addition of the conjuga~e (37 . 5 ~moles) in 100 ,ul o~ hybridization buffer, the mixture of conjugate and Sephacryl bead5 was incubated at 42-C ~or 1 hour wlth occaslonal shaking. The supports wers washe~ with 4 x 1 ml o~ 2x SSC at 37 C. The bead samples and washes were treated with 1 ~1 0.1 m~ ~-nitrophenyl phosphate in 0.1 ~ Tris, 0.1 M NaCl~ 0.
MgCl29 pH 9.5. A~ter development of color at room .: : .. ..
. .
.. ..

WO~/075~2 PCT/US90/00089 .,....p ~ A ~

temperature for 1 hour, the formation of ~-nitrophenolate was measured by reading the absorbance at 410 nm.

d. 'rNB8 te~t for hy~r~zl~es and ~mi~es Filtrates for hydrazide or amine-derivatized supports were tested for ~ree hydrazide or ethylene-diamine as ~ollows. Two ml of filtrate were treated with 1 ml of saturated Na2B4O7 and 3 drops of 3~ aqueous trinitrobenzene sulfonate (TNBS), with thorough mixing.
The appearance of a purple or orange color after onP
minute indicated the presence of hydrazine or amine, respectively. ..

e. DTNB te~t for sulfhydryl an~ bromoaostyl sub3t~tutio~ ~eter~ination A sample of derivatized support (1-20 mg wet weight~ was reduced with 500 ~1 of 20 mM dithiothreitol (DTT) in 0.05 M ~2HPO~, 1 mM EDTA, pH 8.0, for 30 minutes in a l-ml eppendorf tuba and washed with 3 x 1 ml of 0.05 M K2HPO~, pH 8.0, after the supernatant had been removed. The beads were then treated with 1 ml of 1 mM
5,5'-dithiobis(2-nitr~benzoic acid) (DTNB) in 0.05 ~
K2HPO~, pH 8Ø Th~ absorbance o~ the supernatant was ~onitored at 412 nm after 15 minutes (~ = 13,600 ~or released thiophenolata).

Reference Exam~le 2 Prap~rat~o~ o~ hydrazide derivati~es o~ Bio~el ~ 8 Hydrazide derivatives of ~iogel bead~ ~Bio-Rad, USA~ were prepared using ~he procedure of Inman (Meth.
Enzymol. 34(B):30, 1974). Typically, a 20 ml ~olution o~
S M hydrazine at 50C was added to 1 gram of dry r~sin.
After 40 minutes, the excess reagent was removed by .
, . ~
..

WO90t07582 PCT/US90/00089 washing the beads with 0.2 M NaCl until the filtrate gave a negative t~st with 2,4,6-trinitrobenzene sulfonic acid (TN~S). The derivatized beads were sucked dry for 15 minutes, weighed, and suspended in lO mM Tris, 1 mM EDTA
(TE), pH 8.0, or O.l M K2HPO~, pH ~.O.
The non-specific binding of [3 P]-labeled oligonucleotides, alkaline phosph~tase-oligonucleotide conjugates, alkaline phosphatase with BSA, and linker~
derivatized alkaline phosphatase with BSA was determined with the above supports, and the results presented in Table I.

.
NON-8PECIFIC BINDING ON BIOGE~, P-60 ~YD~AZIDB B~AD8 ~ound to Beads Alkaline Alkaline Biogel- [32p] Conju- Conjugate Phosphatase Pho-~phatase-Hydrazide Oligo gate ~ BSA with BSA Linker w/BSA
_________________________ ______________________________ 3 1 micro-mole/gm 0.26 11.3 5.2 1.95 5.90 62 micro-~nole/gm 0 . 23 9 ..8 -~

125 micro-mole/gm 0. 32 13 . 7 4 .1 -- --500 micro-~nole/gm 0.41 13.5 8.6 1.2 ~.49 ., : .: :
., : - .:

WO90/07582 PCT/US90/00~8g 7 ~

The non-specl~ic binding of [32P]-labeled oligonucleotides was low and was not dependent on the level of substitution o~ the hydrazide residues on Biogel. However, background5 from enzyme-oligonucleotide conjugates were much higher (9.8-13.7~) when no BSA was used in the hybridization solutions. This indicated that ^~
the enzyme component of the conjugate was largely responsible for this interaction. The addition o~ BSA to the hybridization mixture was ~ound to be partially lO- effective in eliminating non-specific binding of the enzyme-oligonucleotide conjugate. Parallel experiments using alkaline phosphatase and alkaline phosphatase-linker show that derivatization of the enzyme with ~he linker WdS primarily responsible for the non-specific 15 binding.

Example Covale~t attachment o~ t~liol-oligonucleotide~
to ~romoacetyl ~ogel be~s ~. ~cylatio~ of ~iogel hyarazi~ ~ead-~ w~th N-~u~G~ yl bro~oacetat~

Synthesis o~ N-succinimidvlvbromoacetate 8romoa~etic acid ~20 mmoles) was treated with dicyclohexyl-carbodiimide ~22 mmoles, l.l e~uivalents) and N-hydroxysuccinimide t22 mmoles) in 30 ml CH2Cl2 at 4-C according to the procedure of M.S. Bernatowicz and G.R. Matsueda (Anal. Biochem. ~55:95, 1986) to give the N~succini~idyl bromoacetate as a whit~ solid.
Acylation A l gram ~wet weight) sample of Biogel hydrazid~ beads (50 ~moles/dry gram) was washed with 2x 50 ml 0.1 M ~2HPO" pH 7.0, and resuspended in ~ ~l Q~
this bu~fer. The suspension was cooled to 0-C in an ice .:

~.
:, :
: . , .

WOgO/07582 PCT/US90/000~9 bath, and N-succinimidyl bromoacetate (10 mole equivalents relative to hydrazide groups~ in 250 ~1 N,N-. dimethylformamide (DNF) was added dropwise with ~tirring.
The reaction ~ixture was allowed to come to room temperature over 30 minutes. After cooling to O C again,another ali~uot of N-succinimidyl bromoacetate was added and the reaction mixture was stirred for 30 minutes at room temperature. The beads were filtered through a sintered glass funnel (porosity C) and washed with 50 ml 0.1 M K2HP0~, pH 7Ø The acylated beads were stored in - 0.1 M K2HPOb at 4-C.
To determine the level of substitution, the supports were exposed to a large excess of DTT to effect a quantitati~e bromoacetyl-to-thiol functionality conversion. The thiol groups were then titrated with Ellmanis reagent [5,5-dithiobis-(2-nitrobenzoic acid)~
(see Reference Example le). From Table II it can be seen that the conversion of hydrazide s~roups to bromoAcetyl functionalities proceeded with an efficiency of 56%, 43%, and ~0% for P-10, P-60,~and P-200, respectively, while the reaction with the P-2 support was less efficient.

:' -, : 1'' ,, ' '' . : ;.

WO90/07582 PCTtUS90/000 28 -~
TABL~
RE~CT~9N o~ HYDRAZIDE WIT~ ~RONO~C~IC ACID ~-~
N~B E~T~R PO~YAC~Y~AMID~ ~UPPORT8 Molar ~moles excess ~moles % NHNH2 Support Type NHNH~g NHSBrAc CH28r/g converted _________________________ ._________________________ ___ P-2-CH2Br 50 20 8.0 16~0 (MW cut-off 2,000) P-10-CH2Br 50 20 27.9 55.8 ~MW cut-of~
10, 000) P-60-CH2Br 50 20 21.6 43.2 (MW cut-off 60,000~

P-200-CH2Br 50 20 , 20~3 40.5 (~W cut-off 200,~00 Non-specific binding experiments with oligonucleotide-alkaline phosphatase conjugates (see Reference Example lc) were carried out with t~ree types of P 2 and P-60 supports-tTable III).

~ . ' , , ~: . . , ' . .- . :
., ~
.

. .

W0 90/07582 PCr/US90/00089 TABL~ III

NON--B~ECIFXC BINDING ~I~ 50 FMOLE8 CONJUGAT~ ~% BO~D) Type of -CH2Br -CH2Br Support-NHNH2 -NHNH2 -COO}I
____________ ________________________ _______________ , .

P-2 1.57 1.01 0.71 P-60 7.3 3.5 1.4 -~

A decrease in non-specii.ic adsorption was observed ~or both supports when the hydrazide functionality was capped with bro~oacetyl groups. Since the reaction of hydrazide support~; with bromoacetic acid N-hydroxysuccinimide ~ster doe~ not result in a quantitative conversion ~see Table II, last column), the r sidual hydrazide groups are still available for derivatizatio~

b. G~taryl~t~o~ of ~romoac~tyl Biogel ~o~
A 1. a gram Swet weight) ~ample of bromoacetyl Biog~l beads (50 ~ hydrazid~/dry gra~l was washed with 0.1 M NaCl and suspended in 20 ml of this solution in a glass beaker. To the stirred suspension was added 100 mg of glutaric an~ydride. ~he pH o~ .he suspension was maintained at 4.0-4.2 with 3 M NaOH, whil~ the reaction mixture was stirred ~or 15 minutes. ~hen, another 100 mg of anhydride was addad, and the reaction mixture wa~
~tirred for a further 15 minutes. The beads were then washed with 3x 40 ~1 0.1 ~ K2HPO~, p~ 7.0, by allowing ~he beads to settle in a conical tube and decanting the supernatant.
When the bromoacetyl supports were treated with excess glutaric anhydride, as described above, a hydrazide-to-carboxyl transformation was obtained. The carboxyl group in these bifunctio~al supports pr~vided an additional reduction in non-specific ~inding of the enzyme-oligonucleotide conjugates (Table III, column 3).

c. One-step preparation o~ 5~-~yst~minyl --- pho~phoram~date ~rivative~ o~ oligo~uclaoti~e~
The 5'-phosphorylated oligonucleotide [Maniatis et al., Sup~J in a silanized eppendor~ tube was trea~ed with 300 ~1 of a 0.1 M imidazole, 0~15 ~ 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-Hcl ~EDC), 0.25 M
cystamine dihydrochloride, pH 6. O, and let stand at room temperature overnight. Tha modified oligonucleotides were precipitated with ethanol/LiCl, washed with 300 ~1 H2O, and precipitated again. Yields of 86~ were obtained using this procedure. The reaction is illustrated in Figure 2.

d. D~t~rmi~tio~ o~ leYel o~ cy~ta~in~
att~hme~t t4 5i-pho~pho~ ted oligonucleot~des A 50 pmola sample of 32P-labeled cystaminyl-derivatized oligonucleotide was treatad with 1 ~1 stoc~
cal~ intestine alXaline pho~phatase (Boehringer Man~heim, EI~ qrade) and the volume ~ade up to 100 ~1 with 0.1 M
Tris, 0.1 ~ Narl, 0.01 M ~gCl2J pH 9.5. The reaction was allowed to proceed ~or 2 hours. The sa~ple was then appli~d on a S ml column of Sephadex G-50 and eluted with TE, p~ 8Ø The first and second radioactive p~aks were collectad, corresponding to cysta~inyl-derivatized oligonucleotides and ~ree inorganic phosphate (cleaved from 5'-phosphoxylated oligonucleotide3, respectively.

, .,, , ' ,,: . . . :.

WO90t07582 PCT/US9~/00089 s~

The peaks were counted by Cerenkov counting to estimate the yield of cystamine attachment.

e. Covalent atta~h~ent o~ 5~ oyst~mi~yl-5 derivatized ol~go~u~leotides to bromoacetyl-Biogel bea~
The cystaminyl-containing oligonucleotide was reduced with 300 ~1 0.1 M DTT, 0.2 M HEPES, 0.001 M EDTA, pH 7~7l for 1 hour at room temperature. The product was precipitated with EtOH/LiCl and washed twice with 0.2 HEPES, O.O01 M ~DTA. Typically, 50 mg of beads were ~ treated with 25 pmoies of reduced oligonucleotide in 150 ~1 0.1 M K2HPO~, pH 9.O, and agitated under N2 on a rotary mixer overnight. As a control to detPrmine non-specific . .
binding, a 5'-phosphorylated oligonucleotide was added to a sample of beads under the same conditions. :The beads were washed with: (a) 3x 1 ml O.l M Na2P2O~, pH 7.5, and (b) 3x 1 ml 0.015 M NaOH, pH 12. 32P-labeled oligonucleotides were u~ed as controls to estimate end-attachment efficiencies.
~0 The attachment o~ the t~hiol-derivatized oligonucleotides to the bromoacet~yl-Biogel supports was ~onitored using radiolabeled nucleic acids, and the results are summarized in Table IV.

WO 90/07582 PCI`/US90/0008 ~ ' 32 J- :
q t, c ~ ~ :

U ~
~R
D.3 o o r' ~ O O C-oi o h D~

e ~ ~ ` x : -o o cO,a N ~ o ~ O '~3 ::- h o o ~ u~ C.~~ U, ~ ~ o 0 ~ 2 O ~ ~~ ~ ~ ~ In I
r ~ 0 ~ ~ O
~, o x ~ v a ~ g o ~ t~ ~ O 0 N S
~D O 1'~ 0 1` 01 ~ U
m ~r1 ~ ~ _~ O :~ X
A~~4 . ~
Ll ~ Ll l~t tll 1~ h U ~ .
~ ~ ~ n m N 1~ ~r1X 'Cl O ~ ~;r ~ N ~ O ~ ~ ' O ~ I I I O I I I
I O O O O O O O C~
~ T , . . I . o ~

WO90/07582 P~T/US90/00089 Since the yields of the coupling experiments are percentages of cpms of the total radioactivity introduced in the reaction, the actual yields are in fact higher, due to the incomplete conversion o~ 5'-phosphorylated oligonucleotides to their thiolatedderivatives. It can readily be seen that essentially all of the oligonucleotides are end-attached via thi~ther bonds following this coupling strategy. The variability in the attachment efficiencies (see entries 3-6, Table IV), was ascribed to the susceptibility of the reactive thiol groùp to air oxidation in storage or during the course of the reaction. While the overall attachment e~ficiencies of oligonucleotide to polyacrylamide supports (3-48~) were lower than to Sephacryl (70%), this coupling strategy provides a superior method for obtaining end-attached oligonucleotides.
The hybridization pote~tial of oligonucleotides immobilized on P 60 supports ~MW ~50,000 cut-of~) was evaluated using ~J2P~-oligonucleotide-len~th target and a 7 kb DNA single-stranded plasmid :in a 6andwich for~at, ~he P-60 support was cho~en for the ~tudy, since this matrlx displayed the best attachment efficiencies ~n the coupling reaction with thiol-oligonucleotide derivatives.
The results of the capture experiments are summarized in Table V.

:

W~ 90/~7582 PICI`/U~i90/00089 ~ `h ~, 7 ~ 34 O J~ ~
U ~ ~ ~, ~ o o N al ~8 .

u ~ P~ .
t t~ N

E~ P ~
b~ V I , _ ,,, - ' - -:-- U I ~1 JJ
E~ ~ I OJ ~ u~ U7 J ~ 3 ¦ ~ 3 o CV~ . .

~ ,1 5 X g ' ~

:~;
o . ~_ o n u a ~ ~r ~: I ~ ~ ~ t~ Ul , 'D
~t ~ o~ ~
~ ~ In ~n ~
i ~t o o 1 6\ ~t Y~ C~
~ P. ~ ~
I

WO90/07582 PCT/~S90/00089 As seen in the Table, both types of P~60 oligonucl~otide supports proved to be better than Sephacryl beads f~r direct capture of oligonucleotides.
The support-bearing carboxyl functionalities on the surface displayed a non-specific binding identical t~ the Sephacryl control and were marginally better than the P-60 hydrazide matrix. The polyacrylamide-based support showed a poorer ability than Sephacryl beads to detect long targst DNA in sandwich hybridizations and also displayed higher backgrounds. This result suggested that perhaps a substantial portion of the immobili~ed ~ligonucleotides was in the interior o~ the support, and therefore was not accessible to the long target fragment ~or hybridi~tion. Consequently, only a small portion of the immobilized oligonucleotide would be available on the surface ~or the rate of d~plex fo~ation. At this stage, it was reasoned that it would be bene~icial to reverse the functlonalities in the coupling reaction. An impediment to obtaining better coupling e~ficiencies, and h~ce higher substitution levels, was the susceptibility of the thiol-oligonucleotide derivative to oxidation.
The following Ex~mples 2 and 3 discu s our results with the reverse format.

~xample_~
Co~ t attachme~t of ~romo~etyl-ol~gonuGleotldo~ to B$og~1-8 a. ~ropar~t~o~ of ~ul~ r~ ri~at~ Y~9 O~
3 0 ~ 8UppOEtl~
one gram (wet weight) of Biogel hydrazide beads ~500 ~mole~ NHNH~dry ~ram) was equilibrated with 0.5 NaH~O3, pH 9.7. To ~ suspension in ~ ml were add~d 30 mole eguivalents (rel~tiv~ to NHNHl groups~ o~ N-~`35 acetylhomocysteine t~iulacton~, 3nd the ~ixtur~ was . ;: :- .: , WO~0/075~2 PCT/US90/00089 ,~hlt~.'?

shaken at room temperature overnight. The beads were washed with 300 ml 0.1 M NaCl and stored in TE, pH 8.0, or 0.1 M X2HPOs, pH 8Ø The reaction is illustrated in Figure 1, reaction B. The level of sulfhydryl substitution was determined by titrating with 5,5'-dithiobis(2-nitrobenzoic acid) tDTNB) and monitoring the release of 3-carboxylato-4-nitrothiophenolate at 412 ~m.
~ha conversion was found to be dependent on the substitution level of hydrazide groups in the solid support, as illustrated in Tabl~ VI.

- -~ .
,' ~ ' ,,:, : - . . .
:-,; . ~ , . .

,, WO 90/075~2 PCr/USgO/00~89 ~ 3; `~ 3 TABI~
REAC$TON OF ~IYD~ZID~ PORT~ ~T~
N-ACE~YL~HO~lOCY5TEIN~ ~CTf:)NE

Type of~mol~ molax excess S NHNH2 ymole support NHN~Jglo thiol lactone conversion SH/g~
___________ ________________ ___________ ________________ ~

P-2-SH 50 100 3 . 8 1. 9 2 P-2-SH 500 30 9 . 9 49 . 4 3 P-10-SH 50 100 7 .1 3 . 6 4 P-60-SH 5~ 100 7 . 6 3 . 6 P 200-SH50 100 4 . 3 2 . 2 6 P-200-SH500 30 14 . 2 70 . 3 -SHl .
7 P-~OO 500 30 15.3 76.3 8 P-300-SH500 20 10 . 4 52 . 2 _SH2 9 P-300 500 20 10.0 5~.2 U-TNPH
-SH~
10 P-300 500 20 9 . 2 45 . g i 1. Conversion of hydrazide groups in P-200-SH
support Witsl glutaric anhydride.
2. Conversion o~ hydrazide groups isl P-300-SH
support with trinitroE~henyl sulfonate 3. Conversion o~ hydrazide groups in P-3ûO-S~
supports with glutaric an~lydride.

- . . . . :. , . :. . :: , ...... . . .

W090/07582 PCT/~S90/00089 b. Glutaxylatlon o~ sulfhy~ryl-B~ogel ~ea~s A one-gram sample of sulfhydryl Biogel was suspend~d in 20 ml 0.1 M NaCl, and two 100-mg aliquots of glutaric anhydride were added at 15-minute intervals.
The pH was maintained near 4.0 with 3 M NaOH. After a total reaction time of 30 minutes, the beads were washed with 0.1 M NaCl. Hydrolysis of thioesters was then :
carried out with 10 ml 0.1 ~ Tris-HCl, pH 8.5, for 1 hour at room temperature.
Further conversion of the remaining hydrazide groups in the thiol supports with glutaric anhydrid~ or trinitrobenzene sulfonate provided matrices with mixed functionalit~es (~SH and ~COOH, or ~SH and ~TNP~, respectively). No modification of the thiol groups was observed in these reactions ~Table VI, compare.6 and 7, and 8-10). The rationale behi~d the ~yntheses of these mixed supports was to exploit the anionic or dipolar properties of the carboxyl or TNPH groups in reducing non-specific adsorption of negatively charged nucleic acids in hybridization r~actions. A family o~ thiol-functionalized polyacrylamide supports, spanning a wide range of exclusion volumes, was thus prepared, having thiol substitution lavels varying ~ro~ 2-7h ~moles per gram o~ ~ateri~l. .
c, Prep~ratloa of bromo~etyl derivat$~es o~
ol~gon~ t~
~ 5'-phosphorylated oligonucleotide ~n a silanized eppendorf tube was treated wit~ 300 ~1 of 0.25 M h~xanediamine HCl, 0.1 H methyli~idazole, 0.15 M ~DC, pB 6.0, a~ room temperature for 20 hours. The ~mine derivativ~ was preclpitated twic~ with EtO~/LiCl and redissolved in 28$ ~1 o~ 0~2 ~ HEPES, pH 7.7. A 15 ~1 aliguot of a 10~g/ml solution o~ ~-succinimidyl bromoacetate in ~F was added. After a reaction time o~

W09~/n7582 PCT~US90/00089 ~ J r ~ j 3 3~
1 hour, the oligonucleotide was precipitated twice with EtOH/LiCl. The reaction is illustrated in Figure 3. The overall phosphate-to-bromoacetamide transformation was estimated to be 60-70% by polyacrylamide gel analysis of ~32~]-labelled products, and susceptibility to cleavage of the 5~32po~ group (unreaoted phosphorylated oligonucleotide) by alkaline phosphatase treatment.

d. Covale~t ~ttachme~t o~ 5~-hromo~cetyl-10 ~erivatl3~ ollgonuclaoti~es to sulfhydry~-Biogel ~e~ds Sulphydryl-Biogel support tl gram wet weight) was reducecl with 5 ml 20 mM DTT in 0.05 ~ K2HPO~, pH 8.0, for 1 hour, then washed with 2 x 40 ml 0.05 M K2HPO~, pH
8.0, followed by ~ x 40 ml 0.1 M triethylammonium 15 phosphate, 1 mM EDTA, pH 9.O. Five hundred pmoles of bromoa~etyl-derivatized oligonucleotide was dissolved in 1 ~1 TEAP, EDTA, pH 9.0, and added to the resin in a 5-ml polypropylene tube. A~ter purging t~e tube with N2 and sealing with parafilm, the bead sample was agitated on a 2Q rotary mixer overnight. The beads w~re washed with: (a) 3 x 10 ml 0.1 ~ Na2P2O~, pH 7.5, and (b) 2 x 10 ~1 TE, pH
8Ø Unreacted sulfhydryl groups wera capped by reducing the support with 3 ml 20 mM DTT in 0.05 ~ R2~PO~, pH 8.0, for 30 minutes. After re~oval of exces~ DT~ and 25 equilibration in 0.1 ~ TEAP, 1 mM EDTA, pH 9.0, 3 ~1 of 5 mM iodoacetic acid in the same buffer was added and allowed to react ~or 1 hour. After ~iltration of unreacted reagent through a si~tered glass funnel (porosity ~), the bead samples were ~tored i~ TE, pH 8.0, 30 at 4-C.

Cou~linq e~ficiencies of bromoacetyl oli~onucleotides to thiol ~olyacrylamide suppoXts Th~ immobilizat~on studies concentrated on P-2, 35 P-200, and P-300 matrices, because of their vastly .,. , . ~
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WO 90/07~;82 PCI'/US90/00089 r~ '~ 40 di~erent exclusion volumes. It was hypothesized that the highly cross linked P-2 support ~MW cut-off 2,000) . would have all the oligonucleotides attached ~on the surface, and he~ce be available for capture. The small psre size would also prevent the inclusion of nucleic acids during the hybridization and thereby be beneficial in reducing non-specific binding. In contrast, th~ P-200 and P~300 supports (MW cut-off 200,00 and 300,000) are closer to Sephacryl (MW cut-off 20,000,000) in struckural features in having large pores in the matrix. The r~sults of the coupl~ng rea~tion (see Figure 4) of these deri~atives with these thiol-polyacrylamide supports are shown in Table VII.

~A9~ YII

COUPLING YI~D~ OF ~IOL 8~PP~RT~
~ BRO~OACETYL O~IGON~C~O~ID~B

Support t500 ~mole NHNH2 initial sub- 86-31-CX2Br 86-31-PO4 ~ end ~titution ti~e) ~ attached ~ attached attchmt ____ _______________________ ________________________~__ P-2 -SH 3 . 5 0 ~ 2 94 2 P 200 SH 28 . 6 0 . 5 98 ~S~
3 P-200 29.3 0.5 98 -TNPH
4 P-300-SH 29 . 9 1. 7 94 SH
~; ~~300 22 0 . 5 98 -~:02H

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WO90/075g2 PCT/US90/00089 The coupl ing with the P-2 support proceeded in low yields, even though the thiol concentration on th~
matrix greatly exceeded the oligonucleotide concentration in the reaction. P-200 and P-300 supports provided satisfactory~yields and, in contrast to the reverse format, were consistently reproducible. As was noted earlier, the actual yields are higher than the values reported, due to the incomplete conversion of 5'-phosphorylated oligonucleotides to th~ir bromoac~tyl derivatives. The other factors to not~ are: (1) additional functionalization of the thiol supports with COOH or TNPH groups does not have a significant effect on the attachment efficiencies (entri~s 2 and 3, and 4 and 5): (2) the reaction with bromoacetyl oligonucleotides results in end-attachment of the ~ucleic acid to the support, as evidenced by the minimal binding of the phosphorylated oligonucleotide control; (3) a comparison of the reactivity of bromoacetyl oligonucleotides with P-200 thiol supports indicated that the displacement of bromide by the thiol groups on the support was 30% mor~
efficient than addition t~ the 5' maleimide-derivatiz~d oligonucleotide: and ~4) the opti~i~m pH for coupling was determned to b~ pH 9.0; higher coupling efficiencies are obtained using triethylammonium phosphate as the coupling buffer, compared t~ p~tassium phospha~e.
Hybridization_~haracteristics of t~iol supports The hybridization of oligonucleotides on polyacrylamid~ thiol supports was tested by dir~ct captur~ of oliqonucle~tide targets and in a ~andwich ~ormat with T~5-generated RNA transcripts. Two sets of exp~ri~ents ~re sum~arized in Table VIII.A., whiçh compares P-2 and P-200 ~upports with Sephacryl beads :, ;.: .: :-. : :.: . : ::
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WO90/07582 PCT/U~9~/00~89 f~ 4 Both types of polyacrylamide supports comparèd very favorably with Sephacryl beads in showing excellent ability to captur~ target oli~onucleotides and low non-specific binding. However, in ~andwich hybridizations with TAS products, the P~2 support displayed a reduced ability to capture target, as well as a high background.
Although the P-200 support was better than P-2 in sandwich hybridizations, it suffered in comparisQn to 10 Sephacryl beads due to itff~ 2-fold higher non-specific bindin~. This feature was again seen in a set of experiments comparing Sephacryl beads with P-200 supports .
in a sandwich hybridization to a 7 kb tarqet fragment (Table VIII.B). While the polyac:rylamide support was 15 clearly superior to Sephacryl in oliyonucleotide capture, it showed not only a lower effici~ency in sandwich capture of the long targat, but also an unexpected increase in non-sp~cific binding.
To address the non-specific adsorption problem, 20 the polyacrylamide supports (P-200 and P-300) were treated with glutaric anhydride and/or iodoacetic acid to convert.the hydrazide ~nd sulfhydryl functionalities to carboxylic groups. The hybridization results with these modified supports are shown in Table ~III.C. Consistent 25 with our earlier observations, th~ direct capture o~
oligonucleotides by all of the polyacrylamide ~upports was impressive and superior to Sephacryl heads. While similar levels of sandwich capture o~ the TAS transcrlpts were exhibited by both the polyacrylamide and S~phacryl 30 ~upports, the problem o~ non specific adsorption ~urfa~ed again for tha t~iol-based supports. Capping t~e hydrazide and th~ol groups on the support helped to reduce the background 3- to 5-~old (Table VIII . C, entries 2 and 3 ) .

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W090t07582 PCT/US90/0~089 In direct capture experiments with 3.75 fmole~
~ target oligonucleotide, non-specific binding was - o~served to be ~0.55% for all three types of polyacrylamide supports (Table VIII.C, entrles 1, 2, and 3). However, in sandwich hybridizati~ns in which 5 fmoles of detection oligonucleotide ar2 utilized, a 5- to 14-fold increase was noted.
In order to test the premise that the long target was not captured as efficiently, and perhaps had a role to play in these increase of non-specifio binding, ~ direct capture of a~labeled PC~ product by polyacrylamide and Sephacryl beads was investigated. Table IX
summarizes the results of the experiment.

TABL~ IX
DI~ECT CAPT~RE OF ~C!R-A~P~IFI~D, DOV13I.E--5T~ANDED PRODUC~

86-31 Comple- Non-comple-immobilized Target mentary mentary Signal/
support ~ole) (~ capture) (% capture) noisa ___. ____________________, ______________ - _ ._________ . __ P-300-S~ 1 11 1.1 10 P-300-S~ 1 19.. 5 0.75 26 -COz~
P-200-S~ 1 23 1.7 13.5 Sephacryl beads 1 13.5 0.4 33.8 17.5 0065 2609 ___________ 1. ~verage of duplicates of two ~eparate experi~ents . . ......... , , ; :. ~ i ;, . .
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WO~0/07582 PCT/US90/0~089 As seen in the Table, both carboxylated forms o~ the polyacrylamide supports showed better capture efficiencies of the long-stranded DNA target than Sephacryl beads, while tha bac~grounds were 2- to 3-fold higher than Sephacryl. These results with long target DNA closely paralleled our ~indings with oligonucleotide targets (Table VIII.C.). Thus, it appears that while the polyacrylamide supports can capture long targets as efficiently as (if not better than) Sephacryl beads, there are some other, at present unknown, factors involved in sandwich hybridizations which contribute to the increase of non-specific binding.

~ ybridization of suDport~ ~ith oli~onucleoti~e-e~zyme c~n~u~ata T~e direct capture and background properties of the polyacry~amide support were al~o tested with oligonucleotide-alXaline phosphatas~e conjugates. The supports were incubated with comple~mentary oligonucleotide-enzyme conjugates for 1 hour and then ~ubjected to the standard wash~s to r~mov~ unbound c~njugate. Color development was allowed to proceed for 1 hour using ~nitrophenyl phosphate as substrate, and the release of P-nitrophenolate was determined spectrophotometrically. A non-complementary oligonucleotide-enzyme conjugate was used as a control.
Data from this study are summarized in Table X.

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WO90/075~2 PCT/US9~/0008g The following observations can be made from the results. The average signal generated by hydrolysis of the substrate by support-bound conjugates was ~40~ higher in the case of the Sephacryl beads when c~mpared to polyacrylamide supports. The average of the backgrounds for both supports was nearly identical. This there ore accounted ~or the superior signal~to-noise ratio o~
Sephacryl beads compared to the polyacrylamide supports.
Keeping in mind that polyacrylamide ~upports are msre efficient than ~ephacryl beads in the direct capture of ~ ~~ oligonucleotides, it was therefore surprising that the capture of t:he conju~ates prov d to be contrary.
In order to determine whether the presence of the immobilized oligonucleotide on the support contributed in some manner to non-specific binding, oligonucleotide-bound support was comixed with oligonucleotide-~ree suppoxt in a 1:9 w/w ratio. As s~en in Table Xj entries 3 and 5, no si.gnificant conclusions can be made from the signal-to-noi.se ratio~.
Example 3 Cov~l~nt attao~me~t o~ ~romoR~otyl ol~go~ucleoti~s to ~ri3a~ry1-8 a. Preparatio~ of amlno ~eriYæti~e o~
~r~a~ryl ~ 20~ml suspension of Trisacryl GF-2000 (IBF, Biote~hniGs, VSA) was pipetted into a sintered gla~s funnel, wash~d with 200 ml ~2~ and ~ucked dry for 10 ~inutes. The dried sample (~11 gr~ms) was added slowly to 20 ml of ~istilled ethylene dia~ine equilibratin~ ~t 90-C in an oil bath. A~ter one hour~ the reaction ~ixture was cooled by the addition of 30 ml of crushed ~c~. Exc~ss ethylene dlamine was re~a~ad by washing the resin with 400 ~1 0.2 ~ NaCl, 0.001 M ~Clg ~ol~owed ~y 500 ~1 0.1 M NaCl in a funnel. Washing was ~ontinued .. : :
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b~ Prep~ratio~ of ~ul~hy~ryl ~eri~a~i~e of ~r~sa~ryl The ~risacryl-amine support was eq~ilibrated with 0.5 M NaHCO3, pH 9.7, and the volume was ad~usted to 30 ml in a 50-ml Sarstedt conical tube. Solid N-acetyl homocysteine lactone (1 gram~ was added, and the tube was 10 shaken at room temperature for 2 hours. Then, another 1 gram of reagent was added, and the sample was shaken overnight. ~he beads were washed with 500 ml of 0.1 M
NaCl, and the sulfhydryl group concentration was estimated by titrating with Ellman's reagent ~TNB) (Reference Example le). The reaction is illustrated in Figure 5.
Titration o~ the sulfhydryl groups on the Trisacryl-SH support indicated a Isub~titution level o~
12.3 ~moles-S~ per wet gram of relsin.
c. ~ucc~nylatioD of ~ul~hy~ryl-Trisac2yl A 2-gram ~ample o~ Tri~acryl-SH W?S
equilibrated in 20 ~1 0.1 ~ NaOA~, pH 6.0, and treated with 100 mg sol~d succinic anhydride. After shaking for 30 min~tes, another 100 mg o~ anhydride was added to the ~uspension and shaken for a further 30 minutes. T~e beads were then equilibrated in 40 ~1 0.1 ~ Tri~, pH 805.
After 1 hour, the support was washed with TE, pH 8.0, and ~tored 2t 4-C.
d. ~o~lont attachme~t of 5~-bro~o~cetyl-~er~v~tl20~ ol~go~uGleot~e~ to aulhya~yl-Tr$s~c~yl ~b~ ~ol~d ~upport tl ~ra~) wa~ reduced with DTT
~ollowing the procedllre used ~or Bio~el beads ~Exa~ple 2d) and ~quilibrat~d in 0.1 M TEA~, lm~ EDTA, pH 9Ø

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23 . ~ 3 Five hundred pmoles of bromoacetyl-derivatized oligonucleotide dissolved in 1 ~1 TEAP/EDTA were added to-the support, and th~ tube was purged with N2 and sealed.
After overnight agitation on a rotary mixer, 100 ~moles of iodoacetic acid were added and the ~ixture was left at room temperature for 1 hour. The beads were washed with 4 x 20 ml 0.1 M Na~P~O~, pH 7.5, ~ollowed by 2 x 20 ml TE, p~ 8Ø
` The reaction is illustrated in Figure 6. The coupling reaction with ~32P]-labeled, bromoacetylated oligonucleotides resulted in 12% o~ the label being attached to the sulfhydryl support, and the level of end-attachment was deter~ined to be 97%. The actual coupling yield for t~is reaction was estimated to be approximately 20%, based on a purity of 60-70% Eor the bromoa~etyl ~ligonucleotide. Although the coupling yield:was lower than those obtained with the Bioglel polyacrylamida supports (40-45%), the ~risacryl-resin still contained a huge excess of oligonucleotide ~olecules relative to the amounts of target ~0.5-5 fmoles) used in the hybridization experiments.
Hybridization characteristics of sulfhydryl-Trisacxyl~uPpo~s -The initial hybridization ~tudy (Table X~.A) involving dir~ct capture o~ 3O75 fmole~ of ~2P]-labeled target with ~risacryl-SH (containing oligo 86-31j showed that the non-specif~ binding of non-~ompla~entary target was about three ti~es higher than for the Sephacryl beads ~upport (0.42% v~. 0.14%).

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WO 90/1)75~2 PCI/US90/0~089 ' 52 TABL~ XI ?
~Y~RIDI~ATION STUDI~ ~IT~ ~I8ACRY~-8 A. Direct CaDture ~ Oliqo ~oundl B6-31 Support Target B6-3286-31 S~gnal/Noise (fmoles) (Comp.) (Non Comp) . .
Trisacryl-S~ 375 66.S 0.42 158.3 Trisacryl-SH 375 69.8 0.15 465.3 ( ICH200H-tr~ated) Sephacryl beads 375 54.6 0.14 390.0 . _ B. TAS HIV RNA ~SA~DWICHl CAPTV~
86-31 Support Target 86-3286-31 Signal/Noise moles) (Comp.) (Non-Comp~

Trisacryl-SH 0.5 17.0 0.1 170.0 (ICI120011-treated) Sephacryl beads 0.5 12.5 0.9 13.9 C. Direct C~pture o~ 01inonucl~otide-en~ym~ Coniuaa~ç~
~6-31 Support Target fi6-32-AP 86-31-AP Signal/Noise ~mole~) ~Comp.) (Non-C'omp) ....... .
Trisacryl-SH 5 0.1790.003 61.7 ~ ICH200H-tr~ated) Sephacryl beads 5 0.319 0.0;!3 14.4 WO90/07~2 PCT/US90/00089 It was anticipated that alkylation of the unreacted sulfhydryl grou~s on the Trisa~ryl support with iodoaeetate would reduce the non-~pecific binding by increasing the negative charge-density of the matrix.
Indeed, when the alkylated Trisacryl support W2S assayed in a direct capture experiment, the non-~pecific binding dropped considerably to 0.15%, which was comp~rable to that obtained with Sephacryl beads.
More importantly, the results from a TAS
sandwich experiment (Table XI.B) using 0.5 fmoles of target RNA emphasized the 6uperiority of Trisacryl over Sephacryl beads as evidenced by the higher percent capture of complementary target. Most ~rati~ying was the extremely low non-specific background afforded by thi~
macroporous polyacrylamide suppor~. This waæ in contrast to the-higher background of Biog~l supports, whi~h had ~een the principal cause for the .lower signal/noise ratios in the TAS sandwich format.
Finally, the Trisacryl support was tested with oligonucleotide-alkaline phosphatase conjugates to determine the level of direct capture and backgrou~d properties (Table XI~C.). Incubation of complementary and non-comple~entary conjugates with Tris~cryl a~d Sephacryl beads ~ollowed by ~tandard washing procedures and a colorimetric assay using p-nitrophenyl phospbat~
was performed with S f~ol~s of conjugates. Although the capture of complementary target was higher in the case o~
Sephacryl beads, the non-spacific ~inding o~ the ~ri~acryl was considerably low~r, resulting in a 4-fold : 30 improvemen~ ~n ignal-to-noise compared to Sephacryl beads.

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Claims (36)

CLAIMS:
1. Polyacrylamide supports having covalently attached thereto oligonucleotides substantially at their 5'-ends.
2. Polyacrylamide supports according to Claim 1 wherein at least about 95% of said oligonucleotides are attached to said supports at their 5'-ends.
3. Polyacrylamide supports according to Claim 1 having a molecular weight exclusion limit of between about 2000 and about 400,000 daltons.
4. Polyacrylamide supports according to Claim 1 having a molecular weight exclusion limit of at least about 2x107 daltons.
5. Polyacrylamide supports according to Claim 4 having repeating units each of which contains three hydroxymethyl groups and one secondary amide group.
6. Process for the attachment of oligonucleotides to solid supports substantially at their 5'-ends, comprising coupling thiol-derivatized oligonucleotides to polyacrylamide supports, the amide groups of said polyacrylamide supports being at least partially converted into bromoacetyl groups prior to end-attachment.
7. A process according to Claim 6 wherein said thiol-derivatized oligonuoleotides are 5'-alkylthiol oligonucleotides.
8. A process according to Claim 6 wherein the amide groups of said polyacrylamide supports are first modified with hydrazine and the obtained hydrazide functional groups are at least partially converted into bromoacetyl groups.
9. A process according to Claim 8 wherein said conversion into bromoacetyl groups is carried out with bromoacetic acid N-hydroxysuccinimide ester.
10. A process according to Claim 8 wherein at least about 15% of said hydrazide functional groups are converted into said bromoacetyl groups.
11. A process according to Claim 8 wherein at least about 40% of said hydrazide functional groups are converted into said bromoacetyl groups.
12. A process according to Claim 8 wherein the residual hydrazide functional groups are at least partially converted into carboxyl groups.
13. A process according to Claim 6 wherein the molecular weight exclusion limit of said polyacrylamide supports is between about 2000 and about 400,000 daltons.
14. A process according to Claim 6 wherein the molecular weight exclusion limit of said polyacrylamide supports is at least about 2 x 107 daltons.
15. A process according to Claim 6 wherein least about 95% of said thiol-derivatized oligonucleotides are attached to said polyacrylamide supports at their 5'-ends.
16. Process for the attachment of oligonucleotides to said supports substantially at their 5'-ends, comprising coupling bromoacetyl-derivatized oligonucleotides to polyacrylamide supports, the amide groups of said polyacrylamide supports being at least partially converted into thiol groups prior to attachment.
17. A process according to Claim 16 wherein said bromoacetyl-derivatized oligonucleotides are prepared by reacting 5'-aminohexyl-phosphoramidate oligonucleotides with bromoacetyl acid N-hydroxysuccinimide ester.
18. A process according to Claim 17 wherein the conversion of said 5'-aminohexyl-phosphoramidate oligonucleotides into said bromoacetyl-derivatized oligonucleotides is at least about 60%.
19. A process according to Claim 16 wherein the primary amide groups of said polyacrylamide supports are first modified with hydrazine the obtained hydrazide functional groups are at least partially converted into thiol groups.
20. A process according to Claim 19 wherein said conversion is carried out with N-acetylhomocysteine thiolactone.
21. A process according to Claim 19 wherein at least about 4% of said hydrazide functional groups are converted into said thiol groups.
22. A process according to Claim 19 wherein the residual hydrazide functional groups are at least partially converted into one or more other functional groups.
23. A process according to Claim 22 wherein said other functional groups are carboxyl groups.
24. A process according to Claim 22 wherein said other functional groups are trinitrophenyl groups.
25. A process according to Claim 16 wherein the molecular weight exclusion limit of said polyacrylamide supports is between about 2000 and about 400,000 daltons.
26. A process according to Claim 16 wherein said polyacrylamide supports have repeating units each of which contains three hydroxymethyl groups and one secondary amide group.
27. A process according to Claim 26 wherein said secondary amide groups are first transamidated with homobifunctional amines, the terminal amino groups of which are subsequently at least partially converted into thiol groups.
28. A process according to Claim 27 wherein the unconverted terminal amine groups are at least partially acylated to convert to carboxyl groups.
29. A process according to Claim 27 or Claim 28 wherein after coupling with said bromoacetyl-derivatized oliogonucleotides, the unreacted thiol groups are at least partially haloacetylated to convert them to carboxyl groups.
30. A process according to Claim 26 wherein the molecular weight exclusion limit of said polyacrylamide supports is about 2x107 daltons.
31. A process according to Claim 16 wherein at least about 95% of said bromoacetyl-derivatized oligonucleotides are attached to said thiol-polyacrylamide supports at their 5'-ends.
32. A process according to Claim 16 wherein said bromoacetyl-derivatized oligonucleotides are attached to said thiol-polyacrylamide supports in a sandwich format with TAS RNA transcripts.
33. A process according to Claim 32 wherein said thiol-polyacrylamide supports have an exclusion limit of at least about 2x107 daltons.
34. Oligonucleotides derivatized at their 5'-termini with bromoacetyl groups.
35. Oligonucleotides according to Claim 34 immobilized on thiol-polyacrylamide supports.
36. Oligonucleotides derivatized at their 5'-termini with thiol groups immobilized on bromoacetyl-derivatized polyacrylamide supports.
CA002044173A 1989-01-05 1990-01-04 End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids Abandoned CA2044173A1 (en)

Applications Claiming Priority (2)

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US293,893 1989-01-05
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US5237016A (en) 1993-08-17
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