CA2095212A1 - Incorporation of multiple reporter groups on synthetic oligonucleotides - Google Patents
Incorporation of multiple reporter groups on synthetic oligonucleotidesInfo
- Publication number
- CA2095212A1 CA2095212A1 CA002095212A CA2095212A CA2095212A1 CA 2095212 A1 CA2095212 A1 CA 2095212A1 CA 002095212 A CA002095212 A CA 002095212A CA 2095212 A CA2095212 A CA 2095212A CA 2095212 A1 CA2095212 A1 CA 2095212A1
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- Canada
- Prior art keywords
- oligonucleotide
- phosphonate
- repeating unit
- bound
- represented
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/141—Esters of phosphorous acids
- C07F9/1411—Esters of phosphorous acids with hydroxyalkyl compounds with further substituents on alkyl
Abstract
ABSTRACT OF THE DISCLOSURE
The invention provides reagents and methods for the synthesis of oligonucleotides having single or multiple reporter groups attached for use in detection assays, wherein the reporter groups may be precisely spaced with respect to one another. The invention further provides oligonucleotides having single or multiple reporter groups attached in a manner in which the spacing between the reporter groups is controlled.
The invention provides reagents and methods for the synthesis of oligonucleotides having single or multiple reporter groups attached for use in detection assays, wherein the reporter groups may be precisely spaced with respect to one another. The invention further provides oligonucleotides having single or multiple reporter groups attached in a manner in which the spacing between the reporter groups is controlled.
Description
2 0 ~ j 2 :1 2 WO 92/087~8 PCr/~'S91/083~7 INCORPORATION OF MI~LlIPLE REPORTER ÇROUPS ON
SYNTHETIC OLI~ONUCLEQTID~S
Thc work described herein vas supported by cooperative grant 401 124846 from the National Institute of Allergies and Infectious Discase.
BACKGRQ11~D Q~THE lNVENTlON
1. Field of ~he Invcntion Thc invention relates to modification of oligonucleotidcs for incorporation of singlc or multiple reporter ~roups. More particularly thc inven~ion relates to improved modified oli~onucleotides which are functionally derivatized to ;ncreasc sensitivity of detection. Such oligonucleotides are useful as probes for a variety of nucleic acid-based diagnostic and therapeutic applications based on their hybridization to specific complementary nucleic acid scquences.
2. Summarv of the Belate~rt The preparation and usc of functionalized oligonuclcotides for incorporation of rcporter groups is Icnown in the art.
Agrawal et al., Nucleic Acids Res. 14: 6227-6245 (1986) discloses introduction of biotin and flourescent dyes at either the ~' or 3' end of oligonucleotide. Sce also Agràwal, Tetrahedron Lett. ~Q: 7025-7028 (1989).
Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988);
Agrawal et al., J. Cell Biol. IQ7: 468 (1988); and Haralambidis et al., Nucleic Acids Res. ]8: 501-505 (1989) teach thc introduction o~ flourophorcs into oligonucleotides.
These methods suffer from the limited signal strengsh inherent in the presencc of the single reporter molecule which is incorporated into each oligonucleotide.
Consequently, numerous investigators have attempted to devclop methods which allow the introduction of multiplc reporter groups into each oligonucleotide as a means of increasing sensitivity of procedures which use the oligonucleotide as probes.
Fidanza et al., J. Am. Chem. Soc. I 11: 9117-9119 (1989), tcaches the incorporation of reporter groups at phosphorothioate linkages in thc nascent oligonucleotidc.
Agrawal et al., Tetrahedron Lett. 3L: 1~43-lS46 (1990), and Agrawal ct al., Nucleic Acids Res. 1~: 5419~5423 (199Q), disclose methods for labelling oligonucleotides based on incorporating primary amines at phosphodiester moieties as phosphoramidates.
.
', .: '. . ' 2~52~ ;~
WO ~/08~28 PCI/US91/08347 Nelson et al., Nucleic Acids Res. 17: 7179-7186 (1989), discloses multiple reporter group incorporation at 5' termini of oligonuclcotide using phosphoramidite linkage to the oligonuclcotidc via N-Fmoc-OI-DMT-O2-cyanoethoxydiisopropylaminophosphinyl-3-amino-1,2-propanediol, a fixed-length linker.
Misiura e~ al., Nucleic Acids Res. ~: 4345-4354 (1990), discloscs a method for incorporating multiple reporser groups on oligonucleotides via phosphoramidite linkage using a three carbon glyccryl attachmen~ backbone to which the reporter group is connected by an ether-linked aminopropyl 8roUP~ i~ another rixed-length spaccr.
Haralambidis et al., Nucleic Acids Research 18: 501-505 (1990), tcaches linkage Or multiplc reporser groups solely to 3' ends Or oligonucleotides using polyamide moieties connected to the 3' end of the oli~onucleotides and Iysine residues connccting thc reporter groups to the polyamide moieties.
Althou3h these methods ~rc useful, they have many limitations.
Spacing of reporter groups is a critical factar for increasing scnsitivi~y Or dctection. Thc methods known in thc art do not provide for controlled variation of the spacing of tcporter groups and thereforc may be limited in ma~;imizing sensisivity. In addition, the chemistry in the known phosphoramidite methods is rathcr comple~s. While thc use of polyamide - and amino acid attachment means is amenable to some variation in spacing and involves a somewhat simplcr chemistry, these attachment reagents due to inappropriate spacing can cause quenching of signal, at least with fluorescent reporter groups, thereby decreasing sensitivity.
There is, therefore, a need for improvcd methods for incorporating multiple reporter groups into oligonucleoside. Preferrcd improved methods would utilize a simplcr chemistry than c~isting methods and would allow for controlléd, variable spacing of reporter groups without causing quenching of signal.
Bl~IEF ~U1~ 1ARY OF THE II`lVENTIOI~
The invention relates to means for labelling oligonucleo~ides with single or multiple reporter molecules. More particularly, the invention provides methods and rcagents for incorporating singlc or multiple reporter molecules into oligonucleotides in a manner that allows readily controlled varjation Or the spacing Or thc reporter groups, ~hereby increasing - ' ~ ,., ~, . . . . , , ;
WO 9~/08728 2 ~ ~ 5 ~ 1 2 PCr/US91/083~7 sensitivity of detcction. Thus thc invention also providcs labelled oligonucleotidcs that are morè readily dctectable than labellcd oligonucleotide produccd by e~isting means. The invcntion achieves these impostant goals while utilizing a simpler chemistry than cxisting methods for incorporating reporter groups into oligonuclcotides.
The method of the invcntion allows the attachment of multiple reporter groups to an oligonucleotide of defined sequence. The attachment i3 by way of H-phosphonatc coupling, using two ditfcrcnt types of linker molecules. Thus the invcntion prov;dcs reagents that are useful for producing multiply-labelled oligonucleotides.
One such reagen~ is the fiJst linker which is derived from an alkanediol, and for purposes of the invention is known as a ~phosphonate linker". Another is the second linkcr which is a diaminoalkane, and for purposes of the invention is known as a "diamino linker.~ Modified forms of these linkers~ having one hydro~yl or amino functionality protected by a chemical group are useful reagents for the method of the invention.
Such modificd linkers, for purposes of thc invention, are known as "protccted phosphonate linkers~ and ~protected diamino linkers~, respectively.
The invention provides means for attachment of multiple reporter groups to the S' end, to the 3' end or to the normal or modified ~basic) .- internucleoside linkages of the oligonucleotide, usin~ the linker reagents described above. For labelling of 5' ends Or oligonucleotidcs, support - bound oligonucleotide is bound in consecutive cyclcs to multiples of the protectcd phosphonate linker by H-phosphonate linkage. The protected diamino linkcrs are then bound to the phosphonatc linkers via phosphoramidate bonds to form a support bound oligonucleotide w;th a 5' repeating polymer having multiple protected amino functionalities. Upon relcase from the support using a standard deprotcction step (see Gait, Oligonucleotide Synthesis, IRL Press, O~ford, 1984), an oligonucleotide is liberated that has at its 5' end a repeating polymer with multiple free amino functional;ties. For purposes of the invention such a molecule is known as a ~5' functionalized oljgonucleotide~. Multiple reporter grQups are then added to thc 5' functionalized oligonucleotides via the rree amino functionalities. By similar principles, oligonucleotidcs with multiply-labelled 3' ends may be produced. In this case a phosphonatc linker is first attachcd to a solid support, and a repeating polymer having multiple , ~, ~
WO Q2~08728 ~ 5 2 1 2 Pcr/usgl/08~
SYNTHETIC OLI~ONUCLEQTID~S
Thc work described herein vas supported by cooperative grant 401 124846 from the National Institute of Allergies and Infectious Discase.
BACKGRQ11~D Q~THE lNVENTlON
1. Field of ~he Invcntion Thc invention relates to modification of oligonucleotidcs for incorporation of singlc or multiple reporter ~roups. More particularly thc inven~ion relates to improved modified oli~onucleotides which are functionally derivatized to ;ncreasc sensitivity of detection. Such oligonucleotides are useful as probes for a variety of nucleic acid-based diagnostic and therapeutic applications based on their hybridization to specific complementary nucleic acid scquences.
2. Summarv of the Belate~rt The preparation and usc of functionalized oligonuclcotides for incorporation of rcporter groups is Icnown in the art.
Agrawal et al., Nucleic Acids Res. 14: 6227-6245 (1986) discloses introduction of biotin and flourescent dyes at either the ~' or 3' end of oligonucleotide. Sce also Agràwal, Tetrahedron Lett. ~Q: 7025-7028 (1989).
Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988);
Agrawal et al., J. Cell Biol. IQ7: 468 (1988); and Haralambidis et al., Nucleic Acids Res. ]8: 501-505 (1989) teach thc introduction o~ flourophorcs into oligonucleotides.
These methods suffer from the limited signal strengsh inherent in the presencc of the single reporter molecule which is incorporated into each oligonucleotide.
Consequently, numerous investigators have attempted to devclop methods which allow the introduction of multiplc reporter groups into each oligonucleotide as a means of increasing sensitivity of procedures which use the oligonucleotide as probes.
Fidanza et al., J. Am. Chem. Soc. I 11: 9117-9119 (1989), tcaches the incorporation of reporter groups at phosphorothioate linkages in thc nascent oligonucleotidc.
Agrawal et al., Tetrahedron Lett. 3L: 1~43-lS46 (1990), and Agrawal ct al., Nucleic Acids Res. 1~: 5419~5423 (199Q), disclose methods for labelling oligonucleotides based on incorporating primary amines at phosphodiester moieties as phosphoramidates.
.
', .: '. . ' 2~52~ ;~
WO ~/08~28 PCI/US91/08347 Nelson et al., Nucleic Acids Res. 17: 7179-7186 (1989), discloses multiple reporter group incorporation at 5' termini of oligonuclcotide using phosphoramidite linkage to the oligonuclcotidc via N-Fmoc-OI-DMT-O2-cyanoethoxydiisopropylaminophosphinyl-3-amino-1,2-propanediol, a fixed-length linker.
Misiura e~ al., Nucleic Acids Res. ~: 4345-4354 (1990), discloscs a method for incorporating multiple reporser groups on oligonucleotides via phosphoramidite linkage using a three carbon glyccryl attachmen~ backbone to which the reporter group is connected by an ether-linked aminopropyl 8roUP~ i~ another rixed-length spaccr.
Haralambidis et al., Nucleic Acids Research 18: 501-505 (1990), tcaches linkage Or multiplc reporser groups solely to 3' ends Or oligonucleotides using polyamide moieties connected to the 3' end of the oli~onucleotides and Iysine residues connccting thc reporter groups to the polyamide moieties.
Althou3h these methods ~rc useful, they have many limitations.
Spacing of reporter groups is a critical factar for increasing scnsitivi~y Or dctection. Thc methods known in thc art do not provide for controlled variation of the spacing of tcporter groups and thereforc may be limited in ma~;imizing sensisivity. In addition, the chemistry in the known phosphoramidite methods is rathcr comple~s. While thc use of polyamide - and amino acid attachment means is amenable to some variation in spacing and involves a somewhat simplcr chemistry, these attachment reagents due to inappropriate spacing can cause quenching of signal, at least with fluorescent reporter groups, thereby decreasing sensitivity.
There is, therefore, a need for improvcd methods for incorporating multiple reporter groups into oligonucleoside. Preferrcd improved methods would utilize a simplcr chemistry than c~isting methods and would allow for controlléd, variable spacing of reporter groups without causing quenching of signal.
Bl~IEF ~U1~ 1ARY OF THE II`lVENTIOI~
The invention relates to means for labelling oligonucleo~ides with single or multiple reporter molecules. More particularly, the invention provides methods and rcagents for incorporating singlc or multiple reporter molecules into oligonucleotides in a manner that allows readily controlled varjation Or the spacing Or thc reporter groups, ~hereby increasing - ' ~ ,., ~, . . . . , , ;
WO 9~/08728 2 ~ ~ 5 ~ 1 2 PCr/US91/083~7 sensitivity of detcction. Thus thc invention also providcs labelled oligonucleotidcs that are morè readily dctectable than labellcd oligonucleotide produccd by e~isting means. The invcntion achieves these impostant goals while utilizing a simpler chemistry than cxisting methods for incorporating reporter groups into oligonuclcotides.
The method of the invcntion allows the attachment of multiple reporter groups to an oligonucleotide of defined sequence. The attachment i3 by way of H-phosphonatc coupling, using two ditfcrcnt types of linker molecules. Thus the invcntion prov;dcs reagents that are useful for producing multiply-labelled oligonucleotides.
One such reagen~ is the fiJst linker which is derived from an alkanediol, and for purposes of the invention is known as a ~phosphonate linker". Another is the second linkcr which is a diaminoalkane, and for purposes of the invention is known as a "diamino linker.~ Modified forms of these linkers~ having one hydro~yl or amino functionality protected by a chemical group are useful reagents for the method of the invention.
Such modificd linkers, for purposes of thc invention, are known as "protccted phosphonate linkers~ and ~protected diamino linkers~, respectively.
The invention provides means for attachment of multiple reporter groups to the S' end, to the 3' end or to the normal or modified ~basic) .- internucleoside linkages of the oligonucleotide, usin~ the linker reagents described above. For labelling of 5' ends Or oligonucleotidcs, support - bound oligonucleotide is bound in consecutive cyclcs to multiples of the protectcd phosphonate linker by H-phosphonate linkage. The protected diamino linkcrs are then bound to the phosphonatc linkers via phosphoramidate bonds to form a support bound oligonucleotide w;th a 5' repeating polymer having multiple protected amino functionalities. Upon relcase from the support using a standard deprotcction step (see Gait, Oligonucleotide Synthesis, IRL Press, O~ford, 1984), an oligonucleotide is liberated that has at its 5' end a repeating polymer with multiple free amino functional;ties. For purposes of the invention such a molecule is known as a ~5' functionalized oljgonucleotide~. Multiple reporter grQups are then added to thc 5' functionalized oligonucleotides via the rree amino functionalities. By similar principles, oligonucleotidcs with multiply-labelled 3' ends may be produced. In this case a phosphonatc linker is first attachcd to a solid support, and a repeating polymer having multiple , ~, ~
WO Q2~08728 ~ 5 2 1 2 Pcr/usgl/08~
protec~ed amino functionalities is built upon ~he linker as bcfore. The oligonuclcotidc is then synthcsized upon the frec phosphonate terminus of thc repeating polymer to produce a support bound oligonucleotide with a 3' rcpeating polymer havir~g multiple protected amino functionalities.
Upon release from the support, this produces an oligonucleotide that has at its 3' end a repeatin~ polymer with multiple free amino functionalities.
For purposes of the invention, such a molecule is known as a ~3' functionalized oligonucleotidc." Multiple reporter groups are then added to the 3' functionalized nucleotides via the frec amino functionalities.
Finally, the invention provides means for producing oli~onucleotides that are coupled to multiplc reporter groups at one or more internucleoside linkages. In this instancc, the method is carricd out as described for the synthesis of oligonucleotides with 5' multiple reporter groups, followed by fusion of the repeating polymer to thc 3' end of anothcr oligonucleotide.
Alternatively, the method can be carried out as described for the synthesis of oligonucleotidcs with 3' multiplc reporter groups, followed by release from the column and fusion of the repeating polymer to the 5' end of another oligonucleotide. Those skilled in the art will recognize that functionalized structures analogous to those described for 5' and 3' labelling of oligonuclcotides can bc produced by this method. For purposes oî thc invention, that functionalized structure attachcd to one or more repeating phosphodiester moiety polymers with free amino functionalities is known as an ~phosphodiester moiety linkcd functionalized oligonucleotidc~. Multiply labclled oligonucleotides are then produced by addin8 reporter groups to the free amino functionalities.
In each labelling mcthod of the invention thc spacing of the reporter groups may be preciscly controlled by using phosphonate linkers or diamino linkers of particular Icngths. Thus the invention provides mcans for producin~ oligonucleotides having multiple reporter groups at the 5' end, at the 3' end, or attached to an phosphodiester moiety, wherein the spacing of the rcporter groups may bc precisely varied. Other embodiments of the invention will be madc apparent by the following detailed description, example and claims.
WO 92/08728 2 ~ ~ ~ 2 1 2 PCr/US91/08347 BRIEF DES~RIPTLON OF THE l)RAWIN(~
FIGURE 1 ~ After the assembly of thc rcquircd sequencc, coupllings werc earricd out as described in E~ample ~, using a linkcr molcculc and H-phosphonate chemistry. The o~idation was thcn carried out with CF~CONH(CH2)sNH2, followed by deprotection in ammonia.
FIGURE 2 - Reversed phase HPLC analysis of (a) control 17-mer, (b) L(pN)-17-mer; (c) biotinylated L(pN)~17mer; (d) L(pN)L(pN)L(pN)-17mer; and (e) biotinylated L(pN)L(pN)L(pN)-17-mer. [L = he~tanediol;
(pN) . 6-aminohc~ylphosphoramidate]. HPLC was carried out using a Novapak C18 column and buffcrs were 0.1M ammonium aeetate eontaining (A) 0% aeetonitrilc and (B) 80% aeetonitrile, The gradient was 0% for B
for 2 minutcs; 0-30% B in 23 minutes and 30-100% B in 10 minutes; flow rate 1.5 ml/minute.
.
2~ 2~2 WO 92/08728 PCr/US91/08347 DETAII.ED DESCRIPTIQN OF T~lE SPEC:IFIC EMBQDIMEI~TS
In a first aspec~, the invention providcs rcagentS that are userul for producin~ oligonucleotidcs having multiple rcportcr groups that arc precisely spaced.
S Thcse reagents include ptotected linker molccules having at one end an H-phosphonate functionality and at the other end a protec~cd hydro~tyl group that is not reactive prior to the removal of the protective group. For purposes of the invention, such reagents are known as "protcctcd phosphonate linkcrs". Protccted phosphonatc linkers according to thc invention are charactcrized by the structure DMTr-o-cH2-(c~3)n-o~ oH
whercin DMTr = an `acid-labilc protective group, such as dimethoxytrityl and n = l to 20.
Such protected phosphonate linkers according to thc invention are . 25 synthesized by treating an alkanediol with a salt of an acid-labileprotective group, such as dimctho~yrityl chloride to yield a pro~ected derivativc, followed by converting the protected derivative to the corresponding H-phosphonate by standard procedurcs.
Anothcr reagent according to the invontion is a protccted linkcr molecule having at one cnd a free amino group, and at the other end a protected amino group that is not reactive prior to removal of the protectivc group. For purposes of thc invention, such rea~ents are known as ~protected diamino linkers~. Protected diamino linkers according to the invention are characterizcd by the struc;ure R- I-cHt-(cH2)n-~H2 wherein R ~ a basc-labile group n= I to20.
__ ; .
. ~ , .
, 2~i~32~ 2 WO 92t0872~ PC~/US91/0~347 Such protected diamino linkers according to th~ invention are synthesized by protecting onc amino group of a diaminoalkanc with a basc labilc group under controlled condi~ions. (See Agrawal and Tang~
Tetrahedron Lett. ~1: 1543-1546 (1990)).
Another reagcnt provided by thc invention is an oli~onucleotide of defined nucleotidc sequence having at its 5' end a repeating polymcr having multiple free amino functionalities. For purposes of the invention~
such reagen~ is known as a "S' functionali}ed oligonucleotide~. 5' functionalized oligonucleotides according to the invention are characterized by the structure rO 10 HO (CH2)n-OL ~-O-(CHz)~-O j~ ~-0 D pD~
( IH2)b (~H2)e NH2 ~H2 - whcrein D ~ a ribonucleoside or deo~yribonucleoside, p - a phosphodiester linkage or chesnically modified analog linkagc, n ~ I to 20, a ~ I ~o 20, b Y I to 20, c - I to 20, z, 0 to 20, t - I to 100, and wherein for each repeating unit ~in bracke~s] ~a~ may represent the same number or a different number than that number represented by ~a~ in every other repeating unit, and for cach repcating unit [in brackets] ~b" may rcprescnt ~he same numbcr or a different number than that number representcd by "b" in every other repcating unit.
.
.
~3~2 8~28 PCI /US91 /0334 5' functionalizcd oligonucleotidcs, according to the invention, can bc synthesixed in the following manncr. A protected phosphonate linker is prepared by treating an alkanediol with a salt of an acid-labile protectivc group, such as dimethoxytrityl chloride to yield a protected derivative of the alkanediol, which is then convertcd to the corresponding H-phosphonate by standard procedures. (Sec Frochler and Matteucci, Nuclcic Acids Res. 14: 5399-5407 (1986)). An oligonuclcotide of defined scqucnce is synthesi~ed (ç,~, by standard phosphoramidite, phosphonatc or phosphotriestcr chemistry) to yield a support-bound oligonuclcotide. A
protected phosphonate linker is thcn coupled with the frce 5' hydroxyl group of thc support-bound oligonuclcotidc using an H-phosphonate coupling cycle. (Sce Agrawal and Tan~, Tetrahcdron Lett. ;~: 1543-1546 (1990)). Additional phosphonate linkcrs are added through a scries of deprotes!ion and H-phosphonate cycles, with each cycle adding one phosphonate linker. The total number of cycles (and thus linkers) should cqual the number of reporter groups to be added to the oligonucleotide.
Thc support-bound oligonucleotidc/phosphonate linker complex is then oxidized with a protected diamino linkcr to yield a support-bound oligonucleotide with multiplc protected amino functionalities. (See Agrawal and Tan8, ~.) The support-bound oligonucleotidc wjth multiple protected amino functionalities is then converted into a free 5' functionalized oligonuclcotide by deprotection with a deprotccting agent, such as aqueous ammonia.
Thus the invention provides a method for producing a 5' functionalized oligonucleotide, which me~hod compriscs gencrally the following steps:
(a) coupling with support bound oli~onucleotide a protected phosphonate linker by an H-phosphonate coupling cycle, wherein the protected phosphonate linker is the product of a reaction between an alkanediol and a salt of an acid-labilc protective group, such as dimethoxytrityl chloride, which product has further been converted to an H-phosphonate;
(b) repsating thc coupling of step (a) at least once;
(c) oxidizing thc support bound oli~odeo1tynucleotide with a protccted diaminolinker to produce an oligonucleotide with protected amino functionalities; and wo 92/08728 ~ ~ 9 .~ 2 ~ 2 PCI/US91/083~/
(d) deprotecting the oligonucleoti~te to yicld a 5' function~lizcd oli~onucleotide.
Another reagent provided by she invention is an oligonuclcotide that has at its 3' end multiple free amino func~ionalities. For purposes Or the invension, such reagen~ is known as a 3~ functionalized oligonucleotide. 3' functionalizcd oligonucleotidcs according to the invention are charac~erized by the structure ~0 ~O
D(pD)n OlL P O-(CH2)"~0~ P''(CH2)b'H
NH I~H
(CHz)c ~CH2)d I~IH, NHz wherein D - a ribonucleoside or deo%yribonuclcossde, and p . a phosphodies~er linkage or a chemically modified analo~ linkage, and n~ I to 100, a, I to 20, b ~ I to 20, c . I to 20, d .. I to 20, - z ~ 0 to 20, and wherein for each repeatjng unit lin bracketsl, ~a~ may represent the same number or a differen~ number ftom that represented by "a~ in every o~her repeating unit, and for each repcating unit [in brackets], "b~ may represent the same number or a diffcrent number ïrom that representcd by ~b~ in e~ery ~thcr repe~ting unit.
Such 3' func~ionalized oligonucleotides accordin~ to the invention arc assembled in a manner that is analogous to that for the 5' oligonucleotides, excepr that a support-bound rcpeating structure having multiple protected amino functionalities is assembled using the same steps as before, but it is asscmbled first, beginning with linkage of a free phosphonate linker to the solid support by its frce hydro~yl functionality.
The oligonucleotide is then svnthesi~ed upon the repeating struc~ure.
be~inning ~vith an H-phosphonate coupling between the free phosphon3te terminus of the repeating structure and thc free 3' hydroxyl of the 2 ~ 2 WO 92/08728 PCI/US91/~347 oligonucleotide. Completion of the oligonuclcotide synthesis yields an oligonucleotide couPled to a rcpeating structure ha-dng multiple protected amino functionalities, the repeating structurc in turn bcing bound ~o the support. This oligonucleotide/repcating structurc complex is then converted into a 3' incorporation oligonucleo~ide by treatment with a deprotecting agent, such as aqueous ammonia.
Thus the invention provides a method for producing a 3' functionalized oligonucleotide, which method comprises generally thc following steps:
(a) binding a free phosphonatc linker to a support via thc frec hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least onc more phosphonatc linker, using an H-phosphonate coupling cycle to yield bound phosphonate linkers;
(c) oxidizing the bound phosphona~e linkers with protected diamino linkers t`o produce a bound repeating structure having multiple protected amino functionalities;
(d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalizcd oligonucleotide; and (e) deprotectin~ the amino functionalities to produce a 3' functionalized oligonucleotidc.
The invention also provides useful precursors for the synthesis Or 3- functionalized oligonucleotidcs and for 3' labelled oligonuclcotides. Such precursors comprise the repeating structure of phosphonate linkers and diamino linkcrs described abovc, which are bound to a solid suppor~
suitable for oligonucleotide synthesis. The support-bound repeating structure may simply be an amino functionalizcd repeating structure or protected amino functionalized repea~ing structure, in which case an oligonucleotidc may bc synthesized attached to the structure, and may then be relcascd (dcprotected) to yield a frcc 3' functionalized oligonucleotide.
Alternatively, thc support-bound repeatin~s structure may be a labelled repcating structure. In this case the amino functionalized support-bound repcating structure is synthesized, theD the label is attached to the amino functionalities of thc support-bound rcpeating structure. This embodiment requires the use as a label of a reporser group that ;s stable under ~.
.
WO 92/08728 PCltUS91/08347 I I
oligonuclcotidc synthesis conditions, _~" bio~in, or a labcl that may be protectcd.
Ano~her resgent provided by the invention is an oligonucleotide having multiple frcc amino func~ionalitics coupled to it via one or morc intcrnucleoside phosphoramide linkages. For purposes of the invcntion, such re~gent is known as a ~phosphodiestet moiety-linkcd functional.z~d oli~onucleotide~. Phosphodiester moiety-linkcd functionalizcd olilonucleotides according ~o the invention re characterized by thc following structure lo ro ~1 D (pD),-Ol--~ o-(cH2)c-o~ o (pD)b-D
;~H ~H
2)d( ~H2 t~l H 21~1 H ~
wherein D .. a ribonucleotide or deoxyribonucleoside, and p - a phosphodiester linkage or ~ chemically modificd analo~ linkage, and a- I ~o 100, `
b ~ I to 100, c - I to 20, d - I to 20, e 1. I to 20, f ~ 0 to 20, and wherein for cach rcpeating unit ~in brackets], ~c~ may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unil lin brackets], "d~ may represent the same numbcr or a different number from that represented by "d" in every other repeatinR unit, and for each repeating unit [in brackets~,"c~
may represent the same number or a different number from that represented by ~e" in every other repeatinR unit.
Such phosphodiester moiety-linked functionalized oligonucleotides accordin~ to the invention may be synthesized by carrying out synthesis of a 5' functionalized oligonucleotide and then fusing ano~her oligonucleotidc 40to ~he 5' functionalized oligonucleotide via the free hydroxyl Rroup on its terminal phosphonatc linkcr. ~Iternatively a 3' functionalizcd WO 92/0~728 PCI/US91/0834 oligonuclcotidc can be syn~hesi2cd, thcn fused, via the frec hydroxyl of its terminal phosphorlate linker, to another oiigonucleot;dc.
Thus the invention providcs me~hods for producing an intcrnucleoside phosphodiestcr moiety-linked functionalizcd S oligonuclcotide, comprising 8enarally the following stcps:
(a) coupling with support bound oli~onuclcotidc a protcctcd phosphonatc linlccr by aD H-phosphonate coupling cyclc;
(b) repeatin~ the coupling of step (a) according to the number of amino groups required;
lo (c) oxidizing the support bound oligodeo~tynucleo~idc w;th a protec~cd diaminolinker in the presonce of carbon tetrachloride to produce an oligonucleotide with protccted amino functionalities;
(d) continuing the oligonucleotidc synthesis at a frce cnd of the IS phosphona~c linkcr; and (c) deprotecting the amino functionalities to produce an intcrnucleotidc phosphodicster moiety-linked functionalized oligonucleotide.
In anothcr aspec~, thc invcntion providcs methods for producin~, oligonucleotides having at ~hcir 5' or 3' ends, or attached to onc or more phosphodiester moieties, multiple reporter ~roups which confcr upon the oligonucleo~ide dctectability in a specific assay. Reporter groups according to the invention ~herefore include any molecules which confer upon the oligonucleotide detectabili~y in a specific assay withou~ unduly interfering ~5 with biological functions of the oligonucleotjde, such as hybridization with a complcmcntary nucleotide scqucncc or bindin8 of a specific protcin.
Commonly uscd reporter groups will includc biotin, various flourophorcs, enzymes, and molecules which arc detectable by antibody bindin8 or other Iigand-rcccptor intcractions.
These mcthods of the invcntion ase practiced e~ac~ly as dcscribed for the production of 5', 3', or phosphodicstcr moicty-linkcd functionalized oligonucleotides, followed by coupling of the reporter group to thc functionalized oligonucleotide via linkagc with the free amino functionalitics of thc functionalized oligonucleotide. Thus, in these mcthods of the invcntion the spacing of the rcportcr groups can be manipulated by using diffcrcnt sizes of phosphonatc linkcrs and diamino linkers.
2 ~
WO 92/08728 PCT/IJS91/û83~7 The invcntion contemplates numerous ob~dous modifications that would be apparent to those skilled in ~he art. For cxamplc, structural modification of the repeating units of the invention could be made without undue e~perimen~ation by substituting linear or branched alkcne, alkyne, cycloalkane, cycloalkene~ cycloalkyne, aromatic or heterocyclic spacers for the alkane spaccrs uscd in the phosphonate linkers or diamino linkers.
Also, the diamino linker may be replaced by polyamines. The diamino linker may also be rcplaced by a linker havin~ a protected amino group at one esld and a free hydroxyl group at the other end, whereby the linkcr becomes attached via its hydro~yl functionality to the phosphonate moiety of the phosphonate linker, forming 2 phosphotriestcr linkage. Yet another obvious modification is to use a phosphorothioate linker in place of thc phosphonate linker and, in place of ~he diamino linkcr, a linker having a protected amino group at one er.d and a free sulfhydryl group at the othcr cnd, whereby the linkcrs can bc joined l~ia a disu!fide linkage. An additional obvious modification is to use an aminoalkyl iodidc in place of the diamino linker to rcact with a phosphodiester linkage to form a phophotricster linkage. Still another obvious modification in thc methods of the invcntion is to incorporate reporter groups which are stablc under oligonucleotide synthcsis conditions, e.g., biotin, during thc synthcsis process, rather than thcrcafter.
In addition to embodiments containing th~ above, specifically cnu ncratcd obvious modifications, all embodiments containing obvious modifications arc considered to be equivalents of ~hc invcntion.
Thc following example is provided to further illustrate the invention and is not limiting in nature.
XAMPLE
Incorporation o~ Multlple Reporter Groups ~t th~
S' End Qr 3n Olicod~QxYnuclç~t5de A six carbon non-nucleosidic linker was prepared by treating 1,6-hexanediol with 4,4'-dimethoxytrityl chloride (0.45 equiv.) in pyridine for two hours. The desired product, 6-(4,4'-dimethoxytrityl)- 1,6-hexanediol was purified on a silica gel column in 909~ yield, and was then converted to the corresponding H-phosphonate by a standard tris (1,2,4-triazole) phosphite procedure. To test the efficacy of the linker, a 17-mer sequence, 2 ~ 3 ;~
0 92/08728 PCI /US91~08347 GTAAACGACCiGCCAGT, vas assembled using normal phosphoramiditc ch~mistry, followed by coupling with 6-(4,4'-dimcthoxytrityl)-1,6-hcxancdiol-l, hydrogen phosphonate usin8 a H-phosphonate couplins cycle.
(Sce Agrawal and Tang, Tetrahcdron Lett. 3L: 1543-1546 (1990)). Thc S number of couplings dcpends on thc number of amino-groups to be introduced. At the end of the assembly, support bound oli~onuclcot;dc was oxidized with ~ trifluoracetyl-diaminohcxane. (See Agrawal and Tang, .) Aftcr dcprotection with aqucous ammonia, the amino-functios~alized oliRonucleotide WaS reacted with biotin activc cstcr using a published proccdurc. (See Agrawal es al., ~iucleic Acids Rcs. 14: 6229-6245 (1986)). All oligonucleotides wcre chccked by Cl8 reversc phase HPLC
(Figure 2).
Upon release from the support, this produces an oligonucleotide that has at its 3' end a repeatin~ polymer with multiple free amino functionalities.
For purposes of the invention, such a molecule is known as a ~3' functionalized oligonucleotidc." Multiple reporter groups are then added to the 3' functionalized nucleotides via the frec amino functionalities.
Finally, the invention provides means for producing oli~onucleotides that are coupled to multiplc reporter groups at one or more internucleoside linkages. In this instancc, the method is carricd out as described for the synthesis of oligonucleotides with 5' multiple reporter groups, followed by fusion of the repeating polymer to thc 3' end of anothcr oligonucleotide.
Alternatively, the method can be carried out as described for the synthesis of oligonucleotidcs with 3' multiplc reporter groups, followed by release from the column and fusion of the repeating polymer to the 5' end of another oligonucleotide. Those skilled in the art will recognize that functionalized structures analogous to those described for 5' and 3' labelling of oligonuclcotides can bc produced by this method. For purposes oî thc invention, that functionalized structure attachcd to one or more repeating phosphodiester moiety polymers with free amino functionalities is known as an ~phosphodiester moiety linkcd functionalized oligonucleotidc~. Multiply labclled oligonucleotides are then produced by addin8 reporter groups to the free amino functionalities.
In each labelling mcthod of the invention thc spacing of the reporter groups may be preciscly controlled by using phosphonate linkers or diamino linkers of particular Icngths. Thus the invention provides mcans for producin~ oligonucleotides having multiple reporter groups at the 5' end, at the 3' end, or attached to an phosphodiester moiety, wherein the spacing of the rcporter groups may bc precisely varied. Other embodiments of the invention will be madc apparent by the following detailed description, example and claims.
WO 92/08728 2 ~ ~ ~ 2 1 2 PCr/US91/08347 BRIEF DES~RIPTLON OF THE l)RAWIN(~
FIGURE 1 ~ After the assembly of thc rcquircd sequencc, coupllings werc earricd out as described in E~ample ~, using a linkcr molcculc and H-phosphonate chemistry. The o~idation was thcn carried out with CF~CONH(CH2)sNH2, followed by deprotection in ammonia.
FIGURE 2 - Reversed phase HPLC analysis of (a) control 17-mer, (b) L(pN)-17-mer; (c) biotinylated L(pN)~17mer; (d) L(pN)L(pN)L(pN)-17mer; and (e) biotinylated L(pN)L(pN)L(pN)-17-mer. [L = he~tanediol;
(pN) . 6-aminohc~ylphosphoramidate]. HPLC was carried out using a Novapak C18 column and buffcrs were 0.1M ammonium aeetate eontaining (A) 0% aeetonitrilc and (B) 80% aeetonitrile, The gradient was 0% for B
for 2 minutcs; 0-30% B in 23 minutes and 30-100% B in 10 minutes; flow rate 1.5 ml/minute.
.
2~ 2~2 WO 92/08728 PCr/US91/08347 DETAII.ED DESCRIPTIQN OF T~lE SPEC:IFIC EMBQDIMEI~TS
In a first aspec~, the invention providcs rcagentS that are userul for producin~ oligonucleotidcs having multiple rcportcr groups that arc precisely spaced.
S Thcse reagents include ptotected linker molccules having at one end an H-phosphonate functionality and at the other end a protec~cd hydro~tyl group that is not reactive prior to the removal of the protective group. For purposes of the invention, such reagents are known as "protcctcd phosphonate linkcrs". Protccted phosphonatc linkers according to thc invention are charactcrized by the structure DMTr-o-cH2-(c~3)n-o~ oH
whercin DMTr = an `acid-labilc protective group, such as dimethoxytrityl and n = l to 20.
Such protected phosphonate linkers according to thc invention are . 25 synthesized by treating an alkanediol with a salt of an acid-labileprotective group, such as dimctho~yrityl chloride to yield a pro~ected derivativc, followed by converting the protected derivative to the corresponding H-phosphonate by standard procedurcs.
Anothcr reagent according to the invontion is a protccted linkcr molecule having at one cnd a free amino group, and at the other end a protected amino group that is not reactive prior to removal of the protectivc group. For purposes of thc invention, such rea~ents are known as ~protected diamino linkers~. Protected diamino linkers according to the invention are characterizcd by the struc;ure R- I-cHt-(cH2)n-~H2 wherein R ~ a basc-labile group n= I to20.
__ ; .
. ~ , .
, 2~i~32~ 2 WO 92t0872~ PC~/US91/0~347 Such protected diamino linkers according to th~ invention are synthesized by protecting onc amino group of a diaminoalkanc with a basc labilc group under controlled condi~ions. (See Agrawal and Tang~
Tetrahedron Lett. ~1: 1543-1546 (1990)).
Another reagcnt provided by thc invention is an oli~onucleotide of defined nucleotidc sequence having at its 5' end a repeating polymcr having multiple free amino functionalities. For purposes of the invention~
such reagen~ is known as a "S' functionali}ed oligonucleotide~. 5' functionalized oligonucleotides according to the invention are characterized by the structure rO 10 HO (CH2)n-OL ~-O-(CHz)~-O j~ ~-0 D pD~
( IH2)b (~H2)e NH2 ~H2 - whcrein D ~ a ribonucleoside or deo~yribonucleoside, p - a phosphodiester linkage or chesnically modified analog linkagc, n ~ I to 20, a ~ I ~o 20, b Y I to 20, c - I to 20, z, 0 to 20, t - I to 100, and wherein for each repeating unit ~in bracke~s] ~a~ may represent the same number or a different number than that number represented by ~a~ in every other repeating unit, and for cach repcating unit [in brackets] ~b" may rcprescnt ~he same numbcr or a different number than that number representcd by "b" in every other repcating unit.
.
.
~3~2 8~28 PCI /US91 /0334 5' functionalizcd oligonucleotidcs, according to the invention, can bc synthesixed in the following manncr. A protected phosphonate linker is prepared by treating an alkanediol with a salt of an acid-labile protectivc group, such as dimethoxytrityl chloride to yield a protected derivative of the alkanediol, which is then convertcd to the corresponding H-phosphonate by standard procedures. (Sec Frochler and Matteucci, Nuclcic Acids Res. 14: 5399-5407 (1986)). An oligonuclcotide of defined scqucnce is synthesi~ed (ç,~, by standard phosphoramidite, phosphonatc or phosphotriestcr chemistry) to yield a support-bound oligonuclcotide. A
protected phosphonate linker is thcn coupled with the frce 5' hydroxyl group of thc support-bound oligonuclcotidc using an H-phosphonate coupling cycle. (Sce Agrawal and Tan~, Tetrahcdron Lett. ;~: 1543-1546 (1990)). Additional phosphonate linkcrs are added through a scries of deprotes!ion and H-phosphonate cycles, with each cycle adding one phosphonate linker. The total number of cycles (and thus linkers) should cqual the number of reporter groups to be added to the oligonucleotide.
Thc support-bound oligonucleotidc/phosphonate linker complex is then oxidized with a protected diamino linkcr to yield a support-bound oligonucleotide with multiplc protected amino functionalities. (See Agrawal and Tan8, ~.) The support-bound oligonucleotidc wjth multiple protected amino functionalities is then converted into a free 5' functionalized oligonuclcotide by deprotection with a deprotccting agent, such as aqueous ammonia.
Thus the invention provides a method for producing a 5' functionalized oligonucleotide, which me~hod compriscs gencrally the following steps:
(a) coupling with support bound oli~onucleotide a protected phosphonate linker by an H-phosphonate coupling cycle, wherein the protected phosphonate linker is the product of a reaction between an alkanediol and a salt of an acid-labilc protective group, such as dimethoxytrityl chloride, which product has further been converted to an H-phosphonate;
(b) repsating thc coupling of step (a) at least once;
(c) oxidizing thc support bound oli~odeo1tynucleotide with a protccted diaminolinker to produce an oligonucleotide with protected amino functionalities; and wo 92/08728 ~ ~ 9 .~ 2 ~ 2 PCI/US91/083~/
(d) deprotecting the oligonucleoti~te to yicld a 5' function~lizcd oli~onucleotide.
Another reagent provided by she invention is an oligonuclcotide that has at its 3' end multiple free amino func~ionalities. For purposes Or the invension, such reagen~ is known as a 3~ functionalized oligonucleotide. 3' functionalizcd oligonucleotidcs according to the invention are charac~erized by the structure ~0 ~O
D(pD)n OlL P O-(CH2)"~0~ P''(CH2)b'H
NH I~H
(CHz)c ~CH2)d I~IH, NHz wherein D - a ribonucleoside or deo%yribonuclcossde, and p . a phosphodies~er linkage or a chemically modified analo~ linkage, and n~ I to 100, a, I to 20, b ~ I to 20, c . I to 20, d .. I to 20, - z ~ 0 to 20, and wherein for each repeatjng unit lin bracketsl, ~a~ may represent the same number or a differen~ number ftom that represented by "a~ in every o~her repeating unit, and for each repcating unit [in brackets], "b~ may represent the same number or a diffcrent number ïrom that representcd by ~b~ in e~ery ~thcr repe~ting unit.
Such 3' func~ionalized oligonucleotides accordin~ to the invention arc assembled in a manner that is analogous to that for the 5' oligonucleotides, excepr that a support-bound rcpeating structure having multiple protected amino functionalities is assembled using the same steps as before, but it is asscmbled first, beginning with linkage of a free phosphonate linker to the solid support by its frce hydro~yl functionality.
The oligonucleotide is then svnthesi~ed upon the repeating struc~ure.
be~inning ~vith an H-phosphonate coupling between the free phosphon3te terminus of the repeating structure and thc free 3' hydroxyl of the 2 ~ 2 WO 92/08728 PCI/US91/~347 oligonucleotide. Completion of the oligonuclcotide synthesis yields an oligonucleotide couPled to a rcpeating structure ha-dng multiple protected amino functionalities, the repeating structurc in turn bcing bound ~o the support. This oligonucleotide/repcating structurc complex is then converted into a 3' incorporation oligonucleo~ide by treatment with a deprotecting agent, such as aqueous ammonia.
Thus the invention provides a method for producing a 3' functionalized oligonucleotide, which method comprises generally thc following steps:
(a) binding a free phosphonatc linker to a support via thc frec hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least onc more phosphonatc linker, using an H-phosphonate coupling cycle to yield bound phosphonate linkers;
(c) oxidizing the bound phosphona~e linkers with protected diamino linkers t`o produce a bound repeating structure having multiple protected amino functionalities;
(d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalizcd oligonucleotide; and (e) deprotectin~ the amino functionalities to produce a 3' functionalized oligonucleotidc.
The invention also provides useful precursors for the synthesis Or 3- functionalized oligonucleotidcs and for 3' labelled oligonuclcotides. Such precursors comprise the repeating structure of phosphonate linkers and diamino linkcrs described abovc, which are bound to a solid suppor~
suitable for oligonucleotide synthesis. The support-bound repeating structure may simply be an amino functionalizcd repeating structure or protected amino functionalized repea~ing structure, in which case an oligonucleotidc may bc synthesized attached to the structure, and may then be relcascd (dcprotected) to yield a frcc 3' functionalized oligonucleotide.
Alternatively, thc support-bound repeatin~s structure may be a labelled repcating structure. In this case the amino functionalized support-bound repcating structure is synthesized, theD the label is attached to the amino functionalities of thc support-bound rcpeating structure. This embodiment requires the use as a label of a reporser group that ;s stable under ~.
.
WO 92/08728 PCltUS91/08347 I I
oligonuclcotidc synthesis conditions, _~" bio~in, or a labcl that may be protectcd.
Ano~her resgent provided by the invention is an oligonucleotide having multiple frcc amino func~ionalitics coupled to it via one or morc intcrnucleoside phosphoramide linkages. For purposes of the invcntion, such re~gent is known as a ~phosphodiestet moiety-linkcd functional.z~d oli~onucleotide~. Phosphodiester moiety-linkcd functionalizcd olilonucleotides according ~o the invention re characterized by thc following structure lo ro ~1 D (pD),-Ol--~ o-(cH2)c-o~ o (pD)b-D
;~H ~H
2)d( ~H2 t~l H 21~1 H ~
wherein D .. a ribonucleotide or deoxyribonucleoside, and p - a phosphodiester linkage or ~ chemically modificd analo~ linkage, and a- I ~o 100, `
b ~ I to 100, c - I to 20, d - I to 20, e 1. I to 20, f ~ 0 to 20, and wherein for cach rcpeating unit ~in brackets], ~c~ may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unil lin brackets], "d~ may represent the same numbcr or a different number from that represented by "d" in every other repeatinR unit, and for each repeating unit [in brackets~,"c~
may represent the same number or a different number from that represented by ~e" in every other repeatinR unit.
Such phosphodiester moiety-linked functionalized oligonucleotides accordin~ to the invention may be synthesized by carrying out synthesis of a 5' functionalized oligonucleotide and then fusing ano~her oligonucleotidc 40to ~he 5' functionalized oligonucleotide via the free hydroxyl Rroup on its terminal phosphonatc linkcr. ~Iternatively a 3' functionalizcd WO 92/0~728 PCI/US91/0834 oligonuclcotidc can be syn~hesi2cd, thcn fused, via the frec hydroxyl of its terminal phosphorlate linker, to another oiigonucleot;dc.
Thus the invention providcs me~hods for producing an intcrnucleoside phosphodiestcr moiety-linked functionalizcd S oligonuclcotide, comprising 8enarally the following stcps:
(a) coupling with support bound oli~onuclcotidc a protcctcd phosphonatc linlccr by aD H-phosphonate coupling cyclc;
(b) repeatin~ the coupling of step (a) according to the number of amino groups required;
lo (c) oxidizing the support bound oligodeo~tynucleo~idc w;th a protec~cd diaminolinker in the presonce of carbon tetrachloride to produce an oligonucleotide with protccted amino functionalities;
(d) continuing the oligonucleotidc synthesis at a frce cnd of the IS phosphona~c linkcr; and (c) deprotecting the amino functionalities to produce an intcrnucleotidc phosphodicster moiety-linked functionalized oligonucleotide.
In anothcr aspec~, thc invcntion providcs methods for producin~, oligonucleotides having at ~hcir 5' or 3' ends, or attached to onc or more phosphodiester moieties, multiple reporter ~roups which confcr upon the oligonucleo~ide dctectability in a specific assay. Reporter groups according to the invention ~herefore include any molecules which confer upon the oligonucleotide detectabili~y in a specific assay withou~ unduly interfering ~5 with biological functions of the oligonucleotjde, such as hybridization with a complcmcntary nucleotide scqucncc or bindin8 of a specific protcin.
Commonly uscd reporter groups will includc biotin, various flourophorcs, enzymes, and molecules which arc detectable by antibody bindin8 or other Iigand-rcccptor intcractions.
These mcthods of the invcntion ase practiced e~ac~ly as dcscribed for the production of 5', 3', or phosphodicstcr moicty-linkcd functionalized oligonucleotides, followed by coupling of the reporter group to thc functionalized oligonucleotide via linkagc with the free amino functionalitics of thc functionalized oligonucleotide. Thus, in these mcthods of the invcntion the spacing of the rcportcr groups can be manipulated by using diffcrcnt sizes of phosphonatc linkcrs and diamino linkers.
2 ~
WO 92/08728 PCT/IJS91/û83~7 The invcntion contemplates numerous ob~dous modifications that would be apparent to those skilled in ~he art. For cxamplc, structural modification of the repeating units of the invention could be made without undue e~perimen~ation by substituting linear or branched alkcne, alkyne, cycloalkane, cycloalkene~ cycloalkyne, aromatic or heterocyclic spacers for the alkane spaccrs uscd in the phosphonate linkers or diamino linkers.
Also, the diamino linker may be replaced by polyamines. The diamino linker may also be rcplaced by a linker havin~ a protected amino group at one esld and a free hydroxyl group at the other end, whereby the linkcr becomes attached via its hydro~yl functionality to the phosphonate moiety of the phosphonate linker, forming 2 phosphotriestcr linkage. Yet another obvious modification is to use a phosphorothioate linker in place of thc phosphonate linker and, in place of ~he diamino linkcr, a linker having a protected amino group at one er.d and a free sulfhydryl group at the othcr cnd, whereby the linkcrs can bc joined l~ia a disu!fide linkage. An additional obvious modification is to use an aminoalkyl iodidc in place of the diamino linker to rcact with a phosphodiester linkage to form a phophotricster linkage. Still another obvious modification in thc methods of the invcntion is to incorporate reporter groups which are stablc under oligonucleotide synthcsis conditions, e.g., biotin, during thc synthcsis process, rather than thcrcafter.
In addition to embodiments containing th~ above, specifically cnu ncratcd obvious modifications, all embodiments containing obvious modifications arc considered to be equivalents of ~hc invcntion.
Thc following example is provided to further illustrate the invention and is not limiting in nature.
XAMPLE
Incorporation o~ Multlple Reporter Groups ~t th~
S' End Qr 3n Olicod~QxYnuclç~t5de A six carbon non-nucleosidic linker was prepared by treating 1,6-hexanediol with 4,4'-dimethoxytrityl chloride (0.45 equiv.) in pyridine for two hours. The desired product, 6-(4,4'-dimethoxytrityl)- 1,6-hexanediol was purified on a silica gel column in 909~ yield, and was then converted to the corresponding H-phosphonate by a standard tris (1,2,4-triazole) phosphite procedure. To test the efficacy of the linker, a 17-mer sequence, 2 ~ 3 ;~
0 92/08728 PCI /US91~08347 GTAAACGACCiGCCAGT, vas assembled using normal phosphoramiditc ch~mistry, followed by coupling with 6-(4,4'-dimcthoxytrityl)-1,6-hcxancdiol-l, hydrogen phosphonate usin8 a H-phosphonate couplins cycle.
(Sce Agrawal and Tang, Tetrahcdron Lett. 3L: 1543-1546 (1990)). Thc S number of couplings dcpends on thc number of amino-groups to be introduced. At the end of the assembly, support bound oli~onuclcot;dc was oxidized with ~ trifluoracetyl-diaminohcxane. (See Agrawal and Tang, .) Aftcr dcprotection with aqucous ammonia, the amino-functios~alized oliRonucleotide WaS reacted with biotin activc cstcr using a published proccdurc. (See Agrawal es al., ~iucleic Acids Rcs. 14: 6229-6245 (1986)). All oligonucleotides wcre chccked by Cl8 reversc phase HPLC
(Figure 2).
Claims (23)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of producing a synthetic oligodeoxynucleotide of defined nucleotide sequence, whereby the oligonucleotide is singly or multiply labelled at its 5' end, the method comprising the steps of:
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle (b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) deprotecting the oligonucleotide to yield a 5' functionalized oligonuclcotide; and (e) coupling the 5' functionalized oligonucleotide with a reporter molecule to produce an oligonucleotide that is multiply-labelled at its 5' end.
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle (b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) deprotecting the oligonucleotide to yield a 5' functionalized oligonuclcotide; and (e) coupling the 5' functionalized oligonucleotide with a reporter molecule to produce an oligonucleotide that is multiply-labelled at its 5' end.
2. An oligonucleotide that is singly or multiply-labelled at its 5' end, wherein the oligonucleotide that is singly or multiply labelled at one end is obtained by the method of claim 1.
3. An oligonucleotide that is singly or multiply-labelled at one end with an reporter group, wherein the singly or multiply-labelled oligonucleotide is characterized by the following structure:
wherein D = a ribonucleoside or deoxyribonucleoside, p = a phosphodiester linkage or chemically modified analog linkage, X = a reporter group.
n = 1 to 20, a = 1 to 20, b = 1 to 20, c = 1 to 20, z = 0 to 20, t = 1 to 100, and wherein for each repeating unit [in brackets] "a" may represent the same number or a different number than that number represented by "a" in every other repeating unit, and for each repeating unit [in brackets] "b" may represent the same number or a different number than that number represented by "b" in every other repeating unit.
wherein D = a ribonucleoside or deoxyribonucleoside, p = a phosphodiester linkage or chemically modified analog linkage, X = a reporter group.
n = 1 to 20, a = 1 to 20, b = 1 to 20, c = 1 to 20, z = 0 to 20, t = 1 to 100, and wherein for each repeating unit [in brackets] "a" may represent the same number or a different number than that number represented by "a" in every other repeating unit, and for each repeating unit [in brackets] "b" may represent the same number or a different number than that number represented by "b" in every other repeating unit.
4. A method of producing a synthetic oligonucleotide of defined nucleotide sequence, whereby the oligonucleotide is multiply labelled at its 3' end, the method comprising the steps of:
(a) binding a free phosphonate linker to a support via the free hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least one or more phosphonate linkers, using an H-phosphonate coupling cycle to yield bound phosphonate linkers (c) oxidizing the bound phosphonate linkers with protected diamine linkers to produce a bound repeating structure having multiple protected amino functionalities;
(d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalized oligonucleotide;
(e) deprotecting the amino functionalities to produce a 3' functionalized oligonucleotide; and (e) coupling the 3' functionalized oligonucleotide with a reporter molecule to produce an oligonucleotide that is labelled at its 3' end.
(a) binding a free phosphonate linker to a support via the free hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least one or more phosphonate linkers, using an H-phosphonate coupling cycle to yield bound phosphonate linkers (c) oxidizing the bound phosphonate linkers with protected diamine linkers to produce a bound repeating structure having multiple protected amino functionalities;
(d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalized oligonucleotide;
(e) deprotecting the amino functionalities to produce a 3' functionalized oligonucleotide; and (e) coupling the 3' functionalized oligonucleotide with a reporter molecule to produce an oligonucleotide that is labelled at its 3' end.
5. An oligonucleotide that is singly or multiply labelled at its 3' end, wherein the oligonucleotide that is singly or multiply labelled at its 3' end is obtained by the method of Claim 4.
6. An oligonucleotide that is singly or multiply labelled at its 3' end, wherein the oligonucleotide that is singly or multiply labelled at its 3' end is represented by the structural formula wherein D = a ribonucleoside or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and X = a reporter group, n = 1 to 100, a = 1 to 20, b = 1 to 20, c = 1 to 20, d = 1 to 20, z = 0 to 20, and wherein for each repeating unit [in brackets], "a" may represent the same number or a different number from that represented by "a" in every other repeating unit, and for each repeating unit [in brackets], "b" may represent the same number or a different number from that represented by "b" in every other repeating unit.
7. A method of producing a synthetic oligonucleotide of defined nucleotide sequence, whereby the oligonucleotide has a label attached to one or more internucleotide phosphodiester moieties, the method comprising the steps of:
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) continuing the oligonucleotide synthesis at a free end of the phosphonate linker;
(e) deprotecting the oligonucleotide to produce an internucleotide phosphodiester moiety-linked functionalized oligonucleotide; and (f) coupling the internucleotide phospodiester moiety-linked functionalized oligonucleotide with a reporter molecule to produce a synthetic oligonucleotide having multiple labels attached to one or more internucleotide phosphodiester moieties.
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) continuing the oligonucleotide synthesis at a free end of the phosphonate linker;
(e) deprotecting the oligonucleotide to produce an internucleotide phosphodiester moiety-linked functionalized oligonucleotide; and (f) coupling the internucleotide phospodiester moiety-linked functionalized oligonucleotide with a reporter molecule to produce a synthetic oligonucleotide having multiple labels attached to one or more internucleotide phosphodiester moieties.
8. An oligonucleotide having a label attached to one or more phosphodiester moiety, wherein the oligonucleotide having a label attached to one or more phosphodiester moiety is obtained by the method of Claim 7.
9. An oligonucleotide having a label attached to one or more phosphodiester moiety, wherein the oligonucleotide having a label attached to one or more internucleotide phosphate is represented by the structural formula:
wherein D = a ribonucleotide or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and X = a reporter group, a = 1 to 100, b = 1 to 100, c = 1 to 20, d = 1 to 20, e = 1 to 20, f = 0 to 20, and wherein for each repeating unit [in brackets], "c" may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unit [in brackets], "d" may represent the same number or a different number from that represented by "d" in every other repeating unit, and for each repeating unit [in brackets],"e"
may represent the same number or a different number from that represented by "e" in every other repeating unit.
wherein D = a ribonucleotide or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and X = a reporter group, a = 1 to 100, b = 1 to 100, c = 1 to 20, d = 1 to 20, e = 1 to 20, f = 0 to 20, and wherein for each repeating unit [in brackets], "c" may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unit [in brackets], "d" may represent the same number or a different number from that represented by "d" in every other repeating unit, and for each repeating unit [in brackets],"e"
may represent the same number or a different number from that represented by "e" in every other repeating unit.
10. A protected phosphonate linker represented by the structural formula:
wherein DMTr = an acid-labile protective group, and n = 1 to 20.
wherein DMTr = an acid-labile protective group, and n = 1 to 20.
11. A protected diamino linker represented by the structural formula:
wherein R = a base-labile group n = 1 to 20.
wherein R = a base-labile group n = 1 to 20.
12. A method of producing a 5' functionalized oligonucleotide, the method comprising the steps of:
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cyclic;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities; and (d) deprotecting the amino functionalities to yield a 5' functionalized oligonucleotide.
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cyclic;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities; and (d) deprotecting the amino functionalities to yield a 5' functionalized oligonucleotide.
13. A 5' functionalized oligonucleotide produced according to the method of claim 12.
14. A 5' functionalized oligonucleotide represented by the structural formula:
wherein D = a ribonucleoside or deoxyribonucleotide.
p = a phosphodiester linkage or chemically modified analog linkage, n - 1 to 20, a = 1 to 20, b = 1 to 20, c = 1 to 20, z = 0 to 20, t = 1 to 100, and wherein for each repeating unit [in brackets] "a" may represent the same number or a different number than that number represented by "a" in every other repeating unit, and for each repeating unit [in brackets] "b" may represent the same number or a different number than that number represented by "b" in every other repeating unit.
wherein D = a ribonucleoside or deoxyribonucleotide.
p = a phosphodiester linkage or chemically modified analog linkage, n - 1 to 20, a = 1 to 20, b = 1 to 20, c = 1 to 20, z = 0 to 20, t = 1 to 100, and wherein for each repeating unit [in brackets] "a" may represent the same number or a different number than that number represented by "a" in every other repeating unit, and for each repeating unit [in brackets] "b" may represent the same number or a different number than that number represented by "b" in every other repeating unit.
15. A method of producing a 3' functionalized oligonucleotide, the method comprising the steps of:
(a) binding a free phosphonate linker to a support via the free hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least one more phosphonate linker, using an H-phosphonate coupling cycle to yield bound phosphonate linkers;
(c) oxidizing the bound phosphonate linkers with protected diamino linkers to produce a bound repeating structure having multiple protected amino functionalities; and (d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalized oligonucleotide.
(a) binding a free phosphonate linker to a support via the free hydroxyl of the phosphonate linker to yield a bound phosphonate linker;
(b) coupling to the bound phosphonate linker at least one more phosphonate linker, using an H-phosphonate coupling cycle to yield bound phosphonate linkers;
(c) oxidizing the bound phosphonate linkers with protected diamino linkers to produce a bound repeating structure having multiple protected amino functionalities; and (d) synthesizing an oligonucleotide that is attached to the bound repeating structure to produce a protected 3' functionalized oligonucleotide.
16. A 3' functionalized oligonucleotide produced according to the method of claim 15.
17. A 3' functionalized oligonucleotide represented by the structural formula:
wherein D = a ribonucleoside or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and n = 1 to 100, a = 1 to 20.
.
WO 92/08728 PCl/US91/08347 b = 1 to 20, c = 1 to 20, d = 1 to 20, z = 0 to 20, and wherein for each repeating unit [in brackets], "a" may represent the same number or a different number from that represented by "a" in every other repeating unit, and for each repeating unit [in brackets], "b"
may represent the same number or a different number from that represented by "b" in every other repeating unit.
wherein D = a ribonucleoside or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and n = 1 to 100, a = 1 to 20.
.
WO 92/08728 PCl/US91/08347 b = 1 to 20, c = 1 to 20, d = 1 to 20, z = 0 to 20, and wherein for each repeating unit [in brackets], "a" may represent the same number or a different number from that represented by "a" in every other repeating unit, and for each repeating unit [in brackets], "b"
may represent the same number or a different number from that represented by "b" in every other repeating unit.
18. A method for producing an internucleotide phosphodiester moiety-linked functionalized oligonucleotide, the method comprising the steps of:
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) continuing the synthesis of the oligonucleotide from a free end of the phosphonate linker; and (e) deprotecting the oligonucleotide to produce an internucleotide phosphodiester moiety-linked functionalized oligonucleotide.
(a) coupling with support bound oligonucleotide a protected phosphonate linker by an H-phosphonate coupling cycle;
(b) repeating the coupling of step (a) according to how many amino groups are required;
(c) oxidizing the support bound oligodeoxynucleotide with a protected diaminolinker to produce an oligonucleotide with protected amino functionalities;
(d) continuing the synthesis of the oligonucleotide from a free end of the phosphonate linker; and (e) deprotecting the oligonucleotide to produce an internucleotide phosphodiester moiety-linked functionalized oligonucleotide.
19. An internucleotide phosphodiester moiety-linked functionalized oligonucleotide produced according to the method of claim 16.
20. An internucleotide phosphodiester moiety-linked functionalized oligonucleotide represented by the structural formula:
wherein D = a ribonucleotide or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and a = 1 to 100, b = 1 to 100, c = 1 to 20, d = 1 to 20, e = 1 to 20, f = 0 to 20, and wherein for each repeating unit [in brackets], "c" may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unit [in brackets], "d" may represent the same number or a different number from that represented by "d" in every other repeating unit, and for each repeating unit [in brackets],"e"
may represent the same number or a different number from that represented by "e" in every other repeating unit.
wherein D = a ribonucleotide or deoxyribonucleoside, and p = a phosphodiester linkage or a chemically modified analog linkage, and a = 1 to 100, b = 1 to 100, c = 1 to 20, d = 1 to 20, e = 1 to 20, f = 0 to 20, and wherein for each repeating unit [in brackets], "c" may represent the same number or a different number from that represented by "c" in every other repeating unit, and for each repeating unit [in brackets], "d" may represent the same number or a different number from that represented by "d" in every other repeating unit, and for each repeating unit [in brackets],"e"
may represent the same number or a different number from that represented by "e" in every other repeating unit.
21. A phosphonate linker that is attached to a solid support suitable for oligonucleotide synthesis.
22. A support-bound, amino functionalized repeating structure of phosphonate linkers and diamino linkers suitable for synthesizing oligonucleotides attached to the repeating structure.
23. A support-bound, labelled repeating structure of phosphonate linkers and diamino linkers suitable for synthesizing oligonucleotides attached to the repeating structures, wherein the repeating structure is attached to a reporter group.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61054190A | 1990-11-08 | 1990-11-08 | |
| US610,541 | 1990-11-08 |
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| Publication Number | Publication Date |
|---|---|
| CA2095212A1 true CA2095212A1 (en) | 1992-05-09 |
Family
ID=24445435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002095212A Abandoned CA2095212A1 (en) | 1990-11-08 | 1991-11-08 | Incorporation of multiple reporter groups on synthetic oligonucleotides |
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| Country | Link |
|---|---|
| US (1) | US5510475A (en) |
| EP (1) | EP0556301B1 (en) |
| JP (1) | JP3172178B2 (en) |
| KR (1) | KR930702373A (en) |
| AT (1) | ATE198598T1 (en) |
| CA (1) | CA2095212A1 (en) |
| DE (1) | DE69132510T2 (en) |
| WO (1) | WO1992008728A1 (en) |
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| US4757141A (en) * | 1985-08-26 | 1988-07-12 | Applied Biosystems, Incorporated | Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof |
| US4882269A (en) * | 1985-12-13 | 1989-11-21 | Princeton University | Amplified hybridization assay |
| JPS638396A (en) * | 1986-06-30 | 1988-01-14 | Wakunaga Pharmaceut Co Ltd | Poly-labeled oligonucleotide derivative |
| EP0972779A3 (en) * | 1987-10-28 | 2004-10-20 | Howard Florey Institute Of Experimental Physiology And Medicine | Oligonucleotide-polyamide conjugates |
| US5324829A (en) * | 1988-12-16 | 1994-06-28 | Ortho Diagnostic Systems, Inc. | High specific activity nucleic acid probes having target recognition and signal generating moieties |
| US5157123A (en) * | 1989-03-13 | 1992-10-20 | Georgetown University | S-(2-thiopyridyl)-l-cysteine, a heterobifunctional crosslinking reagent |
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| DE69132510D1 (en) | 2001-02-15 |
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