CA2207011A1 - Discontinuous probe design using hybritope mapping - Google Patents

Abstract

This invention provides a method of detecting or determining a binding oligonucleotide comprising a nucleotide sequence which binds within a known nucleotide sequence of a target nucleic acid using a technique called hybritope mapping. This invention also provides a method of using hybritope mapping to obtain discontinuous probes that bind to a target nucleic acid.

Description

CA 022070ll l997-05-23 W O 96/1795~ PCTnUS9SI15779 DISCONTINUOUS PROBE DESIGN USING HYBRITOPE MAPPING

Terhnic~l Field This invention is in the field of nucleotide probe and ~nticçn~ce probe chf~mictry.
More sperific~lly, this invention provides a method for detell,~.g a discontinuous probe that binds tightly to its nucleic acid target for use in r~i~ostics or as an ~nticen~ce thela~)~utic. Diccol~ o~lc probes bind to two or more non-contiguous regions of a target nucleic acid and include ~nti~-once m()lerllles~ ribozyrnes, tethered probes and branched DNA molec~les.

Bacl~ruund of the Invention Tools for seq~l~n~e-specific lcco3~ ion of DNA and RNA are increasingly ilnpol ~ in the growing fields of nucleic acid probe technology, as well as ~ntic~n.~e and ribozy~me thelap~ulics. Seq~1en~e-specific leCO~-;I;O-~ of RNA is especially challenging due to its comr't colLrollllalional structure and ~,ltl~ n'c~ r hyhritli7~tion.
Cload and Schel)~4 J. Am. Chem. Soc. (1991) 113:6324 6326, disclose a family of sylltll~;Lic molecules for the seq~nce- and structure-specific recognition of RNA called tethered oligonucleotide probes (TOPs). TOPs consist oftwo oligodeoxyribonucleotides separated by a flexible, synthetic tether. The two oligonucleotides bind to non-contiguous, 2 0 single-stranded regions of the RNA molecule, and are tethered by a repeating abasic phosphodiester unit, or a lel)e.,~ polyethylene glycol unit. The authors in this article do not sll~Prst a method for s~1ectin~ superior oligQm-çleQtides to be tethered. Cload and Schepartz, J. Am. Chem. Soc. (1994) 116:437 442, teach the use of a random nlipomlr.leotide library to select superior oli~?s)n~leotides that bind to non-contiguous 2 5 regions of a target RNA to be tethered together in a TOP. The authors, using a known Rev binding site in the human immlmod~.firienr.y virus ~HIV) Rev response element (RRE), se~.,hed for a second oligon~r,leotide by tethering random heptanucleotides to an oct~n-lcleotide that bound to the Rev site, and SClee~g with RNase H to determine the strongest site for secon~l~ry binding.

3 0 Eulopeall Publication 0 138 855 descl;l,es a method of detell~ .il,g antigenically active amino acid seq~lpnçe~ within a known protein by synth~i7.ing a series of overlapping peptides corresponding to sequences in that protein, and then screening the peptides against antibody serum raised against the protein to ~ tk ~ "f~- which peptides react with the serum antibodies The inventors herein describe a method of delP~ g superior sites for binding 5 oli~omlcleotides to a target nucleic acid called "L~ pe Illap~illg ll Hybritope ~,-apping may be used to identif~ improved disco..~ o~s probes with high binding co~ l s Disclosure ofthe Invention In one aspect of this invention, a method of ~le~ or dt;Lel,.,."i"g a binding 10 oli~omlrleotide comprising a nucleotide sequpnre which binds within a known nucleotide sequence of a target nucleic acid is provided, the method co..,~ ing the steps of (a) obtaining a plurality of olipom-r1~Qtides, each of the oligonucleotides comprising a first nucleotide sequence which is co~ lf "~ h y to a seq~1rnre within the known nucleotide seql-rnr,P, and the oligon~cleotirles having o~/e lal~hlg first nucleotide sequences wherein 15 the first nucleotide sequence of each of the oli~omlrleotides in the plurality of oligonucleotides overlaps the first nucleotide se.~ e of another oli~om~cleotide in the plurality of oligonucleotides by from one to four nucleotides; (b) cont~ctin~ each of the olips)n~cleQtides with the target nucleic acid under conl1itionc pe ~-,ilLing specific hybridization of olignm~leotides to the target; and (c) dele.~ or de~ellllil--ng the 2 0 presence or absence of specific o1igo~ rleQtide_target binding between each of the oligonucleotides and the target nucleic acid to intlir~te whether each oligom-rleotide binds within the known nucleotide seq~nre, thereby ~ e~ g or ~letectinP one or more binding oligonucleotides In another aspect of this invention, a method of ~etecting or deterrnining a 2 5 disconlilluous probe that binds to a known nucleotide sequence in a target nucleic acid, the disco..li..uous probe co--llJIisL~g (i) at least two binding oligo~rleotides that are each comple. . .~ h . y to a distinct region of the target nucleic acid, covalently joined directly or by (ii) an organic linker molecl-le, the method co...l ;ci ~ the steps of: (a) d~le--",~ g at least first and second binding oli~omlcleQtides that are each comple~nt~ry to first and 3 0 second region of the target nucleic acid, the first and second binding oligonucleotides binding more strongly to the target nucleic acid than other oligonucleotides that are W O 9611~55 PCTnUS95115779 y to other regions ofthe target nucleic acid, and wherein the first and second regions are non-cont~ ol1~, (b) covalently linking two or more of the oligonucleotides delc~ ed in step (a) in at least two ~."~ ions, directly or through organic linker mole~ s to obtain a set of ~n~ e ~ CQI~I ;. .. ollq probes; (c) cont~cting each c~n~ te 5 ~ cQ..1;. ~ous probe with the target nucleic acid under con~litiQnc pe.,.,iuing specific hybri~ ;ol- of oli~nm-r.1eotidP~ to the target; and (d) ~lel ~,1 ;i~g or deLe, l~ g the p~cs~llce or flbs~-re of specific discc.. ~ o~ probe-target binding b~lween each ofthe COI~ Q11~ probes and the target nuc1eic acid to in~1ic~te wl,cll,er each ~l;ccol~ loue probe binds the target nucleic acid, thereby ~ or ~elecl;,~p. one or more10 ~1iccQl~ ous probes.
In a prere~led aspect ofthe above mPtllod for dele~ a disco~Li"~ous probe, step (a) co.. 'I" ;~Çi the steps of: obl~ a plurality of ~fi~om-eleotides~ each of the o1i~on1lr1eotides colll~ lising a first n~ eotide sequçnce which is comrl~mPnt~ry to a seq~1Pnre within the known nucleotide sequrnce, and the oli~omlr1eotides ha-ving5 .,vc,l~p-,,g first nucleotide sequ~nr,es v~Lel -cil~ the first nucleotide sequçnr,e of each of the ~ligom1r1eotides in the plurality of o1igonllrl~oti~les ovc,laps the first nucleotide sequence of another olig~ m)ç1eotide in the plurality of oli~oml~leotides by from one to four nucleotides; c~nt~ctin~ each ofthe o1i~o~lJcleotides with the target nudeic acid under con-1ition~ pc~ ~--;1~ ;i~g specific hybri~i7~tiQn of oli~n11r1eotides to the target; detecting or 2 0 d~le~ g the presence or absel~ce of specific oligonucleotide-target binding between each of the oligonucleotides and the target nucleic acid to in~iir,~te whether each o1i~om1rleQtide binds within the known nucleotide sequçnce~ thereby dt;le~ fing or delec~ p one or more binding o1if~om1rleQtides; and sPlectin~ at least two binding oligonucleotides that bind most strongly to the target nucleic acid BriefDesc,iplion ofthe D-~wi-,~s Figure 1 depicts a l.~il .ilope ll.appil~g assay of the current invention Figure 2 depicts a method of opl ;...;~ g ~;~CQ.I~ ous probes of the current invention 3 0 Figure 3 shows the results of using the l"il"ilope ,llapping method of the current invention to find the optimal n1igom~cleQtides that bind to the ~V RRE region CA 022070ll l997-05-23 W O96/17955 PCTrUS95/15779 Figure 4 shows the effect of Qli~omlr,leQtide length on the ability of c~n~ te binding oli~omleleQffdes to bind the HlV RRE region.
Figure 5 shows the results of an eA~)e~ nt to confirrn the binding of c~nf~ te binding oligonucleotides to the HIV RRE region by RNAse H cleavage.
Figure 6 shows the design of an experiment to study the effects of discontimlol-c probe design to o~ e target bin.li.~g Figures 7 and 8 show the results of the e .~,e~ shown in Figure 6.

Modes for Carrying out the Invention The invention described herein draws on previously publisLed work and pending patent aprlir~tionQ By way of ~ le, such work collsi~ls of scientific papers, patents or ~e~ g patent applications. All ofthese publications and appliG~I;QnQ, cited previously or below are hereby h~col~ol~led by lerelèncê. St~dard abbreviations for nucleotides and amino acids are used in this spe~ifir~ti~n The practice of the present invention will employ, unless otherwise inrlic~te~
cGIl.~ lteel~ Qoflllolec~ rbiology,..iclo~iology,recollllJill~llDNA,and i... olc)gy, which are within the skill ofthe art. Such lecl~ ues are eYrl~in~cl fully in the Llel~lule. See e.g., Sambrook et al., MOLECULAR CLONING; A LABORATORY
MANUAL, SECOND EDITION (1989); DNA CLONlNG, VOLIJMES I AND II (D.N
Glover ed. 198~); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed, 1984); NUCLEIC
ACID HYBRIDIZATION (13.D. Hames & S.J. ~i~in~ eds. 1984); TRANSCRIPTION
AND TRANSLATION (B.D. Hames & S.J. ~i~inC eds. 1984); ANIMAL CELL
CULTURE (R.I. Freshney ed. 1986); IMMOBTr T7:Fn CELLS AND ENZYMES (IRL
Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984);
the series, METHODS IN ENZYMOLOGY (~ademic Press, Inc.); GENE TRANSFER
VECTORS FOR MAMMALLAN CELLS (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Gl.,s~ , and Wu, eds., respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMlCAL METHODS IN CELL AND MOLECULAR BIOLOGY
3 0 (~ademic Press, London), Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES
AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), and HANDBOOK OF

CA 022070ll l997-05-23 W O96/1795S PCTnUS9S~15779 ~;~KIMENTAL IMMUNOLOGY, VOLU~S I-IV (l~.M. Weir and C. C. Blackwell eds 1986).

Definitions:
The term "~ e" refers to a nucleic acid mqlçclllP, pr~rcl~bly DNA, which is c~n-. ' ntslry to and capable of ~,~ ng double stranded ~s~-~' s with a "sense" strand of an RNA or DNA moleplle Hybr~ 7~ti~n of an ~ ic~ e nucleic acid to mRNA7 for eY~mr~, inhibits its ~ ;nll into p-ol~ls. ~ P~e nucleic acids may be pl~tecli~e (if they comp'~~Pnt a viral protein rnRNA or mRNA l~lscl;bed from an active oncogene, for e ;~ 1 le), or destructive (ifthey complement an essF-~ l host cell enzyme, such as a ho-l~P1~Peping enzyme with no ~ live alternate palll~a~). See also G. Zon et al, EP
288,163, which rli~closes the use of cligo~e~ y~ leQt~ for inh;l ition of retroviral replic~tion and oncogenic proliferation.
The term "label" as used herein refers to any atom or molecule which can be used to provide a deleel~ble (pr~re~a~ly 4~ 1e) signal, and which can be ~ hed to a nucleic acid or protein. Labels may provide signals dete ~ ~ le by fluorescPnçe, r~lio~cti-vity, colo-;...e~.y, X-ray diffraction or absûl~t;on, ~ e~ .. el~ylllalic activity, and the like.
Suitable labels include f~uoropholes, ~.I..ul.,opholes, rA-lin~tive atoms (particularly 32p and 125I), ele~;l,ùll-dense .~g~ s, e-~y---es, and ligands having specific binding pa~
2 0 Enzymes are typically detected by their activity. For example, horseradish peroYidase (HRP) is usually ~letected by its ability to convert 3,3',5,5'-tetramethylb~n7i~inP (TMB) to a blue pi~,.~ , 4u~-.lil~Able with a ~e~;llophotQ~ . It should be understood that the above description is not meant to calego-~e the various labels into distinct classes, as the same label may serve in several di~relt;l-l modes. For ~ rl~ 125I may serve as a radio-25 active label or as an ele~,l-ùll-dense reagent. HRP may serve as en_yme or as antigen for a monol;lQnAl antibody ~.b). Further, one may co...l-;--e various labels for desired effect.
For eYAmpl~, MAbs and avidin also require labels in the practice ofthis invention: thus, one might label a probe with biotin, and detect its pl~sence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP, or with an HRP molecule conjugated to W O96/17955 PCTrUS95/15779 avidin or streptavidin. Other pc~ ;on~ and possibilities will be readily apparelll to those of ordi,laly skill in the art, and are conQ;~ered as e~uivalents.
The term "oli~om-rleQtide" or "polynl~c1eoti~1e" as used herein refers to diagnostic probes, ~ ;c~l~ce probes, oligomer fia~..~ to be detecte~l oligom~r controls or 5 riboymes, and is defined as a mo!e '~ cc~ ;ced of two or more deo~yl ;bollucleotides or ribonucleotides, prer~l~bly more than three. Its exact size will depend on many factors, which in turn depend on the llltim~te fi- ~l l;ol- or use ofthe oligon-~cleoti~le. As used herein, the terms "polynucleotide", "oli~nn~r1eotide'' and "nucleic acid" shall be generic to polydeu~ylil,ol..-r,1eQtides (co..l~ g 2-deoxy-D-ribose), to polyribonucleotides10 (~ 1; ; e D-ribose), to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, and to other polymers co..l ;~ nonn-lcleotidic bac~ones (e.g., protein nucleic acids and ~y--ll,elic se~ e~-re-specific nucleic acid polymers commer-cially available from the Anti-Gene Development Group, Corvallis, Oregon, as Neugene~M
polymers) or nol~ ntl~rd link~g~c, providing that the polymers contain nucleobases in a 15 confi~;u.~ion which allows for base pairing and base sl~rL ;.~p such as is found in DNA and RNA. There is no intentle~ clion in length be L~een the term "polynucleotide" and "oli~nn--r1eoti-l~ " and these terms will be used interrl-~nge~bly. These terms refer only to the pli llaly structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-sL,~ded RNA and DNA:RNA hybrids, and2 0 also include known types of mo~lific~tiQn~ for ~ p'c, labels which are known in the art, methylation, "caps," s~lbstit~lfion of one or more ofthe naturally oc-iw,lllg nucleotides with an analog, internucleotide motlifi~tionc such as, for ~ rl~7 those with uncharged link~ges (e.g., methyl phosphl~n~t.oc, phosphotriesters, phosphor~mid~tes~ c_ll,h...~ec etc.) and with chal~2ed lin~ es (e.g., phosphoroth;o~tec pho~horodithioates, etc.), those 25 co--~ g pend~lL moieties, such as, for c ~ ple, pluleil~s (in~h~(ling nWle~ces~ toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those co.~ P ~h~ o. ~ (e.g., metals, rarlioactive metals, boron, oxidative metals, etc.), those co..l~ alkylators, those with m- tlifi~l link~ges (e.g., alpha al~On~;liC nucleic acids, etc.), as well as unmodified forms ofthe polynucleotide or 3 0 oligonucleotide.

W O 96/1795S PCTnUSg5/15779 ~ 1 will be appl~.,;aLed that, as used herein, the terms "nucleoside", "nucleotide" and "nucleic acid" will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been mo-lifie(l Such modifie~tion~ include methylated punnes or ~ c, a~ ~laled purines or pyrimitlinec, or 5 other heterocycles. Modified mlrl~or ~lçs or mlrleot;tlPe will also include morlific~tion.e on the sugar moiety, e.g., V~LClt;i~l one or more ofthe LYdIUAYI groups are replaced with h~lo~Pn, ~lirh~tir, groups, or are fimr,tiQn~li7ed as ethers, amines, or the like.
As used herein, a polynllr,leotide "derived from" a deei n~ted sequence refers to a polynucleotide seq~lPnce which is cc --~ ed of a sequence of apprc~x....~lçly at least about 6 nucleotides, p.~r~.~bly at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more ~l~rtilably at least about 15-2û nucleotides corresponding to a region ofthe deei n51tP,rl nucleotide se~ r.ç "Co~ ,ol-din " means homologous, comrlemP-nt~ry~ or subst~nfi~lly co~npl~ y to the d~eign~ted seqllPnce The derived polynucleotide is not nece~e~e~rily pl.~;,;eally derived from the nucleotide sequence shown, 15 but may be generated in any manner, in~ for PY~mple, ~hPmir~l synthesis or DNA
replie~tion or reverse transcription or Ll~n.ec, ;I~L;on In ad~litiQn~ co..-l h.~Lions of regions cGllc; ~Glld...~ to that of the dec:P.-~Ied seq~lPnee may be mot1ifiecl in ways known in the art to be cûn~i ~le~ vith an intpntied use.
It will be a~prec;dLed that the binding seq~en~Ps need not have perfect 2 0 cnmple nP~ ily to provide hnn~o~ 'e~rçs In many eit~tione heteroduplexes will suffice wherefewerthanaboutlû%ofthebasesare~ ."~ ci~-o,;.~loopsoffiveormore nucleotides. Accordingly, as used herein the term '~comp'~ ~~Pn~ ~ y intends exact comp'- ..~..~;.. ;Iy wherein each base within the binding region co..es~ollds exactly, and "s~lbs~ ly CG~ lP~k~ y intends 90% or greater holnolopy.
2 5 As used herein, the term l~oli~o.. ~ " refers to both primers and probes and is used ~l-Le cl~ gP;~l)ly herein with the term "polruc~otidG N The term oligomer does not connote the size ofthe molecule. However, typically oligomers are no greater than 1000 nucleotides, more typically are no greater than 500 mlr,lPoti~l~c even more typically are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and may be no 3 0 greater than 75 nucleotides, and also may be no greater than 50 nucleotides in length.

W O96/17955 PCTrUS95/15779 As used herein, the term "probe" refers to a structure comprised of a polynucleotide which forms a hybrid structure with a target seC~ ncp~ due to complc-~.P~ ily of at least one se ~ nre in the probe with a seq~lPnre in the target region. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
Tnr,ludecl within probes are "capture probes" as will be defined below.
The term "capture probe" as used herein refers to a molccllle comprising a single stranded polynucleotide c~ùpled to a binding partner or solid ~.-ppo. l. The single-stranded polynucleotide region is col-lpl~ y to a region of a second polynucleotide, and is ylffir; ~ntly long and m~tr.h~.d to afford s~lffiri~-nt affinity to immobilize the second polynuc-10 leotide to a solid support, directly or through a first binding partner specific for a secondbinding partner bound to the surface of a solid SUppGl ~.
The term "support" refers to any solid or semi-solid surface to which a specificbinding partner may be ancholed. S ~ SUppOl~ include glass, plastic, metal, polymer gels, and the like, and may take the form of beads, wells, ~ipstir~ menlb.~les, and the 15 like. ~rcsenlly plcrcllcdSuppOl~S are provided as poly~yl-,ne beads or microtiter dish wells.
As used herein, the term "target region" or "target nucleotide sequenre" refers to a probe binding region co..lained within the target ~le;l~le. The term "target sequence"
refers to a sequence with which a probe will form a stable hybrid under desired conditions.
2 0 As used herein, the term "detec~ or delclll~,i,illg" an oligonucleotide probe or disco..lill.lous probe refers to a method of se1ectirJn WLt;leill a pool of probes are assayed for a certain biological plopcl~y, such as binding to a target. The object of delell~ alion is which probe or probes c~ the best ~ ..1 le ofthe desired plopclly. For anti~P-n~e probes, the desired prope,~y will p-crclably be strong binding. Probes useful for two-stage 2 5 therapies (where a first drug is added, allowed to comr'~Y and then a second effector m~t~ ~l1e ~;t;led at the complex is added) should exhibit persistent binding, which can be assayed by using low probe concentration for binding and applying increasingly stringent washes to elute the more wealdy bound oli~on~cleotides. For ribozyme probes, the desired prope, ly will plerel~bly be fast bin~ing which can be s~lected for by using low probe 3 0 col~rp~ lions and short hybridization times.

W O 96/179~S PCTrUS9SJ157~9 _ g _ As used herein, the term "~ o~ylllc" refers to a polynucleotide that has the ability to catalyze the cleavage of a target nucleic acid ~hale. In general, a l;I,o~yl.le ofthe current ..~ ion will co~ lise~ a catalytic re~gion, and at least one ~ulu~lale binding region d~t~ P,d by the map~ g lec~ e desclil,ed herein. Ribo,yl.les are described generally inU.S. PatentNos. 5,144,019, 5,168,053, 5,180,818, 5,~s,337 and 5,254,678. Catalytic regions of l;I,o~yllles are known in the art and include h~mmPrhe~, hairpin, hep~titiq delta, and RNAse P ~rpe.
As used herein, the term "ov~lla~ ~~uenrPs" refers to two oligonucleotide sequPnr,es that share a partial c-~.. o~- se~ Pnre For ~ , the oli~omlrleotides 10 GAATTC and AATTCC overlap in their con~..o~ sequPnce AATTC. Because these twooli~omlrl~otides both derive from the seq~çnre GAATTCC, but have a starting point one nucleotide apart, the two nudeotides are said herein to overlap by one nucleotide.
As used herein, the term "discc .~ olls probe" refers to an oligonucleotide having two or more regions corresponding to two or more non-conti~lous regions of a target 15 nucleic acid. The two or more regions are covalently linked, either directly, through an intervening nucleotide sequçnr,P" or ll.lougll an organic linker molecllle As used herein, the term '~ con~ ous probe" refers to two or more oLigonucleotides covalently linked by an organic linker molecule. By "organic linker molecule" is meant an ess~nti~lly linear spacer organic mo~crllle capable of being ~tt, ~h~ at its two ends to two distinct 2 0 oligomlrl~Qtides.

IIyl lilope Mappin~
In one aspect ofthis invention, a method for ~ele~ P or delellllinillg the sequence or sequences of a target nucleic acid which co~c~ e the best target region for an 25 o1i~nllrleQtide probe is given. On the basis ofthis ;..r~ n, ideal probes for diagnostic ~letectiQn of the target, or thelapeulic moleclll.oc, such as an ~ntic~nce probe or ribozyme, may be designecl Accol dil-~; to the present ill~enlioll, the provided method comprises the steps of:
(a) ~y~ g or ol,l~l.lll~ a plurality of oligonucleotides, each of the oligonucleotides CG-~ . a first nucleolide sequence which is COII-~ --P~ y to a sequence within the known nucleotide sequP-nce, and the oli~c)mlrleQtides having v~ell~pillg first nucleotide -CA 02207011 1997-0~-23 W O96/179S5 PCTrUS95/15779 sequences wherein the first nucleotide s~,q~l~nre of each of the oligonucleotides in the plurality of oligonucleotides overlaps the first nucleotide seq~l nce of anotheroli~omlrleQtide in the plurality of oligonucleotides by from one to four nucleotides; (b) cont~ctin~ each ofthe oli~omlcleotitles w-vith the target nucleic acid under contlitione 5 p~ ;.,g specific hybri~li7~tion of oli~onllçleQtides to the target; and (c) detecting or d~ , the presence or s~bsence of specific oli~omlcleotide-target binding betw-veen each ofthe oligonucleotides and the target nucleic acid to inrlic~te whether each oli~o,~llc1eQtide binds within the known nucleotide seq~nre~ thereby dete,~ .fing or detectitlg one or more binding oligomlr,leotides.
A. Test Probe Synthesis The method of the present invention is based on the COllCept that a given targetnucleic acid mo1e ~ , especially an RNA target, has certain regions CO~ nucleotide ceq~l~ncP!e that are more readily available for probe binding than other sequences located elsewllere on the target. By syn~h~ a plurality, or pool of c~n~lid~te oli~omlcleotide 15 probes ("test probes") collespollding to ovellappi.lg se~ ofthe target nucleic acid;
il.c~ each .~ er ofthe pool with the target; and d~lt",lini~lg the amount of probe-target binding, p~c;re~ed probes are dele"l.-,lcd.
The test probe pool, or l.~,;~ope pool, GQ~ of a set of oligonucleotide probes, each of which comprises a first target-binding seqll~-n-~e which is complemrnt~ry to a 2 0 nucleic acid sequence within the known target nucleic acid sequence. The set of oli~omlcleotide probes have ovelldpp~lg nucleotide se~ ences whe~ the first target-binding seqll-n-~e of each of the oli~omlrleotides in the pool overlaps the first target-binding se~l~nre of another oli~omlr~leotifle in the pool by from one to twenty nucleotides, plere.ably from one to four nucleotides, more p-t;re ~l~ly by one nucleotide. The region 2 5 which is compl~~-nt~~ry to the known target nucleic acid sequence is generally ~om 6 to 30 nucleotides, more preferably from 8 to 24 nucleotides, still more preferably from 8 to 16 nucleotides. The test probes are p,~r~lably single stranded DNA molecnl~e, optionally col-~ g one or more phosphorothioate lin~ es or 2'-0-methyl groups.
litio~l~lly, the test probes may contain other seq~l~nres that are useful in the3 0 assay for detecting target binding. For example, the oligonucleotide probes may optionally contain a second sequence which is an adapter probe recognition sequence having a nucleic WO 96rl795~ PCTnUS95115779 acid seq~ence co"~lcmentary to a test probe ~ticog,l,lion seqllP-nc~" which is used to capture the test probe directly or illdifeclly to a solid support. This region is generally from 6 to 30 mlr~1eoti~ , more p.~re ~ly from 8 to 24 m~ oti(llo~, still more pl~r~,~bly from 8 to 16 nucleotides. The test probes may also contain a third seq~nce which is a spacer 5 seql~ence from 1 to 50 n~ eotir~e~ useful in spacing the first target-binding seqll~n~e from the second ada~er probe recogtliti~n sequ~nrç.
The test probes are created by ~"ll,e.. s of individual oligonucleotides by ~ownn~1c BacL~oul,d rer~cnc~s which relate generally to m~.tho-1~ for synthesi7in~
oli~on~ eotides include those related to S'-to-3' syntheses based on the use of ~B-1 0 cyanoell~yl phnsph~te pfOteCti~ groups, e.g, de Napoli et al., Gazz Chim Ital (1984)114:65, Rns~nth~l et al., Tetrahedron Lett (1983) 24: 1691, Belagaje and Brush, Nuc Acids Res (1977) 10:6295, in ~ ces which des~ihe solution-phase 5'-to-3' syntheses include II~l~u and Khorana, J Am Chem Soc (1957) 89:3880, Gait and Sheppard, Nuc Acids Res (1977) 4: 1135, Cramer and Koster, Angew Chem Int Ed Engl (1968) 7:473, and Blackburn etal., J Chem Soc (1967) Part C 2438.
~ d~ ;o~ y, Mt~ cci and C~ull~ , J Am Chem Soc (1981) 103:3185-91 des-cribed the use of phosphochlol;d;les in the ~l~alion of oligomlc1eQtides. Beaucage and C~ulllel~, Tetrahedron Lett (1981) 22:1859-62, and US 4,415,732 descl;l)ed the use of phosphor~m;~it~sforthepl~al;OIIofo~ om~cleotides. Smith,ABL15-24(December 2 0 1983) descl;l,es ~ o~ed solid-phase oligodeoAylil)o.~ eotide synthesis. See also the ;rellces cited therein, and Warner et al., DNA (1984) 3:401-11, the disclosures of which are incol~ ed herein by le~er~nce. T. Horn and M.S. Urdea, DNA (1986) 5:421-25 C1~Y~ e~l phssph~rylation of solid-~u~ polled DNA L~ using bis(cy~n-~etho~y)-N,N-.liisopl~""rl~ul,lnophospl-:~.e. See also, T. Horn and M.S. Urdea, Tetrahedron Lett (1986) 27:4705-08.

B. Test Probe Hybridi~lion and Detection Once the set oftest probes pool has been ~.-11,F ~;,~~1~ the individual test probes are broughl in contact with the target nucleic acid under cQn~ition~ p~ lilling specific 3 0 hybrir1i7~tion of test probe oli~om~cleotides to the target, and then dele.ll~"i,.g the amount of binding for each probe-target pair. Rt;rt t;nces which relate to hybridi7ation techniques W O96/17955 PCTrUS9S/15779 in general include the following: Me l-(th and Wahl, Anal Biochem (1984) 138:267 84 (review of hybrirl;,~l;o~- techniques); Leary et al., Proc Natl Acad Sci USA (1983) 80:4045-49 (bioLIlylaled DNA in conj~ n with avidin-enzyme conj~ t~s for tletectinn of specific olignnur1~otide seq~pncçs); Ranki et al., Gene 21 :77-85 (sandwich hybri~ l;ol- assay). Pfeuffer and Helmrich, J Biol Chem (1975) 250:867-76 described the co~,pl;~ of ~l~no~in~-sl-o-(3-thi~ phosyh~e) to Sepharose g) 4B. R~llm~n et al., J
Histochem and Cylocl-~,.,l 29:227-37 desc il,ed the 3'-1~belin~ of RNA with fluort;sce- ~.
PCT Applir.,l ;OI- WO/8302277 ~les~ ~ il,~l the ~ldition to DNA fr~nPnts of modified ribon--rleoti~e~ for l~ .ling and m~.thotlc for analyzing such DNA fr~nPnt~ Renz and 1 0 Kurz, Nuc Acids Res (1984) 12:3435 44, desc,il ed the covalent linking of enzymes to oligcm~cleotides. WaUace, DNA Reco~ Technology (Woo, S., ed.) CRC Press, Boca Raton, Florida, provided a general bac~roulld of the use of probes in diagnosis.
Chou and Merigan, N Eng J of Med 308:921-25, ~lesrribed the use of a radioisotope-labeled probe for the deleçl;QI~ of CMV. Inman, Meth Enzymol (1974) 34B, 24:77-102, 1 5 des~;,il,ed procedures for linking to polyacrylamides, while Parilch et al., Meth Enzymol (1974) 34B, 24:77-102, descri~ed cU.,p~ nc with agalose. Alwine et al., Proc Natl Acad Sci USA (1977) 74:5350-54 des~;,il,ed a method of Ir~lsr~ling oligonucleotides ~om gels to a solid SllyyGll for hybri~li7~tio~ Chu et al., Proc Natl Acad Sci USA
11 :6513-29, des~il;l,ed a te~ e for derivatizing tt;lllul al nucleotides. Ho et al., Bio-~ y(1981)20:64-67des~ edderivatizingtçnn~ nucleotidesthrough phosphate to form esters. Ashley and MacDonald, Anal Biochem (1984) 140:95-103 r~o,led a method for yley~uing probes from a surface-bound templ~te. Hebert and Gravel, Can J Chem (1974) 52:187-89 and R~il~ ;in et al., Tetrahedron Lett, (1975) 17: 1445-48 desc,;l,ed the use of nll, ophe.lyl-co~ g colllyounds as light-sensitive 2 5 pl~,le~;~ing groups. K. Groegke et al., Helvetica Chimica Acta (1990) 73 :608-617 disclosed the use of t-butyldilllelhylsilyl to protect a L~uxyl functionality.
In a yl~rc;lled embodiment ofthis invention, test probe hybridi_ation and detection ispP.rull..P~d bymo~lifil;~tionofmp~tho~lc ~li~loseA inU.S. PatentNos. 4,868,105 and 5,124,246 to Urdea et al. Such solution phase sandwich hybri~i7~fi~ n assays are performed 3 0 generally as follows and as shown in Figure 1. Ln each assay, single-stranded target nucleic acid is ;~ ed under hybril1i7~tion con~lifions with an excess of two single-stranded W O 9611795~ PCT~US9SJ1~779 nucleic acid probes: (l) a test probe as described above, having a first binding nucleotide seq~1P-nce complPmPnt~ry to the target and a second binding seq~1ençe that is an adapter probe reco nhion sequence; and (2) an adapter probe having a first binding sequence that is a test probe recognition seqUp-nr~e complt"~nl~Jly to the adapter probe recognition 5 sequ~nce on the test probe, a second binding se~l-ie~r,e that is capable of specific L,~ ol- to an o1ipo~ r~leoti~e bound to a solid phase, and optionally co.~ u a spacer seql~çnce of l to 50 nucleotides. The re~s~11ting product is a three compollent nucleic acid cc ~'e~ of the test probe sandwiched ~L~,~,en the target and the adapter probe. The second binding seq1nPnr~e and the optional spacer seq~enre ofthe adapter probe remains as 10 a single-stranded tail.
Under hybri~i7in~ con~itiQnc~ this c~ . ' is then added to (or inr,ub~tecl ~im111t~n~ously with) a solid phase having a capture probe bound to it. The capture probe is a single-stranded o~ on11r1eQtide that is a~lb~ ;Ally CGIllple~ y to the second binding seq~1Pnre ofthe adapter probe. The rçs~1tirl~ product comprises the conlplr-15 bound to the solid phase via the duplex formed by the capture probe bound to the solidphase and the second binding sequPnre of the adapter probe. The solid phase with bound cc ~I~Y is then sep~led from unhol~n~ materials by washing.
The binding seq~1Pnre of the adapter probe that is a test probe recognition sequence s~bs~ 1y compl~ .l; -y to the adapter probe l~co~,-lioll sequPnre on the test probe 2 0 will be of at least 5 ..~.cleolidec, usually at least lO nucleotides, and not more than about 20 nucleotides. It will typically be ~p~v~ e1y lO nudeotides.
The second binding sequence of the adapter probe is se1 ~Pcte~ to be :iub~ lly complt,,,c:nl,~y to the oligrom1r1eotide ~tt~rhe(l to the solid phase and so as to not be e"counleled by endogenous se~lu~ r,P,s in the target. The second binding sequence may be 2 5 conti~1ous to the first binding sequence or be spaced ll.erer om by an int~rmerli~te nol1ço...l le~nPnt~ry spacer sequence of ~clween O to 50 nucleotides. The use of a short spacer region (or none at all) of l to lO nucleotides, hinders P~YrtPn~ion ofthe test probe into the interior of target nucleic acid .nole,~ es when bound to the solid s,lppO, L through the adapter probe, and is thus prerer~ble when ~ g test probes that bind only to 3 0 external s~1rf~ces of target molecu1es The probe may include other noncomplPm~nt~ry sequPnçes if desired. These nonco--.?l~r~ y sequ~Pnçes must not hinder the binding of W O96/1795~ PCTrUS9S/15779 the bin&g sequence or cause l~r,l-ep~;l~c binding to occur. The adapter probe may be prcp~ed by oli~on-~cleotide synthesis procedures or by cl~nin~ preferably the former.
The solid phase that is used in the assay may be particulate or be the solid wall surface of any of a variety of co~ , e.g., centrifilgal tubes, columns, microtiter plate 5 wells, filters, tubing, etc. When particles are used, they will preferably be of a size in the range of about 0.4 to 200 microns, more usually from about 0.8 to 4.0 microns. The particles may be any co~ cll,cllL material, such as latex, or glass. Microtiter plates are a prcrcl~cd solid sllrf~cP~. The oligQnur,leQtide that is ~ ;A11Y co,--~,lç~ .y to the second bindin~g seqUPnre of the adapter probe may be stably ~tt~chP~l to the solid surface 10 lh-~ u~l r....~ ~;o,-~l groups by known procedures.
It will be al)plcc;aLed that one can replace the second binding sequence of the adapter probe and the oligollurloQtide ~tt~chPd to the solid phase with an a~p-upliaLe ligand-recel.lor pair that will form a stable bond joining the solid phase to the first binding sequ~nce ofthe capture probe. rY -..I lP,s of such pairs are biotin/avidin, 15 Ihy~o~e/llly~ le-binding globulin, antigent~llil,o-ly, carbohydrate/lectin, and the like.
The ratio of adapter probe and test probe to %~ e~ moles of target will each be at least stoichiometric and prerclably in excess. This ratio is pr~;relably at least about 1.5:1, and more l~lcrel~bly at least 2:1. It will normally be in the range of 2:1 to 106:1.
Conce..L.~lions of each of the probes will generally range from about 10-9 to 1 o-2 M, with 2 0 sample nucleic acid conr~-.l ~ ~lions varying from 10-1~ to 10 8 M. The hybridization steps of the assay will generally take from about 10 minllte~ to 20 hours, frequently being completed in about 1 hour. Hybrid;~l;QI~ can be carried out at a mildly elevatedLcmpe~ re, generally in the range from about 20~C to 80~C, more usually from about 35~C to 70~C, particularly 65~C.
The hybridization reaction is usually done in an aqueous m~ m, particularly a buffered aqueous ..~edi~.... which may include various additives. Additives which may be employed include low c~nr~ntrations of deler~,c,ll (0.1 to 1%), salts, e.g., sodium citrate (0.017 to 0.17 M~, Ficoll, poly~ ~lloli~Qne~ carrier nudeic acids, carrier proteins, etc.
Non~qlleQus solvents may be added to the ~ueol~e ".~.1;..." such as di-ll~lhylr~ ...ide7 3 0 dimethyl.c -lfoxitle, alcohols, and fc" ? de These other solvents are generally present in ~mrlmt.e ranging from 2 to 50%.

WO 9611795~ PCTJU2,95)15779 The stringency ofthe hybri~li7~ti~n ...sdi~.... may be controlled by telllpeldLule, salt collc~ltlalion~ solvent system, and the like. Thus, dep~ P upon the length and nature of the se.~ e of interest, the s~ / will be varied. Frt;r~,.ed hybrirli7~tion conditions, a~ro,~ physiological cnn-liti~n~ are 37~C, 0.15 M monovalent cation, 16 rnM
5 Mg~, and 1 mM~ e The ~.uce.lule used in the s~alion steps ofthe assay will vary depending upon the nature ofthe solid phase. For ~a,lic,~s, c~ ;r.~ ;on or filtration will provide for separation ofthe particles, disG~ the sllpe~ or i~o1~ti~ the supe.llalalll. Where the particles are assayed, the p~ l;cles will be washed thoroughly, usually from one to five 1 0 times, with an ~p~Vl upli~le luu~el'ed l-~ e.g., pht s~hq~e l,uLrel ~d saline ~PBS) c~ Ai..i~ a delelgell~ such as SDS. When the S~ala~iOII means is a wall or support, the supelll~talll may be isolated or discarded and the wall washed in the same manner as int~ te~1 for the particles.
The co ~Y is then tletected by labelin~ ofthe target in the bound cnmpl The 15 target may be labeled before or after Coll"~l~" rO~ ,.I;nn The labeled target will include one or more molecules ("labels"), which directly or indirectly provide for a detect~hle signal.
The labels may be bound to individual ll,e"ll~el~ of the target seql~nce or may be present as a t~min~l member or t~min~l tail having a plurality of labels. Various means for providing labels bound to the seq~lence have been l~olled in the liLel~lule. See, for example, Leary 2 0 et al., Proc Natl Acad Sci USA (1983) 80:4045; Renz and Kur7 Nucl Acids Res (1984) 12:3435; Richardson and Gumport, Nucl Acids Res (1983) 11:6167; Srnith et al., Nucl Acids Res (198~) 13:2399; Meinkoth and Wahl, Anal Biochem (1984) 138:267. The labels may be bound either covalently or non-covalently to the comp'~ y sequence. Labels which may be employed include rn~ionllrli~3çc, fluoresce ~, r~ ---..;nPSc~ ~, dyes, 25 en,~ll.es, el~yl--c subs~,~tes, enzyme cof~ctQrs, enzyme inhibitors, enzyme subunits, metal ions, and the like. Illu~lr~live specific labels indude fluoresc~in, rhod~mine, Texas red, phycoelylh~,n, umbelliferone, ll~minol, NADP~ a-B-galactosidase, horseradish peroxidase, sl1k~1ine phosph~t~e, etc. In a p~ere -ed embo~lim~nt~ the target sequence is labeled by inco.~ol~liol~ of biotin-UTP for a fraction ofthe natural rUTP or dTTP nucleotides, and 3 0 then il~ ub~ed ~,-vith strepta-vidin-conjllg~tP~ ~ 1ine phnsl,h~ e (AP).

W O96/17955 PCTrUS9511~779 Depending upon the nature of the label, various teçhniqlles can be employed for delev~ ) the p-es~,.lc~ ofthe label. For fluofcsce ~, a large number of dilrt;re..L
fluololl-etel~ are available. For thPmil~ esce~ GI~ or films are available.
With c.~yllles, a fluolésce.ll, rhP~ni~ esc~ or colored product can be provided and d~le~ -.ecl fluorolnéLI;cally~ lll.. ;n~.. l.ically, spe~lluphotolllcL~ically or visually. The various labels which have been ell~luyed in immlm- ~cs~ys and the techniques applicable to as.~ can be employed with the subject assays.
After all hybridization assays have been ~ ru. ~~ .ed with each l-lel~ll)er of the test probe pool, the ~--~l-b~ ~ that intlic~te the highest degree of binding to the target are 10 dele~ ...i~lPd to be the best c~ndid~tP~ probes. Altti..-~ ly, the method is used to identify the fastest hybridizers (by using short in~b~tion periods and low concellLlalion of probes) or most persistent hybridizers (by using low collcellllalion of probes, and increasingly 5~ wash con-litioll~e).

C. TestProbeSi_eDete,.. l."alion Once a test probe has been ide~ e~l by the above nlethot1e to be among the best ç~n~lid~tee~ further c,.~ .. lle,ll~ may be p~ ul~lled to dele.. ~e the ideal size for the first nucleotide sequence ofthe probe complelllel~laly to the target seqUp~nre A pool of seco~ y test probes are synth~ei7Pd by ~ dal.l methods, having a sequPnce 2 0 cc pl - - ~ to the same region of the target nucleic acid and an identic~l first end as the c~n~ te test probe, but each .~ ..bP,r ofthe secon~lh~y test probe pool has a di~el~nL
second end, as shown in Figure 2. Thus, a typical second~y test probe pool has a first nucleotide seq~lPn~e that ranges in size from about 6 to 50, more preferably from about 6 to 18 nudeotides, sp~n~ e the region ofthe target nucleic acid i~lentified by the best 2 5 c~n~ te test probe or probes. The second~ ~ test probes are then screened for the highest degree of binding to the target as above.

Disco--l;...lous Probes The result of h~ Gpe Illap~ing as desc~ ed above is the identific~tiQn of two or~ 0 more c~n~ te probes of ide~li7~1 seq~lence and length. In another aspect of this nlion, two or more of the c~nr1id~te probes are joined covalently together either W O96/17955 ~CTnUS95tlS779 directly or with organic linkers of varying length and composition, and then assayed as above to detP~nin~ those that have the highest degree of binding to the target.
The ~;eCO~ Q11C probes are con~l-u.;led as the test probes l~ opes above, optionally ccs..l~in;..p a region having a nucleic acid s~ enre that is an adapter probe l eco~. ~ se~ e and/or a spacer region as defined above, and also CO~ two or more ~ m)~,leotides covalently joined directly or by an organic spacer -Z-, wherein -Z- is -y3 to the oligoml~leotide lll. ou~l. a sugar, base or phosph~te moiety, and is selected from the group con~ .g of arylene, C6-C~8 arallylene, C6-C~8 aralkenylene, C6-CI8 arallynylene, C~-C30 alkylene, Cl-C30 aLtcenylene, or C~-C30 aLkynylene, co~ g 0 to 6 helcl ~Ja~ s s~lected from the group co~ of O, S, N, Si and Se and 0 to 6 linkages s~ e~l ~om the group con~ . of-CO-, -COO-, -CONH-, -NHCO-, -S-S-, -SO2-, -CH(OH)-CH(OH)-, -CH(ORI)-CH(OR')-, -O-PO(O~)-O-, -O-PO~R')-, -O-PO(OR')-O-, -O-PO(ORI)-R2-, -P~:)(ORl)-O-R2 ~Lc~ Rl is lower alkyl and R2 is lower allylene, -O-~CH2)n-A-)~ and ~ ~NN3> \
~)~ J~

2 0 wherein -A- is -O- or -PO4-, n is an integer from 2 to 4 and m is an integer from 1 to 20.
~ ert;lled embod;-..f .Is of organic linlcers are "rigid linkers" which are linkers that are limited in degree of flPYihility. Rigid linkers are known in the art, and include linkers CQ..lAi.~ P one or more double or triple bonds, or ring structures, inr.lll(linf~ substituted or ~.c~ s~ led a~ylene, C6-CI8 aralkylene, C6-Cls aralkenylene, C6-C~s aralkynylene, Cl-C30 2 5 alkenylene, or C,-C30 alkynylene.
A pool of ~licco-~l ;- .- ~Q~S probes is then constructed sP1ectin,~ di~ere,lL co,-,b~laLions ofthe best cA~ ;d~le test probes and varying lengths and types of linker. For example, if L~,iLop.; n,apping idPntifiP~ four c~n~1id~te test probe oli~om~ 1eotides A, B, C and D, then dual ~nked ~icco~ lJo~s test probes may be constructed as A-B, A-C, A-D,B-C,B-D and 3 0 C-D. Tri-linked ~iccol~ olls test probes might also be constructed as A-B-C, A-C-B, B-A-C, A-B-D, A-D-B, etc., as shown in Figure 2. The identity of the tethers might also be CA 022070ll l997-05-23 W O96/17955 PCTrUS95/15779 varied. Prtire,l~d tether variations are series of ",ono"~c.~ joined together in various len~h~ For c..a"~ple, one tether series is tnethylene glycol, hpy~ethyleneg n9n~ethylene glycol. Another series is o~ ~~ 2 o~ 3 In another embodiment ofthis invention, ~liQGo,.~ o~e probes may be formed by rlling three or more c~n~ te test probe sequ~nc~s in a branched structure, using the b~clled DNA terhnology as set forth in U.S. Patent No. 5,124,246 to Urdea et al. In the "bl~u,ched diCcc~ olls probes" folmed by this teet nology, two c~n~id~te test probe sequences are joined by an organic linker ~lecl~le incl~ in~ a bl~ncl~ g monomer as desc,il,ed in the '246 patent, which pennits the joining of a third can~ te. test probe sequence. An example of a b,al~ched ",ono",~l having three ~tt~rllmP-nf points is ~~

N~O
'0~

o~

The constlucted pool of ~licco~ o~s probes is then screened as above for those with highest binding to the target nucleic acid. The ~licco..1;..-lous probe ~,vith the highest degree of binding is then del~",L~ed to be the best c~n~ te probe. Alternatively, the 3 0 method is used to identify the fastest hybridizers (by using short inc~lb~tion periods and low -W O 96/17955 PCTnUS95115779 Co~ Lion of probes) or most pe,~.sle ~L hybridizers (by using low concentration of probes, and il,e~ ",gly ~ gell~ wash con-litiQne).

5 Use of Discoll~ ous Probes Di3co~ ous probes may be used as t~ nnstic probes, as desclil~ed for example in U.S. Patent Nos. 4,868,105 and 5,124,246 to Urdea et al.
Di~co..l;...lous probes may also be form~ ted as ~ ç~e probes or riboz~nnes. As e probes, the discolllllluous probes may be modified. Dicco..~ ous ~nti~n~e 10 probes will not generally co~ e a region having a nucleic acid sequence that is an adapter probe recognition seq~ence or a spacer re~gion as desclibed above. Additionally, the ~ P-~ce nucleic acid ofthis invention is RNA, DNA or a modified nucleic acid.
F~ es, wi~lloU~ limit~tiQn, of modified nucleic acids are degradation~ L sulfurized and thiophosph~te derivatives of nucleic acids, and polynucleoside amides (PCT
Pu~ tion No. WO91/1633 1 to Stec et al.; PCT Pllbli~ tion No. W088/07~44 to Zon et al.; P.E. Nelsen, et al., Science (1991) 254:1497-1500; M. F.~holm, JACS, (1992)114:1895-1897). Pa~ ally prertll ~d design modifi~tionc ofthe ~ntie~n~e nucleic acids ofthis invention are modifications that are cleeignP,d to: (1) increase the intr~cP~ r stability of the nucleic acid; (2) in-;lt;ase the cellular 1)~ ility of the nuc1eic acid; (3) 2 0 increase the affinity of the nucleic acid for the sense strand, or (4) decrease the to~icity (if any) ofthe nucleic acid. Many such motlifil-~tinnc are known in the art, as described in ANTISENSE RESEARCH AND APPLICATIONS (S.T. Crooke and B. Lebleu, eds., CRC Press, 1993). Thus, the nucleic acids may contain altered or motlified bases, sugars or linkages, be delivered in ~pe~;~li7ed sy~l~ "s such as liposo...es~ or may have ~tt~ched 25 oie~ies Such ~ hed moieties include Lydlol)hob c moieties such as lipids that enhance interaction with cell n~ es, or polycationic .~.oief ;es such as polylysine that act as charge neutralizers ofthe phosl,hale backbone. Particularly p,t;re,led lipids that may be Rtt~h~-d are c~ol~sttorols. The moieties may be Rtt~ d at the 3' or 5' ends ofthe nucleic acids, and also may be ~ d through a base, sugar, or interm~leoside linkage.
3 0 Other moieties may be cappillg groups ~pe~ ;I;cally placed at the 3' or 5' ends of the nucleic acids to prevent e~-n~rle~e degradation. Such capping groups include, but are CA 02207011 1997-0=7-23 wo 96/17955 Pcr/uSg5/15779 not limited to, 11YdI~)AYI pro~e.~ groups known in the art, inrlu-ling glycols such as polyethylene glycols, tetraethylene glycol and the lilce.

A.l.n;.... ....~ Lion The ~ ;e~"~se or ribozyme con,poul-ds of the invention may be Rtlmini~tçred by avariety of methods~ such as intravenously, orally, il~ c~ rly, i~ peliloneally, bron-chially, intranasally, and so forth. The prerell~,d route of ,A,t~ lion will depend upon the nature of the co.ll~uund and the cQn~l l ;OIl to be treated. Compounds may be adminis-tered orally if well absoll~ed and not sul,sl~ ;Ally degraded upon ingestion. The com-pounds may be ~ l-td as phal~7ceutir~l comrositi~ns in co.ll~inallion with a phar-m~ce~t;~.Ally acceptable excipient. Such compositions may be aqueous solutions, emul-sions, creams, o;~ .le, sl~Cl~ç~ ne, gels, liposomal suspPnQione, and the lilce. Thus, suit-able P,YciriPnte include water, saline, Ringer's solution, dcAllose solution, and solutions of eth~nnl, glucose, sucrose, dextran, m~nnnse, ...~ ol; sorbitol, polyethylene glycol (PEG), 15 phosphate, acet~t~, gelatin, collagen, Carbopol~), vegetable oils, and the like. One may additionally include suitable prw~,l valives, stabilizers, ~ntio~cirl~nte, ~ntimi~robials~ and buf-fering agents, for cAa~ !c, BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like.
Cream or o;.~ bases useful in formulation include lanolin, Silvadene~ (Marion), Aquaphor~) ~Duke Laboratories), and the 1ilce. Other topical form~ tions include aerosols, 20 b~n~ çe s~let~inqd-releasep~t~.hPe,andthelike. ~h~ tively,onemayincorporateoren~ps~ te the compound in a suitable polymer matrix or ,.~enl~ e, thus providing a sus-tained-release delivery device suitable for ;.n~ ;on near the site to be treated locally.
Other devices include indwelling c..~ and devices such as the Alzet~ minipump. Fur-ther, one may provide the compound in solid form, qspec;~lly as a Iyophili7ed powder.
2 5 Lyophilized form~ tions typically contain stabilizing and bulking agents, for ~x~mple human semm albumin, sucrose, ...Anl-;lnl, and the like. A thorough tli~c~s~ion of pharma-ce ~ti-~lly acceptable eA r ~ t~ is available in l~min~Qn's Pharm~celltical Sciences ~Mack Pub. Co.).
The ~nti~en~e compositions of the present invention may be prepared for 30 ph~rm~ ltic~l ~dminictration. Injection prtp~alions and suppositories may usually contain l-l0 mg ofthe nucleic acid or nucleic acid analog per ampoule or capsule. For wo 96/l7s5s PCTIUS9511S779 human p~ , the daily dose is about 0.1-1,000 mg, preferably 1-100 mg (from 10-20 mg~g to lO00-2000 mg/lcg body weight). However, the particular dose for each patient dc~ on a wide range offactors, for e-~..rle, on the e~ ,ness ofthe particular nucleic acid or nucleic acid analog used, on the age, weight, general state of 5 health, sex, on the diet, on the time and mode of a~ ..lion, on the rate of e~ l ;on, con.l)ina1ion with other meslic~ jointly used and St;v~liLy of the p~ li~ ar ~ e~S to which the lLt;~ y is applied.
Ph~rm~ce~ltic~l articles of msmlf~lre, withtn the scope of the present invention, include articles whelein the active il1~edie111s thereofare co"l~;..ed in an ~c~ive; amount 10 to achieve its int~n~e(l purpose. A pr~rt "ed range has been described above, and d~ iotl of the most GlIt~,livG ~ ou~ for 1~ .l is within the sldll of the art.
In ~.l.lition to the nucleic acid and their sulfi~ri~ed and pho~horol}lioated analogs of the present invention, pharm~c.o,uti~ 1c~ l;ons may contain suitable eYciri~nt~ and ~I-Yili~ries which f~ itate~l~ce~ , ofthe active conl~ou"ds. The ~ lions, 15 particularly those which can be a~ n ~ ed orally and which can be used for the ple~ d ~pe of r~ alion~ such as tablets, dragees, and c~ps~ and pr~lions which can be ~ Q~ ed rectally, such as supposi~G,ies, as well as s~lit~hle solutions for alion pa~ e1~lly or orally, and co,11posi1ions which may be ~rlminietçred bucally or ~ gually, may contain from 0. l to 99% by weight of active ingredients, together with 20 the; .--~t Ap1~;re.,ed mf.tho~l of, 1."~ ionisp~c;~ 1,especiallyintravenous a~ ali~
.el.it~bl~ formll1~tione for p~t;"1~,al ~ lion include aqueous solutions ofthe active co111l~ounds in water-soluble or water-dis~ ilJle form. In addition, suspensions of the active comro~ln~ls as app,.,p1;a1e oily injection s~pe~.~'ol-C may be ~1ministered.
2 5 .e~ le lipophilic solvents or vehicles include fatty oils, for e.~ pl~, sesame oil, or ~y-1tL~1ic fat~ acid esters, for ~", ple, ethylnle~te or triglycerides. Aqueous injection ~usp~ o~c may contain s~bs~ ce-e which ."~ se the viscos;ly of the ~lsl,e1,~ion, for example, sodium carbo~."~;1Lyl cçll..l~sç, sorbitol, and/or dextran. Optionally, the s--sr~ ;ol- may also contain stabilizers.
~ 1itir~n~11y, the cG.. ,pounds ofthe present invention may also be ~(lmin;etçred ~1 in li~,oso111es, ph~ ce~tir.~l compositions whe1eill the active ingredient is W O96/17955 PCTrUS95/15779 ~ ed either dispersed or variously present in ~-l~uscles con~i~ing of aqueous concP,ntric layers adherent to lipidic layers. The active ingl~lie~ depending upon its so1~lbility, may be present both in the ~(lueo~ls layer and in the lipidic layer, or in what is generally termed a liposomic i,..~.~;on. The l.~.l.opho~ic layer, generally but not 5 exclusively, co..-~.ises phosphnlipids such as lecithin and ~hingomyelin, steroids such as cholesterol, more or less ionic sll~ct~nt~ such as dicelyll-hosyh-~e, stearylamine, or phosl h,.~ ; acid, and/or other materials of a l-~l.l-o~ho~;ç nature. The ~ el~ ~ of the liposomes generally range from about 15 nm to 5 microns. Particu1arly p,t;rel.t;d lipids for the y~ ion of liyoso-lles and/or lipid ~U~yPl~c on~ are DOTMA and DOTAP.
1 0 DOTAP is con~lereially available from Boek~ gri l~nnh~P.im, or may be prepared following the methods described by L. St~m~t~tos et al., Biochem 27:3917-25 (1988); H.
Eibl et al., Biophys Chem 10:261-71 (1979). DOTMA is co....ne~ ~.ially available under the name Lipofectin* (available from BRL, C~ilhel ~u~, MD), and is described by P.L.Felgner et al. Proc Nat Acad Sci USA 84:7413-17 (1987).
The present invention will now be iil~ led by IGr~ ~e to the following eA~Il~lcswhich set forth particularly adv~nt~g~oo~ embo~lim~ntc However, it should be noted that these embocl;...r~ are illustrative and are not to be construed as restricting the invention in any way.
F.~ le ~
EA~..~:,Ie 1 IIy~,ilope Synthesis A h~rl,-ilope probe pool was generated corresponding to a 113-nucleotide 2 5 region in the Rev l~ onse, PlPmPnt ~RRE) of the sf 2 isolate of the human immlmo-dPfiei~P,tl~y virus ~Hlv)g~nome. This region inel~ldes nucleotides 7769-7881 in the ~V s~~
gell~Jllle, and has the sequence (nt 7769) 5'-ATA GTA GGA GCT ATG TTC CTT GGG
TTC TTG GGA GCA GCA GGA AGC ACT ATG GGC GCA GTG TCA TTG ACG
CTG ACG GTA CAG GCC AGA CAA TTA TTG TCT GGT ATA GTG CAA CA-3' (nt 3 0 7881). The pool comprised 96 deox~mbonucleotide probes, each probe being a 28-mer.
The 5' portion of each probe in the pool was idenSi~l and consisted of a 10-mer having the wo 96/l7955 PCTlUS95115779 seq1~nce 5'-CCG ACG GAC C-3'. This GC rich sequent e hybridizes to an adapter probe which is used in binding to a solid ~ul)yOl L.. The 3' 1 8-mer portion of each probe hybAdizes to a portion of the HIV RRE, each portion o~/e,l~ppil.g ~e seql~enr.e of another probe in the pool by one nucleotide. Probe 1, for . rlc has a 3' portion that hybridizes to nucleotides 7769-7786, probe 2 has a 3' portion that hybridizes to nucleotides 7770-7787, and so on, through probe 96, which has a 3' portion that hybridizes to nucleotides 7864-7881. The probe pool was ~ ed by ~L~Id~.l pho~yhol~llidite c~ -r by Genosys Biotechnr logies, Inc. (The Woodlands, 1~.

lC~ le 2 IIyl,liloi~e Mappin~e of the HrV RRE Re~ion A. Probe SY1ILII~;;S
A llybliLope probe pool Collesl,ol,d,ng to a 113 nucleotide region ofthe HIV RREregion was synth~oci7çd as described in Example 1. Adapter probes having the seq~l~n~e 5'-15 G GTC CGT CGG -(N)54-CT CTT GGA AAG AAA GT-3' were also ~"LI,es,~ed using standard methods. The 5' end ofthe adapter probe hybri-li7es to the llyl~lilope probes, the 3' end hybri~i7çs to a capture probe bound to a solid support, and the rniddle is a 54 random base spacer region. The capture probe having the seqll~nce 5'-CA CTT CAC TTT
CTT TCC AAG AG-3' was ~..11-f C ed and bound to - .,LLer plate wells at the 3 ' end.
B. Target Labeling DNA plasmid pRKR~l OS col-l Ail~ a 9.2 kb copy of the geno",e of the HXB2 strain of HIV lacking the LTR region. rRKR~los was cut with Sac I, and full length RNA ~ scli~Ls ofthe ~enome were made using T7 RNA polymerase and the 4 rNl[Ps, inco~yola~illg 1 bioLi"ylaled UTP for evely 30 UTPs, yielding app~v~ ly 75 bioL"~laled l~l~s per full length Ll~5c~iylS. The biotinylated nucleotides are capable of fo....;.-~ a conjugate with alkR1ine phosph~t~e linked to sL,c~ vidin.

C. Mapping Procedure The 96 I,~ ope probe pool of r~.. p~ 1 was used to map the HIV RRE region, using labêled target as ~ed in FY~ Ie 2-B, and 96-well microtiter plate coated with CA 022070ll l997-05-23 W O96/17955 -24- PCTrUS95/15779 the capture probes as ~ ed in F~y~mple 2-A ~tt~rhPA to each well. Target (10 ~ ..~c....ol~s), 500 r~....~o.~.oles of each of the test probes, and 1 picomole adapter probe were sep~alely equilibrated in hybrid; ~ ;Qn buffer (0.1 M NaCI, 0.05 M KOAc, 16 rnM
MgCI2, 1 ~ spermidine, 50 n~ Tris-HCl pH 7.5, 0.2% acetylated BSA) at 37~C for 15 hour. Target and probes were mixed and allowed to equilibrate for an additional hour.
The wells were washed three times with wash P (0.5 x SSC, 0.1% SDS).
Sllc~l~vidin-conj~ ted ~ ine pho~h~ 10,000 t~ fic-n) was added and in~ l~b~e~l for S minllteS at 37~C. Three additional washes with wash P was followed with three washes of 0.5 x SSC.
1 0 T llmiphos-plus'Y (~ migen, So~lthfip~ MI) was then added to the dry wells at 50 ,uVwell, inc~lb~ted at 37~C for 30 ...;..~es and l~minescent light counts dele~ cl The results of this assay on the 96 probe pool are shown in Figure 3. Four Lrl,li~opes, labeled B6, C6, D7 and F8 were selr~led as the best binding 18mers. The HIV RRE regions cQmp~ y to these probes have the 5' end 7787, 7798, 7811 and 7837 respectively.

F,~;....l)le 3 Hybritope Size O~ ;
A. Hybridization Analysis Three ofthe s~lected l~lilol)es in Example 2, B6 (7787), C6 (7798) and D7 2 0 (7811) were scrcened for size o~ ;on. (nU~ Jel S in parPntlles~c intii~te 5' end of comrl~n.e~ ..y HIV RRE region) For each s~le~led (plilll~y) hybritope, a set of sec~n(l~-y hyl~ opes was ~"ll~e~ Each secol-d~y h~ ope contained the sarne 5' GC-rich region as the plilll~Uy hyblilope desc.il,ed in E~ le 1, and conl~led a 3' region hybri-li7.ing to the same region ofthe ~V ME as the ~lilll~ hybritope, beg~ ;..E at the 2 5 same 5' end, but vatying in length from 6-23 nucleotides. These secondary hybritope pools were scree.-ed against HIV RRE target as desc-il,ed in F~ e 2, but parallel screenings were p~,l~ll.led at 37~C and at 53~C. The results are shown in Figure 4. At physiological conr1ition~ optimal hybritope lengths appear to be around 12 nucleotides, while the optimal length at 53~C is around 20 nucleotides. si~.;r.c~ leteGt~ble binding of h~ opes of 10 3 0 nucleotides or less occurs at physiological te~ )el~lur~s~intlic~tin~ a potentially significant W O96/17955 -25- PCT~US9S/15779 source of non-specific hybridization if such lengths were used. Accoldill~ly, RNAse H
analysis was pelÇJ.llled on the shorter secon~ryL~,;lopes.
B. RNAse H Analysis A 205-mer HIV RRE RNA .l~ s.,.;pL was made from pBKBHlOS using the Promega Riboprobe II Core System, incolllol~ling 32P-GTP at a specific activity of 8 x 106 cpm/pmol. The lllilliLI~l~cli~L was run over a Nensorb column and eluted in 50% ethanol, ~-~~ lç~1 r~s..~el-~ledinwaterandstoredfrozen.
EachRNaseHreactionco..~ined 105cpmof~ S~ t,pl~ c~l)a~edat37~C
for one hour in 20 1ll buffer co..~ 50 rnM Tris pH 7.5, 16 mM MgCl2, 1 mM
spPrm~ nP~50 rnM KOAc, 0.1 M NaCl and 1 unit inhibitase (nllclf~z~e inhibitor).
Individual secondary hy~ opes were added at 0.5 ~M and il~ b~(ed at 37~C for one hour.
10 units of RNase H was added to each reaction and incub~ted for 30 mimltf,s 5 Ill of stop buffer (5% SDS, 250 ng/lll pluleinasc; K, 75 m M EDTA) was added to each reaction and ;..c~ ed at 65~C for 15 ...;~ s Cleavage e~icif ncy was c~lc~ te~ from the Ambis scan ofthe product run out on a 5% df---z~ -P polyacrylarnide gel. The results, shown in Figure 5, dPmc)n~trate that ~ignifi~.~nt hybridi~lion of 6- and 8-mers occur at 37~C, but optimal probe selection will perrnit lower probe col ce~ liOllS.

2 0 D;SCOII~ 1QUS Probes Several ofthe secondaly h~il,lilopes analyzed in F~; ...l le 3 were used as the basis for a dieco~ ous probe design as SCh zl i~ed in Figure 6. A 37-mer region of the HlV
RRE was studied using secondary L~rl,lilol~es of varying lengths collesponding to B6, C6 and D7, either joined contin~o--ely or with a 2 5 -o-(CH2CH2-O-CH2CH2-O-CH2CH2-0-)2 spacer (NZ_z spacer") where there are intervening UIlL~ ~led nucleotides as set forth in the chart in Figure 7. For ~,Y~mple, probe #1 in Figure 7 is a cQ~tim~ous probe which hybridizes to nucleotides 1-2S of the target shown in Figure 6. Probe #8 is a ~iecontinl~ous probe CO~ three oligonucleotides colre~ûndillg to nucleotides 1-7, 14-23 and 26-3 0 34, each se~~ joined by a Z-Z spacer. The disco.lLI.l.lous probes also include the 5' GC
rich region described in Example lA. The results of the use of -iiec~ ;. ..-Qus probes in the =
CA 02207011 1997-0~-23 W O96/17955 PCTrUS95/15779 assay of F~mrle 2 are shown in Figure 8. The results ;..~ ,A~e that continuous probes with no spacers p~;lr~lllled poorly, while probes ~ CC...I;-.-~Q11~ with 4-6 nucleotide spacing between hybridiz;ing regions showed higher hybri~ ioll effi~;~ries than continuous probes with the same number of nucleotides.
Example S
Use of Hyl"ilope Mapping to Find Lead Com~uu"ds With anti-HIV Activity A. Transient Transfection Assay HeLa cells were plated in six-well plates. The culture ~ 'A~ were removed the next day, and mP~tillm cc,..lA;.~;n~ either 0.2, 0.4, 0.6, or 1 mM of the phosphorothioate oligomers (16-mers) des~rihed in Section S-B below was added todllpli~tP- wells. C~1ci~lm phosphate-p~ A~rd HIV-1 provi~al (and carrier) DNA was then added to each of the wells. The following day, the cells were washed, and fresh 15 me~illm co.,l;~ g the a~ ia~ oligomer was added. The culture m~dinm was harvested three days after the ini*~Ation of tr~n~fection, and the levels of HIV-l p24 de~ ~ed. The HIV-l p24 levels were co,l,pa~d to those of the cultures that did not receive oligomer, as well as those that received a yl~yA~ ;on of completely randomized oligomers of similar length, (N)17, An oligon~c1eotide was considered an inhibitor in 2 0 this assay if it reduced the p24 level at least two times as much as the random oligonllcl~otide control.

B. Test of anti~en~e efficacy of selected probes from the hybl;lope maps A total of eleven probes to the HIV RRE de~rihed in Example 1 were 25 ~y.~lt.e~ d as 16mer phosphorothio~ted oligon~ tides. Since the position of the S' end of t'he probe is the most crucial for effective hybridi7~Ation, two nucleotides of the 18mer probes used in the screen de~ribed above were deleted from the 3' end. This mo~tifi~-~Atic-n helps to reduce nonspecific effects. Each c-An~ t~te probe was incubated with HeLa cells at concentrations between 0.2 and 1 IlM and the cells were transfected 3 0 with HIV-1 DNA. Three days later, after continuous incub-Ation with probe, the p24 levels were determined.

W O96/17955 PCTnUS95115779 Table 1 below shows the results. An oligon-lrl~oQtide was cc n~idered an inhihitQr in the p24 bi-llo~ l assay if it lcduced ~e p24 level at least two ~mes as much as the "..l~lcj... oligonucleotirle control; it was c~n~i~Pred an effec~ve hybndizer in the mapping assay if it ~n~ d at least two times the RLU of the average probe.

Table 1 Phosphorothioate Probe Number Binding: Binding: Fold Anti-RRE T~pt n~l RNA ~hibition Effecti~e Probes:
GCGCCCATAGTGCTTC 7807 0.4 4.2 9.1;19.0 CGCCCATAGTGCTTCC 7806 O.S 1.9 4.2;3.0 CACTGCGCCCATAGTG 7811 2.0 0.4 3.1;5.4 AATTGTCTGGCCTGTA 7843 0.6 2.5 2.7 Ineffective E~ol)es:
GTCAATGACACTGCGC 7819 0.6 3.5 1.1 AGCGTCAATGACAC-G 7 22 0.~ r.l 0.7 GACAATAATTGTCTGG 7Q49 0.6 ~.2 0.4;0.8 AAGAACCCAAGGAA~A 7780 0.~ .9 O.S
GCTCCCAAGAACCCAA 7786 2.4 0.4 0.5 CAAGAACCCAAGGAAC 7781 0.2 0.5 0.3 AGACAATAATTGTCTG 7850 0.4 2.4 0.1 The 'Number"in Table 1 above is the 5'position on the HIV SF2 target to which 1 0 the 3' end of the 18mer parent probe binds. The binding to both the tr~n~criI)t and the viral RNA are c;~lessed as RLU bound with the test oligo/RLU bound with the average oligo. Both were d~l,.ined with 18mer DNA probes. The fold inhibition (I) was calculSIt~?d as: I= p24 random oligo/p24 test oligo, using 1 ~M probe.
Four probes showed some biological effeetiveness (7806, 7807, 7811, and 1 5 7843). I'wo probes did not hybridize to either the ~ or the viral RNA
(7781,7822) and they were in~rrt~cl ;ve biol~i~1ly. Two probes hybridized effectively to the I . ,..~c.~. ;l'~ (7786,7811) and one of them (7811) was effeetive biologically. The other seven probes hybri-li7~A effectively to viral RNA (allowing that 1.9 ~ 2.0) and three of these were biologically effeetive. By ser~~ g probes that were 'winners~in 2 0 the hybri~li7~tion assay to either the tr~n~ript or the virus, all four biological winners would have been dete~t~ with just nine probes tested.

W O96/17955 PCTrUS95/15779 The e~act 'hit rate" of probes against HrV is not known (hundreds of probes would have to be tested biolog~ y to know this ..~ ), but it is highly unlikely to be ~Iy~L~c near 4/9. One skilled in the art will realizP that the use of ribom~-le~,v~in co~ es ieol~t~l from cells (rather than d~ ul~;n;~:d viral RNA or in vitro S ! .ili~ ) should provide a more ~ ;u.,~le Hyl~ e Map of the ~-~eeihle sites with even higher hit rates.

The present invention has been desclil,ed with rt;rt;le.lce to specific embodim~nte However, this application is int~nded to cover those ç~ ,es and substitutions which may 10 be made by those skilled in the art without depcu l,llg from the spirit and the scope of the appended claims.

Claims (14)

Claims WE CLAIM:

1. A method of detecting or determining a binding oligonucleotide comprising a nucleotide sequence which binds within a known nucleotide sequence of a target nucleic acid, the method comprising the steps of:
(a) obtaining a plurality of oligonucleotides each of the oligonucleotides comprising a first nucleotide sequence which is complementary to a sequence within the known nucleotide sequence, and the oligonucleotides having overlapping first nucleotide sequences wherein the first sequence of each of the oligonucleotides in the plurality of oligonucleotides overlaps the first sequence of oligonucleotide in the plurality of oligonucleotides by from one to four nucleotides;
(b) contacting each of the oligonucleotides with the target nucleic acid under conditions permitting specific hybridization of oligonucleotides to the target; and (c) detecting or determining the presence or absence of specific oligonucleotide-target binding between each of the oligonuc1eotides and the target nucleic acid to indicate whether each oligonucleotide binds within the known nucleotide sequence, therebydetermining or detecting one or more binding o1igonucleotides.

2 The method of claim 1 wherein the first sequence of each of the oligonucleotides in the plurality of oligonucleotides overlaps the first sequence of another oligonucleotide in the plurality of oligonucleotides by one nucleotide.

3. The method of claim 1 wherein each oligonucleotide in the plurality of oligonucleotides further comprises a region having a nucleic acid sequence which is an adapter probe recognition sequence complementary to a test probe recognition sequence in an adapter probe, and further wherein step (b) comprises the steps of:
(b1) providing: (i) a capture probe oligonucleotide bound to a solid support, the capture probe having a nucleic acid sequence complementary to a capture probe recognition sequence; (ii) an adapter probe oligonucleotide comprising a region having the capture probe recognition sequence, a variable spacer region from 0 to 50 nucleotides, and a region having a test probe recognition sequence;
(b2) combining, in a liquid medium under binding conditions for complementary pairs, each oligonucleotide with the target nucleic acid, capture probe and adapter probe to form support-bound probe-target complexes; and (b3) washing the solid support to form support-bound probe-target complexes subsequently free of unbound target.

4. The method of claim 3 wherein in step (c), the presence or absence of specific oligonucleotide-target binding is performed by steps comprising:
(c1) labeling the target nucleic acid; and (c2) detecting the presence of support-bound probe-target complexes by detectingthe presence of label in the complex, thereby detecting specific oligonucleotide-target binding.

5. The method of claim 4 wherein in step (c1) the target is labeled by incorporation of biotin-UTP, and the label is detected in step (c2) by steps comprising:
(c2a) incubating the support-bound probe-target complexes with alkaline phosphatase (AP) covalently coupled to avidin or streptavidin to form support-bound target-AP complexes;
(c2b) washing away unbound AP; and (c2c) incubating the support-bound target-AP complexes with an AP substrate and monitoring the conversion of the substrate.

6. A method of detecting or determining a discontinuous probe that binds to a known nucleotide sequence in a target nucleic acid, the discontinuous probecomprising (i) at least two binding oligonucleotides that are each complementary to a distinct region of the target nucleic acid, covalently joined, optionally by (ii) an organic linker molecule, the method comprising the steps of:
(a) determining at least first and second binding oligonucleotides that are complementary to first and second regions of the target nucleic acid, the first and second oligonucleotides binding more strongly to the target nucleic acid than other oligonucleotides that are complementary to other regions of the target nucleic acid, and wherein the first and second regions are non-contiguous;
(b) covalently linking at least the first and second binding oligonucleotides determined in step (a) in at least two combinations, optionally with organic linker molecules to obtain a set of candidate discontinuous probes;
(c) contacting each candidate discontinuous probe with the target nucleic acid under conditions permitting specific hybridization of oligonucleotides to the target; and (d) detecting or determining the presence or absence of specific discontinuous probe-target binding between each of the discontinuous probes and the target nucleic acid to indicate whether each discontinuous probe binds the target nucleic acid, thereby determining or detecting one or more discontinuous probes.

7. The method of claim 6, wherein step (a) comprises the steps of:
(a1) obtaining a plurality of oligonuc1eotides, each of the oligonucleotides comprising a first nucleotide sequence which is complementary to a sequence within the known nucleotide sequence, and the oligonucleotides having overlapping first nucleotide sequences wherein the first sequence of each of the oligonucleotides in the plurality of oligonucleotides overlaps the sequence of another oligonucleotide in the plurality of oligonucleotides by from one to four nucleotides;
(a2) contacting each of the oligonucleotides with the target nucleic acid under conditions permitting specific hybridization of oligonucleotides to the target;
(a3) detecting or determining the presence or absence of specific oligonucleotide-target binding between each of the o1igonucleotides and the target nucleic acid to indicate whether each oligonucleotide binds within the known nucleotide sequence; and (a4) selecting at least two binding oligonucleotides that bind most strongly to the target nucleic acid.

8. The method of claim 7 wherein the first sequence of each of the oligonucleotides in the plurality of oligonucleotides overlaps the first sequence of another oligonucleotide in the plurality of oligonucleotides by one nucleotide.

9. The method of claim 7, wherein step (a) further comprises, following step (a4), the steps of:
(a5) for each of the first and second binding oligonucleotides selected in step (a4), obtaining a plurality of secondary oligonucleotides, each secondary oligonucleotide having a 5' end of its first sequence identical to the first sequence of selected oligonucleotide and having a sequence complementary to the same region of the target nucleic acid, wherein the length of the first sequence for each secondary nucleotide that is complementary to target nucleic acid is different than the first sequence length for the other nucleotides in the plurality of secondary nucleotides;
(a6) contacting each of the secondary oligonucleotides with the target nucleic acid under conditions permitting specific hybridization of secondary oligonucleotides to the target;
(a,) detecting or determining the presence or absence of specific secondary oligonucleotide-target binding between each of the secondary oligonucleotides and the target nucleic acid to indicate whether each secondary oligonucleotide binds within the known nucleotide sequence; and (a8) for each of the first and second binding oligonucleotides, selecting the secondary oligonucleotide that binds most strongly to the target nucleic acid.

10. The method of claim 6 wherein the organic linker moieties are selected from the group consisting of arylene, C6-C18 aralkylene, C6-C18 aralkenylene, C6-C18 aralkynylene, C1-C30 alkylene, C1-c30 alkenylene, or C1-C30 alkynylene, containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of-CO-, -COO-, -CONH-, -NHCO-, -S-S-, -SO2-, -CH(OH)-CH(OH)-,-CH(OR1)-CH(OR1)-,-O-PO(O')-O-, O-PO(R1)-,-O-PO(OR1)-O-,-O-PO(OR1)-R2-, -PO(OR1)-O-R2 wherein R1 is lower alkyl and R2 is lower alkylene, -O-((CH2)n-A-)m and wherein -A- is -O- or -PO4-, n is an integer from 2 to 4 and m is an integer from 1 to 20.

11. An antisense probe comprising a nucleotide sequence determined by the method of claim 1.

12. A discontinuous antisense probe comprising a nucleotide sequence determinedby the method of claim 6.

13. A ribozyme probe comprising a nucleotide sequence determined by the method of claim 1.

14. A discontinuous ribozyme probe comprising a nucleotide sequence determined by the method of claim 6.