CA2145750A1 - Applications of fluorescent n-nucleosides and fluorescent structural analogs of n-nucleosides - Google Patents

Abstract

Structural analogs of the six non-fluorescent N-nucleosides commontly found in RNA and DNA, which are inherently fluorescent under physiological conditions, are identified and methods for their preparation provided. Such analogs may be incorporated into DNA and/or RNA oligonucleotides via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides having prescribed sequences. Such analogous sequences may be identical to, or the analogous complement of, template or target DNA or RNA sequences to which the fluorescent oligonucleotides can be hybridized. Methods of preparing either RNA or DNA oligonucleotide probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in oligonucleotide amplification, detection, identification, and/or hybridization assays are also provided.

Description

-~WO 95/05391 PCT/US94109316 214~7~0 DESCRIPTION

APPLICATIONS OF FLUORESCENT N-NUCLEOSIDES AND
FLUORESCENT STRUCI URAL ANALOGS OF N-NUCLEOSIDES
Bach~loul.d of the Invention A. Field of the I~ ic n The present invention relates to fluorescent ~llu~ lul~l analogs of the non-nuu~cc~n~
"".1~ iPC C~n~m~ nly found in DNA and RNA, methods of their del;v~li~lion and sul,s~-,_nl use in the ~ulh~-~ of lluul~cclll olignn~lrlPoti~lpc~ and to their new and useful app1i-~ both as ~luu,~cenl ~ nn --...~ and in n.. ~nl oli~."u.. lc~lides having y~esc~ cd se~u~nces itinnq11y, it relates to a~y1i.~1inn~ in which n.,o,~cel.t ~llu~lul~ analogs are, ~ n-P~ for specific non-nuul~ccnL n11r1Pn~ PC in ~lt s~,il,cd DNA or RNA sc~lL_nc~s and to methods of using lluo~esce~L n1i~.n, 1rolides as L~i.1i" ~inn reagents and probes for tl:a" - - and th. ~ se~nif ~,u~oscs and as ~ ; n~1;. and t~ "i~ research tools.

B. General D~,~ ,lion of the Art The six cnmmnnly ocr-rrin~ N-n~ Irn~:~lPc which ~ . in the ~. ~pn~;linii of DNA and RNA from all sources have the :~llL_IUl~ shown in Figure 1 wherein R6 is H for inosine and NH2 for ~ -, Rg is H for uridine and CH3 for thymidine. Furthermore, Rl2, RI4 = OH for li~ PC~ R12 = OH, Rl4 = H for 2'-deoxy m~lPoti~lpc~ RI2 = H, RI4 =
OH for 3'-dco~ ec and R12, Rl4 = H in did~r ..",~
The six; 1y oc.-- . . i..g "~ r~1 i". c do not absorb light at ~ hc >290 nm and are cill~;li~.,l~ non-ll,,o,~c~l under phy~in1OgirA1 C~n~1i1in1~. D~1iYali~w of the çnmmnn1y oc~1rring N-llu~l~lidw for a variety of synthetic, .~ ;,.nclir, and tk.. ,.r.~ r ~u~l~oses are cnmmnn, -r - - ~ "~in;~ on both the k ~ o~iy-ilic base and the fu,~osc ring. These can be made at the loci shown in Figure 2 in which R4 is a reactive group de.i.~_~il,'- with a d~,t~lable label (NH2, SH, =O, and which can include an optional lin~ng moiety ir~-ly-~ing, but not limited to, an amide"l-in~,lh~." or disulfide linlcage or a e ul.lbil,alion thereof with r~ io-~ ~1 variable reactive groups, Rl through R3, e.g., Rl-(CH2)X-R2, or Rl-R2-(CH2)C-R3-, where x is an integer in the range of 1 and 25 iu~,lb.,ivt;; and Rl, R2, and R3 can be H, OH, alkyl, acyl, amide, thinethrr, or disulfide); Rs is H or part of an etheno linkage with R4;
R6 is H, NH2, SH, or =O; Rg is _~u~,cn, methyl, bromine, fluorine, or iodine, or an alkyl or aromatic - 1t11Pnt or an optional linking moiety i -- h 1ii.g an amide, thineth~r, or disulfide linkage or a ~.. I.i--~in ~ thereof such as Rl-(CH2)X-R2, or Rl-R2-(CH2),~-R3, where x i integer in the range of 1 and 25 i lel~ivt;, Rlo is l~d1u~n, or an acid s_~il;~_ base stable blocking group, or a ph- .il~ -rous derivative, Rll=R12=H; Rl2 is 1~ 1ru~n, OH, or a ~hn~phnrous d~,~ivalivt;; R is H, OH, or OR where R is a protecting group or ~ i1in1-A1 WO 9S/05391 PCT/US94/09316 ~

fluorophore. The letters N and C in the N-~n ~ lf c and C-m~rlf~c~ -c df cigrr~te the atom at whieh the gl~_usi-lic eovalent bond eonneets the sugar and the ~t~lo~y~lic base. In the eases of the eommonly ocenrring rn.. ~ PC the bases are either adenine, guanine, eytosine, inosine, uraeil, or thy-mine. The bases are attached to a furanose sugar, a general ~IIUCIUI~;; of whieh is shown in Figure 3. The sugar ~ for the n~lescent analogs share the same -u--.1~ J
system for all R groups, but the .. h.. i.. g system for some of the heterocycle analo~c may differ.

I. Known MPthntlc of T ~bPIinP Nn-l~oti-lPc Nu~l~lidc sequenees are rnmmnnly utilized in a variety of app1ir~tionc i...~ i..
~ and t~ ~ ~ probes whieh h~liLli~ target DNA and RNA and ~.................... plir~ of target SC~1L_~IL~C. It is often ne~a-y, or useful, to label lUi- leulide SC~
A. T ~PIin~ of oliLJ~ lcolide probes with ra.lio~o~?es. Hyb- i.~ if n of specifie DNA or RNA sequenees typically involves ~nr P~ling olignnuf lP~tides of lengths whieh range from as little as 5 bases to more than 10,000 bases (10 kb). The majority of nlil")n~ iP. probes ~ull~,.llly in researeh use are . ~lina~ l;v~,ly labeled; however, beeause of (a) the short half lives of the isotopes in ~mmnn usage, (b) the safety l~uil~lu~,nts, and (e) the eosts of hqntl1ine and disposal of ~f~ probes, w~ and sensitive non-isotopic methods of drl~P~ .. are required for h~ .n~ methods to achieve ~.id~l~ ad a~pl~ff~- and ~l ' ~i B. Non-isotopic methods of labelin~ oliF.ollL~ leuli.le probes. In general, all of the non-isotopic methods of ~et~prting h)~ lin:~ probes that are cull~ tly av,lilabl_ depend on some type of deliv~ in n of the ..-- l~ Pc to aLlow for det~rtinn, whether through antibody binding, or e~ .liC pl~Cf-~.~..g, or through the ~uolcsee.læ or ehPmi~ f~ of an attached "1~U1~ f~llP In most cases, c-li~...nJ- lr~lidcs have been delivàli~ to illwl~,ulale single or multiple mf'~ of the same reporter group, generally at specific cyclic or ~ -1;. p~ Techniques for qttq~hing reporter groups have largely relied upon (a) fin~rtinnqli7~qtinn of 5~ or 3~ termmi of either the .- -- ... - . ;.^ ~ lcnc ~1PC or the oligo.. "~ l~4lide strands by uus ^hPmir-q~ ,a~,liOn5 using de~.~ t~led olig ~ ides in aqueaus or largely aqueous media (see Cardullo et aL [1988] PNAS 85:8790-8794); (b) srthP-ci7in~ mo~lifiP~
Ps cnnl;~ (i) protected reactive groups, such as NH2, SH, CHO, or COOH, (ii) a~ alablc "lln~- -1 linkers, such as NHS esters, aldehydes, or h~ c, or (iii) affinity binding groups, such as biotin, attached to either the ~ u~-lic base or the fllr.qnnce moiety.
Mn.1ir~ havebeenmadeonintactnl~ F~l;~p-lcorto - ",.. l~.-i~iP~cwhichhavelly been ~,u.ated inta nli~ 4~ F5 during rhPmirql synthesis via terminal I .,...-f~ ..,.ce or "nick i -lqtinn" (see, e.g., Brnmhq-ugh et aL [1988] PNAS 85:5610-5614; Sproat, B.S., ~L T q~n/~ B. Beijer, P. Neuner, P. Ryder [1989] NucL Acids Pes 17:3371-3386; Allen, D.J., P.L. Darke, S.J. Benkovic [1989] P~ hy 28:4601 4607); (c) use of suitably ~n~t~led c1~mirq~ ^s, which can be coupled at the 5' t'-- --;----C of protected oligunl- Icolides during rhPmirql ~lLe~cis, e.g., S' qminnh~yl-3' 0-rhn~l-ho~ TAr.qlqmhidis~ J., L. Duncan, G.W.

~ WO 95/05391 PCT/US94/09316

2 1 ~ ~i 7 Tregar [1990] NucL Acids Res. 18:493-499); and, (d) addition of r ~ in~ql groups on the sugar moiety or in the phosphr~ pctpr ba~ Ll,o..e of the polymer (see Conway, N.E., J. Fidanza, L.W.
McT ,Anghlin [1989] NucL Acids Res. S;.,.~,o,.~". Senes 21:43-44; Agrawal, S., P.C. 7~mP~ik [1990]
NucL Acids Res. 18:5419-5423).
At the Cimrl-ct~ non-nuAlPn~i~e linkers and labels have been attached to the 3' or 5' end of existing nli~n~ potides by either ~ alic or chPmir~l Athn~c Mo~lifiA~ fi- n of ~"~ P
residues internal to the sequence of a DNA or RNA strand has proven to be a difficult plU~lUl'~
since the reaction condilions must be mlld enough to leave the RNA or DNA ~li~mPnc intact and still yield reaction p.ud..~ L~ which can participate in normal Watson-Crick base pairing and 0 stacking i--1~ ns (see Figure 4).
C. D-,livali~liOI s of the I A~ o- y. lic base (B). Nu~.ous methods for both cyclic and ~,AU~,y~,liC dc~livnl i,,.ti.... of the N-m~rlPn~ P base have been dP-crlihe~ n-1;ng the following:
(1) Hapten labeling. DNA probes have been a_ino mn-lifiP~ and s~se.lu~ ..lly d.,l;vaLi~cd to carry a hapten such as 2,4-~lhlillu~h_..ol (DNP) to which enyme-colljh~ d anti-hapten antibodies bind which `ou~SC~lu~ y can be l~uceOo;~d using a cnlo~
tt. as a label (Keller et aL ~1988] Anahytical Fj~ y 170:441~50).
(2) Amino- and thiol-de.;v~.li~d oli~ r;)l i~iP.C Takeda and Ikeda ([1984]
NucLAcidsResearchS~.,.~Oul,,.SeneslS:101-104)usedphoO~hol.icO~erd~,.i.,_liv~ofp..l..,O~;u~
liuc for the plCp~ inn of amino-derived nli~mP.nc Ruth and cnllp~gl~p~c have ~lP~crrihe~
methods for s~ g a deuA~.. lidi~c analog with a primary a_ine "linker arm" 12 carbons in length at Cs (JDh1~ i et aL [1986] NucL Acids Res. 14:6115-6128). These were later reacted with nuo~cO~lu to produce a nuoreCOc~ - 1P Urdea and Horn were granted a patent in 1990 (U.S. Patent No. 4,910,300) covering ~ ^ derivatives on which the 6-amino group at C4 had been mniifiPA 3' and 5' amino luodiryil,g phncphr..n...i.lilPc have been widely used in rhPmi synthesis or d~livali~d olig~ ~lides and are cul~ idlly available.

(3) Labelin~e with pholob:oli.. and other b OLiujlàlill~! a~ents. The high affinity of biotin for avidin has been uced to bind e~yll~àliC or ~ reagents to delivali~d DNA probes (Foster et aL [1985] NucL Acids Res 13:745-761). Biotin conjugated to other linkers has also been widely used, i~ in~ biotin-NHS esters (Bayer, E~, M. Wilchek [1980] Methods in R~ rn~Ana~ysis 26:1), biotin :",~. ;I-~---i iPC (Lee, W.T., D.H. Conrad [1984]
J. E~. Med 159:1790), and biotin m~lPimi-lPc (Bayer, E.A. et al. [1985] AnaL P c~rh~n 149:529).
Reisfeld et aL ([1987j BBRC 142:519-526) used biotin hydrazide to label the 4-a_ino group of cytidine. A patent was granted to Klevan et aL in 1989 (U.S. Patent No. 4,828,979) for such d~.iv.li,.l~ at the 6-position of adenine, the 4-position of cytosine, and the 2-position of 35 guanine. These d~lliv~ l innc illlelf~le with hydrogen bonding and base-pairing and have limited uses in plUdL_~Ulg nliL,.. ~ for use in hJhli-~ n

(4) dU-Biotin labeling. N.. ~ P 5'-i phc-p~ or 3'-O-phrlcphr- were ~ fiPd with a biotin moiety conjub~.ted to an ~'ip~ aino group at WO 95/05391 ~ PCT/US94/09316 214~75~ 4 the S-position of uracil (Langer et aL [1981] PNAS 78:6633-6637; Saiki et aL [1985] Science 230:1350-1354). The nucleotide triphncphi te derivatives are eLrc~liv~ ;u-~,o-ated into double stri.~ led DNA by standard techniques of "nick translation." Once in an nli~nnrlPotide, the residue may be bound by avidin, ~llc~lav;din, or anti-biotin antibody which can then be used for S det~P~tinn by ll, o,~c~ce~ cl- .. ,ih.. i-,f c~ or en~ymatic pru~;~ g.(5) 11-di~oxi~enin-ddUrP labelin~. The en~ne, terminal ~ r~lrc~P has been used to add a single ~ -i---11-dideoxyU I~ to the 3' end of nli~ "~ ~Lides. Following L~ ;.l;, I inn to target nucleicacids, DIG-ddUTP labeled h~.idi~tion probes were detected using anti-DIG antibody conjugate.
(6) AAIF. T~ nnl~nl~cf~ detectioncanbedoneusingmnnnrlon~lFab~
fri.~Pnt~ which are specific for RNA DNA hybrids in which the probe has been delivati~d with, e.g., biotin-11-UI~ (Bobo et aL [1990] J. Clin. ~f.~,~ioL 28:1968-1973; Viscidi et aL [1986] J. Cli~
MicrobioL 23:311-317).
(7) Bisulfite. n~lir~ of cytosine. DraperandGold([1980] Pin{~",;~hy 19:1774-1781) iuL o~lhced alirh7<tir amino groups onto cytidine by a bisulfite catalyzed t~ ;n~
r ,a lion, the amino groups were :,uI,se ~ , labeled with a ~uorc~nt tag. In this ~)-u~lu-c, the amino group is attached directly to the ~ - base. Like the dcliv~ n~A of uracil, these de,iv ~ interfere with hydrogen bonding and base-pairing and are not ~~ Iyuseful for p~u~lu~ iug efflcient h~ nlig (8) Fluorophore dcl;vaLi~ed DNA probec. Texas Red (Sulf~rhlnro-~hn-li minP.)df,ivaL~ dprobe~carecù"~ allyavài~blcwhichh~li~li~tospecifictargetDNAs and which can be detected using a liow ~uluet~,l or a ~ ,.u~ ul,c. Nu-..~,.uus authors have reported coupling ~UUIU~hOl~ to rhr.mir;-lly srt~ - ~ oliL ,~ otid~ which carried a 5' or 3' terinal amino or thiol group (B~ lgh et aL [1988] Nucleic Acids ~es. 16:4937-4956).
(9) Direct enzYme labeling. ChPr--ir~l coupling of an enyme directly to a rh~mir~ lly syTlthP'ci7P~ probe has been used for direct detection through sllhstrate p.u~ g For PY mrlP Urdea etaL dFC~;l-C~an nlign..~ idesa"d~ hassayinwhichmultipleDNAprobe hyl~ ;, .I;n,.~ were u_ed to bind target DNA to a solid phase after which it was further labeled with s~ 1itinn~1, alkaline phns~k~ls.~e dc,ivali~;d h~- ;.1;, ~linn probes (Urdea et aL [1989] Cl~
Chen~ 35:1571-1575).
(10) ~- ;.1;";,"" ester labeling. A single phenyl ester of methyl ~- ~ ;.l;,,;,,,~
is attached at a central position on an RNA or DNA probe. Hydrolysis of the ester releases an ~rri-lnnP C02, and light. Because the ester on u -h~b-idi~d probes hydroly_es more quickly than the ester on probes which have l-~-idi~d to target RNA or DNA, the cher~ c~F .~r~ of the L~-idi~d probes can be di~ ;~h_d from that of free probes and is used in a "h~ l;"~
protection assay" (Weeks et al. [1983] Clin~ Che~ 29:147~1479).

~WO 9S/OS391 2 1~ 5 7 5 PCT/US94/09316 :- i D. Dclivali~alions of the furanose ring (E~. MPthn-lc for de.ivali~dtion of the filr~nnse ring (Rll through Rl4 in Figure 3) and at the phosphn~lip-ct-pr ba,~l,olle of nlig~,.~.,. I~olides (Rlo in Figure 3) have been reported.
(1) IL,lt;,ll~.clc.,lide linka~e reporter ~roups (Rlo site). Phosphoro-thioate esters have been used to provide a binding site for n~ opholes such as monol.ulllol)i~ne (Conway et al. [1989] NucL Acids Res. Symposium Senes 21:43~4). Agrawal and 7~n Prnik ([1990]
Nucl. Acids Res. 18:5419-5423) reported methods for hl~l~ol~li.,g amine specific reporter groups (e.g., monobr mnbin~nP,) and thiol specific reporter groups (e.g., ~ar~s~ isothincy~n~t-P-) through ll.o.liryhlg the ph~ iP~ctP~r b"'~l-f)nr of DNA to phnc~ nln~ Pc and phosphu,ulllioat~ diesters, respe-,li._l~.
(2) Cl~. ~lir reporter groups (Rll throu~h Rl1 sites!. Smith, Fung, and Kaiser ([1989] U.S. Patent No. 4,849,513) (lp~c~rihed syntheses for an assortment of d~ aliv~c and labels on the ~C4' ;~1 ;r moiety of ~n~ P~ and nu ' ~ analogs through the introdllrtin~ of an ~liph~ti( amino group at Rlo. The authors did not report or claim any uses or applir~tinnc of inherently ~ - ~nl nlig.,~ p~liAp-c~ either made ~hPmi~ ly or c~lllàli~lly or using the Ll..a~nl ~ n~:~e analogs or their d~,livàliv~.
E. T,;...i~ ns of non-isotopic methods for labeling oli~ lr~lides. In order to create non-~ in~ctive types of APt~P~ P nlignnll~lP~tides, it has been n~a,y to chPmi~lly modify the n... ~ l, c typically used in DNA and RNA probes, which has made such probe in,. eA~ and labo~iu~, in many cases the dpt-prtinn . h~ -C have also proven - - and e~A~ nS;~_ to use, which has largely been rP-~p~ - lc for their failure to find ,;..iri.- -~ in~ in cliI~ical l~hn.~.~(.. ;, c In their a~ in-~ to h~ inn, other li...il7 fin~c Of rhPmif-~lly dcliva i~d probes have also become appàre.~t.
(1) ~hPmif~lly d~livaLi~d dNTPs are generally not cost-effective for use as stock d~A~ r,a~;f~c r ~ in PCR ~mplifif~tinn hence, labeling of amrlifi~ DNA is limited to (i) ~ ir~ inn using p-~v;o~ly labeled primers, or (ii) ~nrP~Iing with labeled h~- i.l ;,~ ~ i.." probes. Use of the former L~L~ Y results in false posilivc~ during ~ ...l.liri. l inn owing to (i) non-speci_c ~nnP~Iing of primers to non-target scE,.~ of DNA during~mrlir~ l inf~ or (ii) cn~ l in~ by ~mpl - - present in the la~olalo-y ~ llvil~ which are residual from previou-c ~ ir~ in" ~ EApense and terhnif~l fliffif~ tiPc in post-Lyl,-i-li,~-lin,~ plU~i~ g have largely limited the appli~ nl~ of labeled h~ , lin~ probes to research.
(2) Base pairing is hindered for many nli~mPrc made with derivatiz_d . lr,~ Pc through the il,l.o~b-~;linn of bulky or non h~ hfi~,_l. bonding bases at illa~l~liale sites in a sc~l -- Owing to the inherent ba~gluulld h.. i.. ~-~-.-e of many clinical samples, even the a .;.li";"", ester probes have failed to achieve their th- ~,Li~l levels of se~iLiviLy. The l~UUCi - for post h~b. ;fli,~l inl~ processing have ~ aine~d a li--,i~Lion to such mf~tl~nflc 2 1 4 5 7 ~ Q t, , (3) It has proven difficult to provide non-r~lio~ v~ Iabeled probes which may be ;~ ;VG1Y ~lud~_Gd in large qn~ntitiPQ~
(4) ~hPmil....,i...~..,l probes are short lived and samples so tested are difficult to quantify or to l~ pn~b~" a,~ cl~.

(5) IIyl - ;-~ n in most cases is only inferred, is non-quantitative or only semi-4ual-liL~Iive, and is non-~ hle.
These l i... il; l ~ have hindered applir~ti~ nc of DNA and RNA hyh- i.l;,,. l inn probes to clinical l~hn".lo.y testing and tk- ~ uses.
F. Fluol~n~ N-nu~1Pn~ P-Q and lluulG~cGllt ~llu~Lulal analogs. Formycin A
(generally referred to as Folllly~ h~), the pluLu~ al nuolescc.. l n~ P~Q~ P analog, was originally isolated as an ~nlil.. nl antibiotic from the culture filtrates of Nocardia ~.t~,r~".,,a (Hori et aL
[1966] J. A~ ir~, Ser. A 17:96-99) and its ~L~ IU1G i~PntifiP~ as 7-amino-3-b-D-~ r --~ yl (lH-pyrazolo-[4,3d] ~yliuudhlc)) (Figures S and 6). This anlil)iolic, which has also been isolated from culture broths of S~IL~ CC~ lav. ~lae (Aizawa et aL [1965] Ag~. BioL Chem. 29:375-376), and S~ c.. gummaensis (Jqr~r~P~e Patent No. 10,928, issued in 1967 to Nippon Kayaku Co., Ltd.), is one of nu~elUUS ui~,lol;;~l C-l ;l~o~n.. lr~ lp. analogs of the N-nl-- lr~ P~ i Iy found in RNAfrom all sources. The other naturally-o~rrin~ C-,ibo.n~ lrn~ P~ which have been isolated from microo~ (Figure 4) include r.,~ ~ B (Koyama et aL [1966] T~
Lett. 597-602; Aizawa et aL, supra; Umezawa et aL [1965] A ;~;~nr~ Ser. A 18:178-181), uAuru~ ~ B (Ishizuka et aL [1968] J. ,4nhhi~hr~ 21:1-4; Sawa et aL [1968] A ~ c~tir~ 21:33 339),1,s~..doulidiL~c (Uematsu and S~q~ olnik [1972] P;~L -" -hy 11:4669 4674), sLowdh .~
(Darnall et aL [19671 PNAS 57:548-553), ~JlaLVllly- hl (Sweeny et aL [1973] Cancer Res. 33:2619-2623), and ~ c~ e et aL [1972] J. Antih~ h~-cs 25:11 ~17). Formycin, formycin B, and oAoformycin B are ~yla~ulh-~ylill~il~ c nl lrll-i~PC and are ~llu~lulal analogs of ~' - e, inosine, and 1~ -C~ ,ly; a ~yl~u~yliLuidLLc ~,L,I I analog of g~ inP
obtained from natural sources has not been reported in the lil~ .alulæ A thorough review of the biosynthesis of these CUlUJJUUU~lS iS available in Ochi et aL (1974) J. A ~ jr~ xxiv:909-916.
Physical properties of the nllrlPnci~lP, analogs. Because several of the ~n~ nC ~ .c were known to be active as antibiotic, antiviral, or anti-tumor .; ,.-~pù~--lc their 1 dC~ ali~LiOIl and physical properties have been extensively studied and w-upal~i to the ~LIU~IU1~ and syntheses of the N-~ iAe-s cnmr~nnly found in DNA and RNA. In the late 1960s, several u~;lul~l analogs of the six cnmmnnly or~rring N-m.~ lPc. were found to be n.,Ol~c~,~
under phy~ h~ n~l;l ;O~c~ lluol~ a ill the a}lalogs results from a lnt~le~ r rigidity Of the het~,.u~-,lc b~ ule itself, not all the ~llu~ulal analogs of a given type, e.g., the ~u~lP,~ c are nuûl~c"~, nor is lluol~ccuce an ~Acl~wiv~ or inherent ~,~o~c.ly of any particular class of u~ lul~l analogs. Our s~ CP~qllPnt studies have shown that only a few of the pyrazolo and pyrolo ~,yl;l.~ c and purines are nuolescenl, and that the pluptlly is shared with a few other r-:~e dc~iv~liv~ and ~Ilu~,lulal analogs inrh-(ling, but not limited to, several ~ ul~d N-~ 214~7~1;1 1PC ~ 1PC ethF ~ P~C;~1P-C and l1F_,~"". 1.~ 1P.C, the ~ u-,lul'~S of whic_ are shown in Figures 5-11. Those ~llU~,lUl~ in Figures 5-11 which are shown ~u l~ uL,ded by boxec have been either ~ v;~U~ly reported or found to be fluorescent during dc.elopLucnt of the present - iu~ iol~.
S Ulu~ ri7F~4 nlignmPrc c~ lg fluo-c;sccn~ analogs were pl~ d by Ward and colleagues for physical studies using then available nnrlFnci~le pol~,l..el~se en_ymes (Ward et aL
[1969] J. BioL Chem 244:3243-3250; Ward et aL [1969] loc cit 1228-1237). There have been no recent reports in the lile.~luie of attempts to combine the use of nuorcs~u~ '1PC or their ~LIu,lu-~l analogs with the synthesis or hyl~ lifm tPlhl~ of mr~ r biology or tosynthesi~e nuo.~,s~nt ~ n.. l~ti~lP.c thc eL.,Lu.

Brief Summary of the Invention The subject iu~_nlion pertains to l~u~ e analogs which are nuoies~nl. Thes l1UOr~ U~ lr~ :t1e analogs are useful as m(l~ r.c in synthpci7ing and l~hP.lling n~rleoti-1e se lL_IIces. The iU~ ioLl further pertains to the use of these r~ t l.. -- lr~~ P.c which can be ~l~h~ilule4 for naturally or~lrring n.ll~nc:~lPc in the synthesis of ~lig~ lide probes.
When used as h.~h-i~ lion probes, the nuu.cs~.-ce of such o~ nu~lroti~l~pA can be used as a tool to detect and identify specific genetic s~..,uoes. This :- n~lnlr~gy is distinct from other non-~ io.-~ ' ivti methods of probe ~1P.tPrtinn in that it does not utilize ~---- lr~~ -c which have been coupled to en_ymes or other reactive proteins and does not require post h~l-- i-li,_ I i--for the ~l~P,t~.,l jn" of h~hl i.li,~t;.~
As ~P-~ .e~ in the Ba~vl ' section, there are many sho.~ to the methods and c~ ""l"";~ innc ,ull~,nlly used in DNA and RNA probe technology. It is an object of the present ~ _ulion to ov~,.w~c these sho.l~ c of the prior art through the use of nuoleswllt nnrlPnci~Pc and their nLo~P~w -t ~ 1 analogs which can be directly iLco-~u-~led into a ilJcd s~ as (i) specific ~b~ It'FA for a given nonlluv.~cenl ~ P which appear at defined lnr~tinnc in the rnmrl~ y sc~l, to tPmplotP or target DNA, and (ii) as labels for the 1~ ;ril -tin~ and ~etp~ti~7n of specific sc~ll - of tpmplote~ product~ l;rip~ or target DNA and/or RNA.
It is another object of the present invention to provide novel, inherc~ lly nuol~l.
",-1- ~- ~1P and - - analogs and the novel trirhn5p~otP. and phn~l~h. -,~ itP~ forms thereof, which are useful in the ~l.lLesis of labeled pol~l.L_l~lide probes, ~.--1.1;-- ~ ..~, ~1i,~gnnctirc~ and th- ,; ~ It is a further object -of the present hlv~,l.lion to provide methods of making lnn.~O~w~ll oligo.. lr~l;,lP.c capable of specific Watson-Crick base pairing with ~ il,ed SP~LPI1.X5 of target DNA or RNA.
It is another object of the invention to provide methods of using ~ t ,.1.~ P
analogs and nlig.. ~ r~l;~1P.C made theleliul" and s-ynthp-ci7p~ according to the methods of the pre,sent il.~ ioll to identify, detect the pl~;S~,llW of, and/or alter the function of known nucleic WO 95/05391 PCT/US94/û9316 ~
21~75~ ` 8 acid sc~u~m~s of DNA and RNA. ~.1..'iti.n~l1y, it is an object to improve and simp~fy the methods of ~ t~P~ti~ n and to simpli~ the appli~ ti~ m and uses of DNA and RNAhyl~- ;Ai".~i. n techniques.
In another aspect of the i~ nlion, c~lu.r~ methods are provided for making nucleic acid probes which are cnmp~ y to, and will bind to, only the sense or only the anti-sense, but not both, strands of a DNA duplex (fDy Ill"l,ic syynthesis). It is an il. pG.Ianl aspect of the invention that .~ l....e.lic synthesis is the necessaly c~-.n~-~'iti n for creating rapid and ~uallli.aliv~-nucleic acid probe tests, assays, .1i~gn.-.~ti.g, and tk~ .r~ A ~ lt aspect of ~V~..n.cl~i~
synthesis is its .~'~ep~-n~ n~-~ on the &~ u~ic use of promoters, primers, or inker m~.-.1ifi.-~l primers to direct the synthesis or i~ ti.-.n of oligon~ ~1ides or oligomers using only one of the t vo strands of a duplex as the t~?mpl~t.-It is yet another aspect of the invention that .V~ ic synthesis makes possible the directed use of multiple different t~ for C~JIICUI1~ synthesis of a "cocktail" of .r~
probes which can h~.idi~ cou. ul.~ ly to iml~Ppen~1.-nt and unique target sites on a single piece of nucleic acid, genomic DNA, or _L-I-- s..... r It is an ilu~ol.~lll aDpect of the iu~ llion of probe ''~ u_L.~il.'' that if multiple copies of the same target se~l are present on a single genome, such aD the multiple copies of the tandem repeat ;~ ."~ --ir sequences ~ cf-1nge~ in F~mrlP 7, a single .~.. lu~,L~i-, probe template can be used to create a "cocktail" which will bind to many targets on a single genome which are identical in sc~lu~ e but widely di ,I-il,uted in locus on the genome.
In one aDpect of the iu~ .ion, nU. r~ nt ,l~ 1 analogs of the Cl~mnlnnly oc~nTTing nn~ Qi~PQ~ and their dclivali~ useful in the synthesis, labeling, and .~enp~tinn of .~ ~lides are provided having the :,I.u._lu..~l f.- rmnl~P. of Figures 5 through 11. The ~)mm.nly occurring cn~ Ps .l.~"~ lly form hydrogen bonds in a specific rc.--nr/~ lor ~ io~ ip, ;g--~n-d Watson-Crick base pairing aD shown in Figure 4. Where ap~lo~lidte, speci.ic ~:lu~ ~p~ analo~g capable of ,~-~ o~~ g the pattern of watson-crick Ly~u,~-~ bond rulu.a.iou analogous to that of a particular c Imm~-nly ox-~lrrin~ n. ~ le are provided, as in~ 1ed for, e.g., A:T and L~ T in Figure 4 by the lo - r/<,(~p~-~l patterns.
In another aspect of the iu.~ uLion, methods of making and deliYaLi~g the Llhulw~nL
~LIu lul~l analogs of the c~mmnnly o~--. - ;.. ~ n~ F~ are provided i,-- ~ li.. E the steps of d~.ivaliLi..g the Rlo, Rl2, and Rl4 moieties to be (i) reactive in DNA or RNA synthesis, and/or (ii) reactive in RP.~ n~ Energy Transfer of the nuor~s~u~e from the ~IlL_Luldl analogs.
In still another aspect, methods of ~..11.~.,;..g and using pGl~ AF~ probes are provided using one or more of the rlholes~..~ ~ll, I analogs and/or their dc~ivali~d forms.
Such probes can be used to screen a sample ~----~;~i--i--~ a plurality of single sLranded or double str~n~lP,d pol),uh~ l~Lide chains and will label, detect, and identify the desired s~h~nce, if present, by hyh- i.~ ir n It is an ilU~ UI1~lll aspect of the i. v~..liuu that the nuul~n- lig..~ le probws can be used with "solution hjh. i.~ ir)n" methods as depicted in Figures 12 through 18.

_WO 9S/05391 PCT/US94/09316 21457~Q --In a~;u,dallce with the foregoing objects, the present invention ~,,,,,p, j~, c inherently fluorcsoent nnrlPnc;~lPc which can be uscd to label, modify, or identify o!ignnurlPotides made the.cLu..., the uscs of such inherently nuorescent oligonurlP~ti~1P~s as L~ inn probcs, and methods for f1etPrting nuclcotide se lu~ l.ces.
An illl~JUllalll aspcct of the h.~ liOII is the stable Lll,olesccllce ~ nl~ of the nuo.upholc_ and the use of time-resolvcd spe~;llus~u~y or photon counting to detcct and to quantify the amount of a Lluoluphore prcsent in a sample.
~ l(litinn~l r." ,.. ~ r., advantages, methods of use, and novel fcatures of the invention will be set forth in the dP-crrirtion which follows, and in part become a~pdlenl to those skilled in the art after .-,.,i,-~lin~ of the following, or may be learned by practice of the invention.

Brief Description of the Drawin~s Figure 1 shows the six commnnly-oc~llrring N-nur~Pnsi~ip-c which ~ o~ te in DNA
and RNA.
Figure 2 shows the general ~IIU~IUiCS of the ~r.. ,- nl~ly~ g N -n.. 1P~ ~1PC and their de~iv~ - I inn sites, Rn~
~igure 3 shows the general :~LIU~Iule of the fi-r~nce ring of both the purine and ~J~ id -le ~ flP,n- ~lPc and the rommnn sites, Rn for del;~ nl~
Figure 4 shows ~ ~ Crick base pairing between the normally oc~lrring N-~ Ir~ PC
A:T and G:C and base pairing between Çul~_ --;T, rul uy~;U, 2~6~1iall~lnu~ illc.T~ and S-amino-~,....~ B:C.
Figure 5 shows :.1. u~,lu~ analogs of the ~mmnnly~ef~lrring N--~- It~- 'IPC derived from binlngir~l sourc~s.
Figllre G shows the pyrazolo [4,3d] ~ylil, ~d~c ~ Ic~ analogs.
Figure 7 shows the pyrazolo [3,4dl pyrimidine nl.. lt~ ~P analogs.
Figure 8 shows the pyrazolo [1,Sa]-1,3,5-triazine - ~i,lf. analogs.
Figure 9 shows the a~JJ - and ~ r, nllrlPncidp~ analogs.
Figure 10 shows the df~a~y ~' - and f1f ~ "rl~ci~l9 analogs.
Figures llA-llB shows e-~ P1P-C of some nu(~ IIU~LUI~I analogs which are (llA) non-H-binding, and (llB) nuu~- lf-~ fe energy transfer (E;~ET) analogs.
Figure 12 is a diagram of ~ - RNA sy~nthesjs using FI~P or ATP.
Figure 13 is a diagram of promoter directed &~y~.lic RNA probe sy-nthesis using viral p.. - t~ and viral RNA pol~l--e.~cs.
Figure 14 is a diagram showing an example of the method for one-step labeling of ssDNA
inserted at the EcoRI site of pUC/M13 plasmid vectors and using dFl05.
Figure 15 is a diagram showing the necessity of using ~ ll.,L ic DNA or RNA probes for rapid and l~allliL~Live h~,;.l;,_tinn of the probe to target DNA. As shown, ~ LliC

WO 95/05391 ~ . PCr/US94/09316 _~
~ 4~0 probes provide cignifir~nt h~ s in L~.idiLaLion ~rri. ~ when ~I~ alc;d with probes.
Figure 16 is a diagram showing the collvt;l:,ioll of the libo~ reti-lP analog, formycin A, to its 2'-deoxy triphncph~tp~ or ph(.~hn.~...i-litP. form_.
Figure 17 is a diagram of ~PtP~tinn of a target DNA sequence in genomic DNA
hyb~ inn with nuu.~c~ probes.
Figure 18 iS a diagram of detection of an amplified DNA segment by solution hybri~i7~tinn of a nuolcs~.lt probe.
Figure 19 shows a flow chart ~ ~mming the sep~r~ti- n scheme u ed to separate reaction products from unreacted reagents following the e~ylllalic .ub~ lin~ reaction of ~1 for ATP in RNA probes.
Figure 20 shows a s~ l ir of the mP~ n i~ for il.~ iug dPt~orfir n sc~;liviLy by the u e of a probe '~cocktail" which contains multiple probes of different se l, -Figures 21A-21F-l show specific nuuiesc~nt nU~lPnQ;~P analogs which have been irlPntifiPA
and h~ d as to their class, :,Ll u~ Lul~;, chPnni~l name, abs~,.l,dnce spectra, emission spectra, and methods of synthesis.
Figure 22 shows the 5' universal end label cu-..r-i~i--e four distinct r... fi(. ~1 groups which include: region A, a non-base-pairing homopolymer from 1 to about 50 nuol~scen~
~"" lrn~ P, analogs long, region B, an optional non~ nc :~le rh~ .ho.li~ , "tether" c~ -region A to region C; region C, an e~.~ à~iC synthesis primer, çomp' y to a promoter in the target seqnPnrP; and region D, a -,J. 1~ P chain from about 40 to about 20,000 ~u I ~ PC
in length ~ y to the target nu l~ide sc~l..ence. Regions A, B, and C which are typically 20 to 60 bases in length can be rhPmil~lly srthPAi7PA Region D can pltir~labl~ be ellL~Ua~i~lly srthPci7pA The synthesis of the S' universal end label is dpcrriheA- in F -mr - 8.
Figure 23 shows a 5- h .. ~-li(' Ic~lc~ nl~ of the method for srthPci7ing a highly fluulcs~nt 5' labeled probe. The method c~ P-c the following steps: (1) Ir~l-irl;..g, with a specific lc~l . i. l inn enyme, a sc~, having a known PIULUUI~I site and known 1~ inn site du..~DIIcalll from the known pluluote site, (2) inserting a unique target sc~, at the ,. site, (3) Lyl~ ;.-g a ~luul~enl ~ n- ~1e analog probe ~-....l.,;~;--g a sc~ul,.-ce comp' y to the plUllli:)ttil of the inserted target sc lu~llce, and (4) P~tPn~ling the probe sr~ r, from the L~li~l~d l)lUlU~ region, using a nucleic acid pol~ ase, to s-ynthesi7e a specific probe ~.--.p~ . - ls..y to the inserted target se~- ~nc,.
Figure 24 shows the nnrn~li7PA D~e~ IIulu profiles compared for Fl8s (ethPnn~(1PnncinP) and Flos (rulL .~. ). The spectra were ~le~ " j~.r~ between 250-405 nm for the ~I - po ~ , using a 2.5 nm slit.
Figure 25 shows a method for iL~ lg senDi~ivi~y of ~etPctinn or di~. rcn~ial labeling using multiple copies of a 5' universal end label. Shown is an example of a target s~uence~ using ~WO 95/05391 2 1 4 5 7 5 0 ; ~ PCT/US94/09316 .

of the probe is the 5' universal end label on each of a plurality of probe se.~u~ ces where each of the probe scq~.~nces is c~mpl y to a different fragrrttnt or segment of the target sequence.
- Figur_ 26 shows the sequencing application of 5' universal end label. The unique probe (which can be pluduced by the method shown in Figure 24) can be employed using DNA
pol),,llelase to produce a plurality of dideoxy r.,.~ having different lengths.
Figure 27 shows the S - ~ Signal ~mplifit ~tinn ylo~ule. The example in the figure employs the genome from the Hepatitis B virion having two unequal lengths of DNA forming its double~ ~ genome. The method shows the steps: (1) . . n ,nr nn of the shorter DNA strand using ml~lcolides or phnspl~trylated ~ ie analogs; (2) the s~ --nl inn of the two -strands and the addition of two primers, A and B; (3) c,~t~,~ion of the DNA strand to which the A primer is h~liJi~d using reverse ~ n~ (4) ~tili7~ttion of the syrtt~ ~ double-stra ~ied sequence in an amplir~ ftl~ cycle which r~ (a) production of anl;~ e RNA t~mp~t~ from thesrthA-ci7~A DNA using an RNA pol~ el~se, (b) synthesis of DNA from the RNA l~ ---pl ~tA using primer B and a nucleic acid polymerase, and (c) synthesis of double stranded DNA (DNA
replicate) from the B primer-DNA. The replicate DNA can then repeat the signal amplifiratinn cycle (step (4)).

Brief Dc;.-liylion of the Sequences SEQ II) NO. 1 is a synthetic nli~nnt~l~otitlt according to the subject i~ tlLiomSEQ m NO. 2 is a synthetic oligo........ Ieoli~l~A, and the cu r~ t of SEQ ID NO. 1.
SEQ ID NO. 3 is a synthetic oliL.n.. - ~oLide and a ll-,o.~,.t analog of SEQ ID NO. 2.

Detailed D~s~ lo:,ulc of the Invention Disclosed and claimed are novel n~.JIc~cc ,,t n~ AA, analogs and methods of use of the Lluoles~.ll nl,- ~ APC in, for A~Tnr'-, nucleic acid probec and ~lia,, - :- kits. One plcf~ .lcd e ~~Aim~t pertains to the use of inh~",ll~, n.,O~ e analogs in the I and e~Lic synthesic of DNA h~ n~ probes in~ ~ g solid phace synthesis, template directed e~ - pol~,. -- ,. j,_l i~m and ~ n~ using pGl~.,.cl,.sc chain reaction mPt~n~c Another ' - - relates to the use of ~.. l.~nu.. ,~c~.lL DNA hyl~ -ti~m probes in the r~ n of specific DNA sc.~,._ æs, e.g., gene l"a~l)hlg and the detPrtirm and ~ g - of i..r,. lin-.~ and genetic diseases.
Specifically, the subject i~ _nli(,-- pertains to - - ' ~ analogs which are nuolcsc~ ..l and which can be 5..1,~l il u~Pd for naturally occllrring nllrlP~Qidpc in the synthesis of olig n - I~Lide 35 probes. When used as l.yl-- i~ probes~ the nuo,es~.ce of such o~ Pc can be used in a variety of ~lU~lUl~ to detect and identify specific genetic s~_ - This In~thnd~-logy is distinct from other non-,~ in~ ivc methods of probe detP~ti~n in that it does not utilize ~ ~ which have been coupled to en7ymes or other reactive proteins. Thus, ~k ~ ihc~

WO 9S105391 PCT/US94/09316 ~
21~ 5 7 5 0 herein are aprlir~tinng of inherently nuolescen~ nuclPnci~iP, analogs in developing l~ idi~;ation techniques for routine, ~ n~ hle clinical ~i~ncic The ~luOl_SCc;ul analogs of the subject hlveulioll are of three general types: (A) C-nnrlPnci~ie analogs; (B) N-nul lenc:~lP, analogs; and (C) N-~7~nllrleotide and N~ m~ lPoti~e analogs. All of these compounds have three features in commnn 1) they are 5l1U~LU1d1 analogs of the cnmmnn mlrlPnci~ipc capable of replacing naturally or~lrring n~lrlP~rirlPc in e~lllaLic or 1 synthesis of oligomlrlP{~tides; 2) they are naturally nuolcscenl when excited by light of the app.uplidte ~ length(s) and do not require ~Mitinn~l rhP.mir~l or en7ymatic p-u~s~s for their ~leterfinn and 3) they are spectrally distinct from the n~ C ~IPC c~mtnnnly ~----tr~ed 0 in naturally or~rring DNA. At least 125 specific ulupOull~ of the subject invention have been it~entifiPA These c~lllpuunds, which have been rh~r~rtpri7rd according to their clacs, sllu~;lul~, rhP~ir~l name, abscll,ance spectra, ~mirrinn spectra, and method of synthesis, are tabulated as shown in Figures 21A-21F-1.
Derluilio,ls. The following de~ilions are provided for ease in nn-lPr.ct~n-iing the desuli~liuu;
"CnmmonlyOr~l ringN...~ :1Pc"arethesixmnl-n...~.i. N-n Ir~ii~lp-cshowninFigure 1, which p~ te in naturally or~lrring DNA and RNA, enter into classical Watson-Crick base pairing, and are e~liv~ l~ non-llL~.l~nl under phycinl~gir~ in-~c The r~~
one-letter symbols in Se~lL_UCC; shorthand are A, C, G, T, U, and I for a~lPnncinP~ cytidine, gll~ni~linP. lh~ i liuc, uridine, and inosine, lcsp~liv~
"Structural Analogs" of the . ly occllrrin~ nn l~ P.s are slluululally related ~nnlPr.llPc that mimic the normal purine or ~lhuidillc bases in that their Sllu-;lulf~ (the kinds of atoms and their ~rr~n.L, ~) are similar to the cnmmnnly occllrrin~ bases, but may have certain mn-lifir~tinnc or ;1 h ~ lin- r which do not affect basic biological activily or bin~ hf....;.~l r~.. finnc Such base analogs include, but are not limited to, imirl~7nlP. and its 2,4- and/or 5-D--b~ ed delivalivcs~ indole and its 2-, 3; 4-, 5-, 6-, and/or 7~Ub;~lilu~p~i deliYaliv~, bPn7imi~l~7nlP and its 3-, 4-, and/or 5-~..h51il..l~ delivaLve~, inda_ole and its 3-, 4-, 5-, 6-, and/or 7- ~,h,lil..lPd d~,~ivaLv~ pyra_ole and its 3-, 4-, and/or 5-sllhctit--tP-d derivatives; triazole and its 4- and/or 5-snhctitl~tPli dc~ivalivc;D~ tetra_ole and its 5-D-~hDIil~lrd dt;livaLvt;D~ b~ ^ and its 4-, 5-, 6-, and/or 7-snhctitntf~i d~ivaliv~, 8-~7~ pninp and its ~ lu~-p~l d.,livalivt;s, 6-aLal~ ll~ille and its ~ v~ d~livaliv~s~ 6-~7 ~llr~ l and its svbstitllt~pd dc;livaliv~ 5-aLd~y i - ^
and its ~ub.li~ p~d d~livaLv~ 8-azal~ lhi--r and its D~b~ delivaLv~
pyTazolu~yliJ~ ilLe and its ~ul~lilu~ed d~ivaLv~s~ 3-de~ ;1; orotic acid; 2,6-dioxo-1,2,3,6-tetrahydro-4-~ylhllidil~c~l,u~ l;cacid;l)all~ilulicacid;u,ica~idi ethpnn~l - ';eti' ^ ylidi an allopurinol (4-hydroxy-pyra_olo 13,4dl ~ylill~ c); or their ~lul~;led d~,livaliv~S as dP-c- . il.~
below. Base analogs can also be any of the C-nurlPn~ pc such as are shown in Figures 4 and 5 in which the normal C-N bond between the ba e and the furanose ring is replaced by a C-C bond;
such bases include, but are not limited to, uracil, as in the C-~ e pseudouli.linc; 1-~ WO 95/05391 214 5 7 ~ ~ . PCT/US94/09316 hylul~cil;s(4)-~allJn~.rthfrl~y-l~2~3-tri-77~ ç~i(4)-f-a~ Aalllido-l~2~3-triazole;
3(s)-callJuA~ lclllylyJ~aLvle; 3(5)-~ albolllelhfJAy~ylaLole; 5-carboethoxy-1-ul~ aLole;
TnqlP.imi~7~ (in the C-~ 7~c ~7-p~ showdomycin); and 3(4)-~all)uAd~llido-4(3)-h~ A~JylàLole (in the ~nurlPn~i~7e ~ aLo.,"~ ); and any of the other analogs listed or inferred in Figures S t_rough 11; or their protected de,;~alivQ.
"Fluolupholc`' refers to a ~ ---e or portion thereof which is capable of emitting fluol~s~nce in a detectable range. For the nl.orf soG~It ~L, uc~ul~l analogs of the nl~rlP,otif7~ c, t_is nuo~csce.l~ typically occurs at wavelengths in the near ultraviolet (>300 nm) through the visible n~hc Preferably, lluo,~ce..~æ wil occur at ~ _~_lengths between 3'J0 nm and 700 nm and 0 most plf f~,~aJl~ in the visible ~ C between 3J0 nm and 5'J0 nm.
"Flb~ lt Structural Analogs~ are synthetic or binrhDmir~lly derived ~nnnnmDrir ~llu~,lul~l analogs of the Si~A CC~ nly occllrring N-nl_- lrn-i~7Dc (Figure 1), such as are depicted in Figures S through 11, which may or may not be capable of classical Vla~o Crick base pairing ~.1~----7i-~guponthe-..n---~ u,t;and/or~ P,inwhichtheyareused,butwhich are spectrally unique and di_tinct from the rx~mmf)nly orrllrrjn,~ ,L C in their C~'r,'ritiPA for selective f -- ;Iz.li~n and e - under physiolngir~l co ~ lilinnc For f~mplP the C-n-,- ~ 1P
rul~ . A is a ~1,, 1 analog of ~lpnncinp that can form equivalent donor/~ nl h~dlu~
bonds, but which has an -- ;I;.linn 'Yimnm in lig~n~ lr~f~c at 303 nm and an i-..-.... at 40S nm (Stokes Shift = 102 nm).
"Dc.i~a~iL~I" m lfn~i~e analogs are Lluo~ ~nL ~l1L_~U1~l analogs in which reactive or protective r~.-- 1;-- -~l groups are bound, covalently or otherwise, at the R4 through Rg p~ ~
of the h~ t~,.u_,lc and/or the Rlo(SI), the Rl2(3'), and Rl4(2 ) p~- ~i of the ~ly~sidif moiety.
Del;~alivQ at the 2' glycosidic position may include rluolQ~..ce ,~ energy transfer (E~RET) ar~:e~l~-.. or donors which enhance or accept and re-emit at longer ~ ,ngf~s the inherent nuorQce ~ -.r. of the ~luo,wcen~ IU1~1 analog itself.
A~JO1~ u- l~lide," "O1;E,, ---- l~ e," or "olig is a nucl~lidc chain ~ IU C;
c~ l~i.. i.. g at least two c nn1YOr~;ng~ -C or ~ lulal analogs. The "rl..ul~ce.l~ olig m. l.~lirlP probe" or nuule3~--l h~ lirll probe" provided herein is a nucleotide chain ~I~u~u~, as above, c--l.l i--il~g at least two ml - at least one of which is flu~)rcs~~
II~I..i-li,~li.-.~ is the pairwise ~nnP~ling through Watson-Crick base pairing of two ~r...,~ Y~Sing1e_Sf~n~P~mr~~ 1PA (see Figure 4), which may be DN~DNA,DNARN~or RNARNA, and in which the two strands may come from different sources. The ~nnP~lin~ is specific (i) for ' , ' y base pairs in which the h~dlu~,_.. bond donors and ~e~t~ are 35 oriented as in Figure 4, and (ii) for the cc ~' - y genetic se.~, of the specific gene, target DNA, or target RNA(~,C;illarle~ "target DNA/RNA") to which the probe is to be L~lidi~d. Compare, for f~mple the hydrogen bond pattern of ~lPnncir~e and ~ .~_iu (Figure 4).

WO 9S/05391 PCTIUS94l0~316 21~57~ 14 "DNAIRNA Melting Tl ..p~ c" and ~Tm~ refer to the t~ ...OI---e at which the .llo~n bonds between hybridi_ed strands of DNA or RNA are disrupted and the strands tP. into single strands, thereby di.-u~ling the 7Llu~;Lult; of the duplex or hybrid.
"Analogous nuol~F~ se ~ nce" refers to the nnrlP~ei~lP sequence of a polynucleotide which has been synthP-ci7p~ by any of the enzymatic or chPmir~l methods dP-~rrih~Pd in the present invention, but in which fluorescent nl~.lrnc.1r, analogs have been explicitly sl~hctitlltprl for particular ~ nly orrurring ~ P~ P~, e.g., the .~I.s~ irJn of formycin A-5~-triphn~phqte (E;'I~) for ~ ~cinP 5'-tripho~pl e (ATP), when using RNA pGl~ hse to produce RNA probes cornl' y to a ~les.clil)ed DNA tPmpl~tP In an analogous nuo~ cn~ sequPn~P the nuoles~nL nllrlP~ e analog has been .~ pA in the oE.~.,~ Lide chain at some or all pn~;l io~ in which the cu~ n.,-l;" g cnmmonly oc~lrring l~v~ lr~ e would have occurred in the sequence as dictated by, e.g., the I l' ", in the case of e~lL.ylllaLic synthesis. Similar prog-~...-..rA 5..1,,~ "~ canbe made using 3'-O-phn~phu~ P~ of the individual nll~.,cs.c l--analogs during standard phosphotriester synthesis. Thus, for; . 1~, the ~o~ y S~L'j1~ of the Chlam~ che~,.~ !('t7'~ MOMP gene, or its n~lol~.oen~ analogous Se~L_n~;, can be synthP-~i7P~ e~y~Lik,lly using dATP or dFTP, l~ ly, in the presence of DNA
pol~ -~P" dCTP, dTlP, and dGT~':
MO~'GENESEQUENCE(SEQ ~ NO.1):
AAC GTT CGA GAC GGA CAC CCC TTA GGA CGA CTT GGT TCG
COMI'LEMENTSEQUENCE(SEQ ~ NO.2):
TTG CAA GCT CTG CCT GTG GGG AT CCT GCT GAA CCA AGC
ANALOGOUSFLUORESCENTSEQUENCE(SEQ ~ NO-3):
TTG C$~ GCT CTG CCT GTG GGG ~-1 CCT GCT GFF CCF FGC

wherein the n..ulcAFc 1l~ deu ~l.lly~,i.l A C~ residues nr lPrlinp~ in the ~n~lngol~Q se~ ~~ are the :~ll U lUl;dl analogs of the deu~l n~;~-r ~ residues in the same relative pn~;Lio~ in the ~I~ *'Y S~
"FRET a~e~lol" or ''FlLol~uce 12~ Energy Transfer a~c~,lul refers to a rp ~yl~ Jf'.nt cLu,l,u~ ~re, or r~ ~uphore, e.g., a dansyl, naphthyl, anthryl, pyrenyl, lucLL~!h~ -nif-prone~ or ~u~ - moiety, which is capable of absorbing emitted light from ~-.olcs~n~ ~LI u~lulal analog donors and re-elui~liug that energy at other, longer ~ -' ~hQ- In the context of the present i..._,.lion, such sc~ y nuulu~hores may be 5cl~lil,~,l), excited as a second label, or may be used as a n OI~C~ ,c acceptor to broaden and enhance the primary nuulc~c~l.cc of the ~Llu luldl analog energy donor.

A. Sll u~,lul-~. Sources. Synthesis, and Dc,iv~ iOll of the Fluulc ,cenl NUrlPnQi~p Analo~s Briefly, the present ill~_ulion includes the heterocyclic pyrimidine or purine ~ Luldl analogs of the cnm~nnly oc~rring ~m~ l nn~ P, bases (B) which are ~uol~.,L under phyQ;nlng~r~l cui~ilions and which are linked by a carbon-carbon or carbon-nitrogen bond to the set of ~ 21~$7~

~lr~nnce rings (~lP-ci~ted F in Figures ~9) of ribose (Rl2=Rl4=OH), de~cyribose (R12=H, Rl4=OH, or R12=OH, Rl4=H), or dide(,l,yli~ose (R12=R14=H) and their dcliv~.livGs such as are de~crrihP~ below, and/or are apparent to one fa~iar with nnrlPoti~P rl- y.For the present invention, formycin, 2-amino purine ;ho~ r~ ~P and 2~6-diamino S libo~ P, all of which can (i) form the same or related base-pairing hydrogen bonds as ~APnncinP, and (ii) ,~h~ e sperifir~lly for a~PnncinP in Watson-Crick base pairing as well as in a wide variety of e~y.lldlic reactions inrln~ing nucleic acid replir~tinn~ ligation, and phncphrrylation, are used as r~lesGulaliVG" of the set of nuolGscenI ~n.. I~ c and n~. Ir~ e analogs (Figure 4). Related properties and parallel claims obtain in the present iuv~,..Iion for all other Ih.olG;,~.II analogs of g~ , cytidine, IL~,I,~dil c, uridine, inosine, and their d~ivalivG.7.
(1) Sl1U-,IU1GS of the llu-~lr~ le analo~s. The generic purine and ~,illi.lile SLI U~,IU1G., of each type of .711 U~lUl~ll analog to the c~mmnnly o~ I l il.g n-lrlPn~i~P-c are given at the top of each of Figures 5 through 11, below which are lcp.~GI.l~ltive . ~ f F~ Of each class of analog. Only ~,~lupl~F, of the purine analogs are given in Figures 6 and 7, since the known ~ idi.. c analogs have already been illnctratP~ in Figure 5. With the GJ~IiOll of the N-' ~ '^ analogs, which have only sl~ " i~ l lc at R4, R6, and Rg, the generic ,I, u~w~F, at the top of each page show an oval encircling the part of the .7I~U-,IU~G where ,~b.,lil"~in~e to the hetcI.~.,lic base llictin~lich the analog from the; - -ly oc~lrring N- '-clpirlp-c shown in Figure 1.
(2) Furanose moieties common to the nuor~s~llt nl.. I~ le analo~es. The nnmhering of the sugar carbon atoms in rll-~ ce is 1~ to 5' as ir~lir~tP~ in Figure 2; thus the base, B, is r~?nn~ to C1 of the sugar. The filr~nneP moiey of any nuoleF7c~llt heIelu~,J.'~
claimed in this invention has, in c~mmo}l with all other analogs, the set F, of glycosides and ,,--h~ lllP~ glycosides, as follows: s~ lionc can be made, in pliU~ilJIe, at any of the 5 sugar carbons; the subset F is defined by derivatives and/or ~- k ~il 11l io--c at posi~ions R1o, Rl1, R12, R13, and Rl4 which (i) are a~a-~,nL to one skilled in the art, and (ii) are the fu~ano~,yl d~livaliv~c of all the n..o,~,s~n~ r analogs claimed in the present iu~_nliou. These include all phn~l.hn~uu5 s~,l,,,lill-lin~c (e.g., triphncphqtP, lhio~,hn~h~tp~ a_inoph~,pl~ P" etc.) and all protecting ~k~lilulil-l~c (e.g., ~ Yytrityl) at position Rlo. For all glycosides, F, in Figures 5 through 11, R1o7 R11, R12, Rl3, and Rl4 are defined as follows: Rll and Rl3 = H, Rl4 = H, OH, or ORi; Rl2 and Rlo are either H, OH, ORm~ or NHRp wherein (a) Rj p,~,t~ling groups are ypically lower aryl or alkyl ether, e.g., methyl, t-butyl, benzyl, o-niI,~be~l, p-niL.ubc~yl, o-nitrophenyl, or lli~h_llyl~_lh~l, or a lower alkyl or aryl ester such as acetyl, benzoyl, or p-nillube~vyl~ or an alkyl; acetal such as tetrah~ro~.a~l, or a silyl ether, such as trimethylsilyl or t-butyl-di.. ,_lh~ilyl, or a sulfonic acid ester such as p-tnlr- lfnnyl or I ~ Ih~ .. 1fnnyl; or halide such as bromine, fluorine, or iodine. A~ tin~1 c p'.~ of suitable blocking groups may be found in Green, T.W. (1981) P?.,.~. c Groups in O7~anic ~vnthesis, New York: Wiley & Sons.
Alh,~ali~ Rl4 may be a FRET dt;li~aliv~ inrlllAing, but not limited to, such ~luo~ùphorec as 7-[3-(chlorodimethylsilyl)propoxy]-4-methylcoumarin, 0-4-methylcoumarinyl-N-[3-triethoxysilyl)propyl~ll,allldle, and N-3-triethoAysilylpropyl)dal~ylalllide; (b) Rm represents an app.ùplidte pluL~Ih~g, s~ i,-g, or reactive linker group inr~ ;ng 2' or 3'-amido, 2' or 3'-azido, 2~3~-ul~alulaled~ and the subset of phosphorous delivc-lives involved in I or S enzymatic syntheses of olig~ rc having a phosphate ester, thiopho~l,hale ester, or aminophosphate ester b~rl~hont (c) R~ is any common, standard nitrogen ~lole~ g group, such as those r~mmonly used in peptide synthesis (Geiger, R., W. Konig [1981] In l~e Peptides:
Ana~sis, Synthesis, Biology, Vol. 3, E Gross, J. M~ nhnf~.r, eds., ~r~ienlir Press, New York, pp.
1-99); this ir^l~lr.~, but is not limited to, acid-labile protecting groups such as formyl, t-13ulylvAycallAJ''yl~ bc~ A~ ûllyl, 2--chlolul,~ luA~_ IJVIIYI~ 4--chlolul~cllL~lu~_allJullyl, 2,4--dichlolu~.~ylu~. I,u~l, fulrulyluA~ llyl, t-~hl;lo~_arlJu~l, ~ m~ntyluAy~lbl)llyl~ 2-ph~,llyl~lu~JI-(2)-~A,~ bU~ 2-(4-biphenyl)propyl-(2)-uAy~ll,ul,yl, lliph~ ylll-~,ll~l, p-a~ diph~ Lyl~ di-p-anisyl ~ l, 2-nitrophenylsulfenyl, or di~,h~llyl~hosphinyl;
base labile protecting groups such as L,in~-ol~acelyl, 9-nLale~ Ih~lUA~ l,o,.~l, 4-toluene-sulLu~l~ loAycarbonyl~ h,Yl~ulrù~ lLyl~lbonyl, and 2-cyano-t-bul~luAy~lbuJlyl~ as well as others, such as chlolc,acelyl~ yl, 2-nitro-benzoyl, ~lilhi~ yl, maleoyl, i~olliwlill,Yl, 2-bromoelhYlu~ IJU .,YI, and 2,2,2-trichlolu~ A~ l,u,.~l, alternatively, R~ may also be any reactive group d~liYali~ le with a detectable label (NH2, SH, =0, and which can include an optional linking moiety in~- ' G an amide, thi-~ethrr or disulfide linkage, or a romhir~fit~n thereof with ~d~ - -I variable reactive groups R1 through R3, such as R1-(CH2)X-R2, where x is an integer in the range of 1 and 8, ._, and Rl, R2, and R3 are H, OH, alkyl, acyl, amide, thit~ethPr~ or disulfide) or any linker or spacer r ---- ~ --i-.g as a homol~ir ~ 1 or hetelubir, i----l linker i- I-..li"g, but not limited to, such reactive groups as hydr~7~
m~ ,r'~ ,.hlr. diols, and ~ ;-.;-,,y~lyl groups. At most only one of R12 and Rlo may be NHR,~.
The ill~_alion further includes novel ph~lcpht~ "i.lil~.~ having the formula:

5, o 3' 2 ' O
p R14 R150 N(R16)2 ~WO 95/05391 2 14 5 7 5 ~ PCTIUS94/09316 ~r wherein B is any of the " w~en~ nllrlp~Qi~le analogs ~ipc~ihed herein and Rlo, Rll, Rl2, Rl3 are as defined for the set of gly~ Pf; F, as above, and Rl4 may be either H or OH. Rl6 =
lower alkyl, pl~,f~ bly lower alkyl such as methyl or isopropyl, or heterocyclic, such as morpholino, pyrrolidono, or 2,2,6,6-lci : ylyy~lulidono; R15 = methyl, beta~y.-nr~ l, p-nitrophenyl, o-chlolululr~phc~l, or p-chloluphcuyl. All other R groups are as before in.-ll-.lin,~
those identifying spacer or linker arms of from 1 to 25 carbon atoms in length. Prior to the synthesis of the r ~ ~ at R12 in order to (i) preserve any reactive ~uh~ on the heterocycle which are ihllpol.~llt to its partirip~otinn in Watson-Crick base pairing, and (ii) render the amidite cn~ le with the DNA or RNA chain .~clllbly ~ y, the base moiety B in the phn;,~.k--".. -~ ilP can be plu.~led, which generally involves a~,yla.iol or ~mi.10tinn of the ~,AU- y~ lic amino groups and 1P-c but is not limited to, acetyl, benzoyl, isobutryl, ~u~iuyl, phthaloyl, or p-anisoyl; such amidine groups include, but are not limited to, ~liluelL~lr~ r, di-n-bulylr(~ ",i~ or diluelllyl~r~ , if B is ~-b~l il Ul-P~ with other reactive groups such as carboAyl, hydrûxyl, or l.._lcdplO, these are applupliately protected as well.The present iu~,~,nlion e -- ~ pA ~f~; the synthesis of oli~ -- leolides on a solid phase support, wherein the nlil iS reacted with the protected I1UG1C~ 1I n~ lr analog pl- - r as ilhlctr~ted in Figures 5 through 11 and d~,livali~d as in the ~ ule, above.
iitinn~lly, the present i,.~ _u~Oll includes the novel " rwc~nl nli~ ' S lides having included in their sf.~ ,c at least one lluulw~ P~ e analog d~ ali~ as the phn~-k"""--i-l;lc in the ~ lulc, above. Moreover, it is yet again another aspect of the present invention to provide ~Lolf-s~lll nlig I~- l'~Lid~C made by the reactions of the arur~- -- -.lir l-rd n..olf,s~ut analog 3~-O-phn~hn~ ,,--i.lilPc which are bound to, or have been bound by, a solid support.
(3) Sources and other l~lc~-.,.lionc of the fluorescent ~IlL_lulal analogs.
rOl Ul.~_in A is isolated as the, ;l,. ~ ide from the culture broths of Nocardia i,.~,f~".,.a. The antibiotic is also isolated from culture broths of Sl~c~/c",.. ~ lavend~lae and Sl,~l"".~e~
gun2m~l~nQ-i~A, and is one of nUll~,lUus llli,lUbldl C~ n~ L~ P- analogs of the N-nn^lPnQ;~lPc c~)mmnnly found in RNA from all sources. The other naturally or~lrring ~- il ~ " lr~ PC which have been isolated from microolga~c (Figure 5) include fulluy~ in B, uAurùlll~_iu B, pseudou,idi~_, shu.. .. u.~_ ~yla~uluy~ill, and ~LU~ . Formycin A, rullu.~. B, and uAur~Jlluy- in B are C- - - '^ or pyrazol~ " lr~ p-c of the class shown in Figure

6 and are ~Lll - 1 analogs of ~ r-~i~r,, inosine, and h.~ , a I~Jla~u~Jlilludi~c ~Ll~_lulal analog of g - obt^inP~ from natural sourccs has not been reported in the lit~ lule but can be lly synt~- ~' from the 2-chloro-rul~.~ B or its deoxy form. A thorough review of the biosynthesis of these COlupOu~lb iS available in Ochi et aL
35 (1974) J. A~. ~;' . tir-Q x~iv.:909-916. Synthesis of the N4 and N6 de.i~LLiv~ of the C--II-- 1F~OL;r1PA are ~1P ~. . il.cd in Lewis and T~ ~ ' ([1980] J. An~ ChemA Soc. 102:2817). Corresponding syntheses for the isomeric ~uo~l~Jlo-[3,4d]-~.~.i---i~li~ c are in Wi~,~u. Iri et aL (all others are Culll.-.~ .~ ially available in ribose, and several in deoxy and dideoxy forms, in^ll~rling the WO 9S/05391 2 I 4 5 7 ~ d PCT/US94109316 coti~lps and dea-a nucleotides~ or can be synthpci7~A de novo~ e.g.~ 7-dpc~7o~pninp (Gerster et aL [1967] J. Med. Chen~ 10:326)). C-nnrlPnci~p analogs of the pyra_olo-s-tria_ine class (e.g., pyrazolo [1,Sa]-1,3,5-tria_ine) were prepared from amino pyra_ole-C-nllrl~onci~1P as originally r~p~crrihed (Fox et aL [1976] J. HeterocycL Chen~ 13:175).
S Production of the deoxy. dideoxy, and phosphorvlated forms of the n~.O~ribon-lrlP~ci~lP, analo~es. ChPmir-ql syntheses are available in the literature for the dPlivdliLalion as 2'-deoxy forms and 3'-deoxy forms of N-nllrlP~ci~1pl~ ethP~n~ PQ~ as well as the C-~m. lr~ P~ c (Robins et aL [1973] Can. J. Cher~ 51:1313; Jain et al. [1973] J. Chg. Chem. 38:3719;
DeClerq et aL [1987] J. Med. Chem. 30:481). Similar ~ c~u,es obtain for the deoxy forms of the q7~nllrlP~Oti.l~P$~ (IP~".U,.. l~lides and are found in the same and ati~itintlql sources (e.g., Robins et aL [1977] Can. J. ChenL 55:1251; DeClerq et aL, supra). Protocols and p,o~lu.~is for synthesis of the 3'-azido, 3'amino, 2',3'-~ tPi~, and 2',3'-dideoxy analogs are as reported (Lin et aL
[1987] J. Med Chem. 30:440; S~ldlill[J.. ~', P. [1987] Synthesis 10:879). Protection or derivqti7~tif)ll of the 2'-OH with silyl or FRET moieties can be done as by Peterson and Anderson ([1989] Silicon Compounds: Register and Review, Petrarch Systems, In, pp. 60 70).
Reported herein is the novel 5.~ I ;nn of a cyclic protection proc~lule from the ribose to the deuAylilJosP~ Cu~ of C-~ n~ by which only the 2'-deoxy form of the analog is p,o-luced, and by means from which high yields can be o~i - d without the difficult p.., ;ri. .~ "
r ~ ~ y to separate the two isomers p,udl,ced using the a~tu~ob _Iylyl halide ~,u~lul~s cited above.
For cnLylllalic syntheses, mono- and triphosphate forms of the nurl~nQi~lf, analogs can be prepared by en_ymatic phosphoiyldlion with, e.g., poly~ e kinase using PQ~tqhliQh~d pluCC-~Iulc~5, or by rhf~ ql phn~ ,ylalion. In general, the 5~-~uonorhnQp~qt~A are ~l~al~d rqlly by the POC12 (Smith and Khnr.q~o [1958] J. ArtL Chern. Soc. 80:1141; ynQhil et aL
[1967] T~.. '.e~,. LetL 5095). The wllQ~onding trirhnSphqt~-Q can be ~ lly ~ - - 1 according to the same authors or Mi. hrl~o,- ([1964] P:~chi)n. Biophys. Acta 91:1); or Hoard and Ott ([1965] J. Am. Chem. Soc. 87:1785). That is, the ll-onuphn~ it~-Q are treated with carbo~iimi~le (CDI) followed with llibulyl ~ pyrophnsph~qte tO give the ttirhn~lJllnl~ylâted form. Where it is dQired to phn$l~hf~lyldlt; analogs with exposed amino groups, such s~ stit~ tQ
can be IhinS~tylàled by ll~all.. _l.t with ethyl trifluorothinq~cetqt~ acw,di lg to the ~lluc~luie of Thayer et aL ([1974] P ~h~J77 J. 139:609).

B. Svnthesis of Fl~lole,cenl Oli~Jnu-l~Lides The prQent i{.~ lLio,l presents synthetic methods for the intro~ctinn of one or more of 3~; the Lluol~s~nL 1~ e analogs of the c~mnnly o~ g nucleotidQ into synthetic Ol;L~ )LideS.
(1) Use of lluul~ PhOSPhOt~m;~l;tf'-e F1UO1~~ L Fh~ .hO~ ..;tl;t~-e can be ~WO 95/05391 214 5 7 5 0 ~ ~ ~ PCT/US94/09316 According to the present invention, I:luu~cenl residues are introduced into ~h.o.mir~llysrth~.ci7~d oligo...~ ~lides by first srth~Ai7ing the protected 3' O phocphnr~mi~lit~ of a nllrlPn~ analog, e.g.7 2'-deu,.~.,..~_h, A7 the ph-~cphor~mi-lite is then substitut~d for the c~ cpn~ g standard - pkn~ho".. ,,;-1it~. in this case deoxy-~ nocinf~-3' O phncphor~mi~1ite and reacted with the olien~ e being srth~-ci7~ on a solid support using standard phosphotriester ~h~mir~l synthesis. The,B-cy~rloethyl dc-livaliYcs may be scl~;li-Y~ly inserted at any desired position in a cl ~mi~lly srth~ci7p~ oli~,.----,. l ~lide to produce olignm~M of prescribed sul..c,l~7 of 60 or more bases in length and carrying any pr~et~rmin~d number of nuol~x7cent bases.
For ~ r~e~ non-self-L~lidi~g olit,oln~.lr~tides were srthf~ci7p~ which had the pclf~lly alt~rn~ting sc~ n~es, [AC]X and [FC],p where x is the number of AC and FC dimer pairs and x had values of x=1û, 15, 2û, 25, 30, gave nearly irlPnti- ~I values for both l~,~eLiLivt; (>98%) and overall synthesis yields, and pl.,d-,ced oligomers which differed only in that [FC]X was l:luores~"l, whereas [AC]xwas not. Both olignomers hylJlid~ed sperifir~llywith ~ rl y ~lt~rn~ting oligom~.rc of the sequence [TG]X but not with th~ sch_s or with nn~..-u i-' Y
s~u~.~ces such as [AG]X and [TC],~ as ir~ t~d by (i) e~ li.. bromide staining in agarose gels and(ii)themeltingbcL~.y;..lofthehybrids. Equivalentvaluesofthemeltl-~n~;linl~ es in 0.075 M NaCl for the [FCk:[TGk and [AC]X [TG]X hybrids varied by less than 1C for a given value of x (length of o!ig - s Lide). Spe~ifiA--q-lly, one aspect of the present i~ nlion involves the synthesis of 3'-O-phncrhnrqmirlitPc of the nuol~ t nucleotides and of their llLo~cs~4nl ~Llu~lul~l analogs, the use of amidites to synthesize highly nuol~,s~nl nlig.,ln~ lri!l;~lp-c having plcsclil,ed sc~u~ce;s and the uses of such olig.--~4~lr~lides as qmrlifiA-qtinn primers, nuul~,S~ -l olil,..n- l~Lide "tags," and h~-;.li~ ~in~ probes.
(2) Use of nuu,~ce..~ pc,l~,.il,ol~u~lGolides and polydeuAv-il,ollu~l~Lides.
Fluol~nt pol~ n--~ P~Lides and polydeoxy-- ih.~ ) id~s of pr~ lil,cd sequences can be synth AAi7P~ C~lllaLically using DNA trmrlqt~-c from a variety of sources inA~ ing those p~ cd by 1 - 1 synthesis, cloning l~h~ L~i, or obtained from genomic DNA. R~-~,.,~livcsyntheses of RNA OIigJ ~ PC using three such DNA tc,..plalcs, E. coli RNA pol~ _-ase, the rNTPs cytidine, uridine, and ~ i.nC.i~.r, together with the ribose trirh~-p -'A of either formycin A or ~ n~;--P are ilhlctr.qt~d in Figure 12. A l'Cpl~C~ livci ~,,..llll~,lliC synthesis of an RNA
probeusingat~- .pl~bearingdil~;~llalviralpromoters,theviralRNApol~,.~cs,therNTPScytidine, uridine, and ~;, A together with the ribose triphncp~hste of either rullll~_iu A or q~lPnncinP is illl-ctr~qted in Figure 13. S3~ ic polydeuA~libo~n~ OI;~1PA have been rnade by "~2'-deuA~ hlA-5'-triphngp' ote (F~) fordeu~ d - ~ ~;l.h.~ ,h~l~; (dATP) in standard DNA polyerase syntheses and in DNA ~up'~ -- using th ~ ~'^ DNA
pol~_.~c enzymes and the pGl~ ,.Ase chain l~a~,liOIl, the CUIl~ UlheS~
have been achieved using the same reagents and plu~ulcs but with the follo~nng ...O-Iir;. ~l ;n..~
(i) syntheses using such DNA pol~ -- as Klenow r ~ l or ~n~lirif~l T7 DNA POI~ ASC

WO 95/05391 2 1 4 S 7 ~i O PCT/US94/09316 ~

inw~ tJulal~ d into one strand of a duplex at the beginning of the se~ .I"e that was to be u ed as the t~mrlq-te and the corresponding primer was used to initiate all syntheses; (ii) primers coll~ple cnlaly to only one strand of a template were used in qmplifirqtinn as is commonly d~-srrihed as asy~ ic PCR; or (iii) paired primers in which one of each pair of primers wa~s S coupled to a linker such as biotin were used in standard DNA amplifirqtinnc such as PCR, but one strand was pl~,f~ ,rlliàlly removed by subsequent icolqtinn such as by use of an .lYidill~/lal~ d column or ~ g. lir beads. Comrq-rq-hle syntheses can be made by other s..~ i....c inr~ inr, e.g., the nuolr~.ll N-~-u- Ir~ P.c 2-amino purine, and 2,6-amino purine (also sllh.ctitl~t~ for q~d~nncinr-5'-triphosphate) and either of the r- ~ esc,-.lt C-nllc1el ci~le ~ hOipàlcs of formycin B or 5-amino-formycin B (~ ted for inosine trirhncphqte and gllqnncine~trirhncphqtte, r~e, ~ ly) in either their ribose and deuAylibose forms.

C. Labelinr of Fluol~c~ Polynucleotides RNA and DNA can be e.,~ylllali~lly labeled by several methods inrl~lr1ing, but not limited to, (i) 5' DNA end-labeling using both the forward phosphorylation reaction (Ri.hal~on, C.C.
[1965] PNAS 54:158) or the ~Yrhqnge kinase reaction (Van de Sande et aL [1973] P L i~ hy 12:5050); (ii) miAed primer labeling by . ~ fr mixed se,~h-, _e hr~q~le. .-,YIlu, l~lides qnr~ql~l to rectrirtinn fr.qgmrntc (~,;ul)e-~, A., B. Vogelctpin [1983] Anal. Biochem. 132:6; Feinberg, ~, B. Vogelstein [1984] AnaL BiochetrL 137:266); (iii) 3' DNA end-labeling using the en~yme, terminal dCoAy~ CICOIidyl l-i~ f .. ,-ce, to catalyze the ~ addition (Okayama et aL [1987]
Methods F.~ )L 154:3; TT. ~ . L- " G., J. Messing [19871 Methods En?yrnoL 154:28) of - ~- units of the deu~-i~ ,h~C, or single ~ innc of deoAytrirhr cphqt~c of several of the ~ ,~cc..t n---l~n-i~1e analogc to the ternunal 3~-h~.llrJA~I of DNA i~ ol~, i, l...l;"g nonnLol,_,c~ probes of pr~il~ed sc~l.,,.._e, e.g., the Chlamyd.a tr~chomn~ MOMP
gene probe srthP.ci7 ~ as ~P5f rihed below; (iv) ligase labe ing in which non-fluoreswnt "sticky-ended" or "nicked" RNA or DNA nlig.,~ 4I;-IPC are labeled by ligation with the a~lop.i~le IlI.OIf ;~Wlll RNA or DNA nlignmPr.c (Pl- ~ LKB [1989] Analects 17.2; Helf~nan, D.M. [1987]
Metho.ls En2ymoL 152:343); (v) nick ~ cl ~ in~ in which DNA polymerase is used to i~cul~ol~l~
the trirhr.~ . c of the nuol~wn~ analogs randomly in an eAisting DNA strand in a duplex (Meinkoth, J., G.M. Wahl [1987] Methods En~moL 152:91).

D. ~h~ IU l ;~-I iO n of Fl ,-esceul Oli~onucleotides of Plcs~ ed Se~u~nw-c IIyl,~ inn, thermal melting, agarose gel f hqr~ctl -i~liOu and nuo., ~,'et~tinn studies were used to ,1- " l~ . i,,~. nli~nnf~ 4tif1f~c of ~ il,ed sequenws. In some ca es, the n-,o.~wnl nlip~.n.. l~olif.'P.c were wlu~' - y to known se4.. n~ of target DNA from clinically ilul,ol~nl p~thf!gf~nc or mutations, e.g., the MOMP gene s~u~ w from Ch~ Ain trn~7wmnt~'~. In these studies, the tf---~ used for e~ u~lic synthesis of the nuOlCS~
olig.---... k~lides were the cloned ~ ~ a so intP.n~'P~ for use later as the target DNA in _WO 95/05391 PCT/US94/09316 ~ ~14~7~Q

subsequent h~ ion studies. Hyl ~ ;".finn of the nlig~ln.. lr~tides with target DNA results in ~ f...~ -g of the nLol~,scellce of the ~llu~,~ul~l analogs in a nuor~nt probe, which Lluoles~.lcc is recu.~.cd upon dPn~t-lration of the hybrid, thereby proving that hyb~ tif)n has occurred. The seLf~ linl~ of the synthetic nli~nnrlPoti~e poly(rFrU), which is liec~ceed at length, below, is ~cscnlalivc of the results obtained in such ~ .pelil.. ~,nL~ and is ~-----"-~ A
in Table 1.
A ~lcf~ cd process according to the subject i~ ,..Lion involves four basic steps. InitiaLly the nuOlw~ llu-;Lul~l analoge are rhPTr ir~lly or binlr)gir~lly srt~A~i7PA and, where app.up.idle~ further delivaLi;Gcd as required to synthesize a nLJl~sccnl olig~n~ P probe.
0 Second, a DNA or RNA probe m~ lPrlllP cnml ~ -~t~ry to a nucleic acid sample of interest is cu~llu~led to have ll~,orcscenl nllrlPn~ e analogs which can be (i) distributed randomly or at specific lnr~ti~nc throughout its length, or (ii) placed as terminal labels as ~PArrihP~A below. Third, the nucleic acid sample is then s~ PA from unreacted ~ and can then be cl-~,~- t~
directly, ueed as an PYtrir~;r non-specific label for tagging specific hyl-- ~ ,.l inn probes, or used directly as a h~ n probe. In the latter case, hybri~li7~tilm may take place on a soLid phase to which either the target DNA/RNA or the nuolcs~nL probe has been immobiLized such as in SouthPrn blot l ~ f - ~, or "Dot-Blot" t~l lliqll~ or it may occur in solution (herein, "solution h~/l,- i~li,~l i- l~"), ~ft~,l whicL probe/target hybrids are sep~r~tPA from unL~ idi~cd probes by simply washing or filtr~ti~n FinaLly, the lluu.es ~ of the ~lig~u~ ~lides L~lili~d to the target DNA/RNA is detected and qll:~ntifiPA

E CU~ lion of F~UOre;S~.I Probe Molecules In a~lddllce with the present invention, a pl~ PIe~ IpA ~ r~ JL ;~1P, analog or mixture of nuo-~enl analogs is s-~h~ lPd spP~ifir~lly for one or more of the non-n~ ~ccnl commonly ol - -. - i.. g ~ Pc and is then iu~ol~olated into DNA or RNA ~ ,roti~lpc to create p~ il,~l se ~U~IA~S~ The ~ lil,ed S~L_nC~ may be chosen to be equivalent in their Watson-Crick base pairing to a lu"l~lif~e sc~ ,..cc constructed from normaL~y o.~ .i..g : ~- ~ and complementary to a given target DNA or RNA seaA. ~, such nLJl~cent probP~c are said to be 7r~qlf gOllc to the ~ -y Se~Lf ~ of the target DNA or RNA. The n~olc~ce.ll probe may be synthPci7PA- by either e~ylualic or rhPmir~l synthesis for ~b'f~ t ap~ lif~lc such as (i) I~~ probes, (ii) ~mrlimPrc for direct f~ m of ~mrlifiqhlegene s~7Avpnrp-c ;; ' ~ to a gAven set of prirners, or (iii) as non-specific ulli~ dl" labels which can be 7tt~rh~7A to specific L~. i.li, .~if7,n probes by, e.g., ligation.
F1~JIL~CenL I~L I~A~ci~e analogs of the commonly oc~lrrin,o ribo-, deoxy-, or didc~ ~Lides can be h co.~ .aled into nucleic acid polymers using one of severalotherwise Cu..~_llLiOllqAl Cl~ aLic and rhPmir~71 t~ LAcs i-~ li. fo, but not limited to, those flP-cr iheA here.

WO 95/OS391 2 1~ 5 7 ~ O PCT/US94/09316 ~

(1) E~ .alic svntheses. Enzymatic syntheses include:
(a) the use of the enzyme DNase I to introduce small "nicks" into one strand of a double str~n~e~ DNA duplex. The holoenz~yme folm of ~. coli DNA polymerase I can then be used to extend and repair these nicks using a mixture of fluorescent nucleotide analog triphncph~tPc e.g., deu~Lul,lly~ 5l-trirhncph~te (FTP), with commnnly oc~lrring deoxynucleotide trirhncrh~t~ c in the reaction mixture. This method ihlLluduC45 a large number of rl-lolu?hores randomly throughout the DNA polymer, in~ ing both strands of the double heli~c In practice, the cx)mmnr~ly oc~lrring nucleotide, in this case d~t~ S' triphncph~t~
(dATP), can be ~ entirely, and the dF l ~ suhctitllte~ in its place, without ci~nifil ~nt loss of synthetic yield, loss of Lyh. ;ll;".~ inn ~e~ ;r~ y, or strength of duplex form~tinn as ~.~uled by the values of the DNA melting t~
(b) the use of a variety of enzymes, i..- h--li..,e the Klenow r,. ~5,. ..~
of DNA pol~.~.elase I and the T4 DNA poly~_.~se, to fill in ovPrh~nging single stranded regions of DNA produced by the prior actions of ~ linll enzymes. This method cor~rpntr~tp-c the nuOr~SCCn~ analogs at the end of each DNA strand. Siilarly, nl,or~nt DNA oli~ f~~ Pc c~...pl~ -y to a specific DNA template can be srth ' (i) by using DNA fr~gmPntg and E. coE DNA pol~el~ie, or (ii) by CO~Ll ..~ Ling a l~--...hi.-~ plasmid ~o~ e the primer site for a specific primer such as the M13 forward primer i~--...~.l;~l~l~, 5' to the desired DNA template sc ~ ~. The DNA pol~ se will synthesize a compl y DNA mr~ using deu~lil,onucleolides or other deoxyanalogs ir ~ hll1ing, e.g., d~ l~ a~s a sll~ stit~lte for dATP, present in the reaction mixture;
(c) an incol~olation method which also plu~ ~ a terminal C~ f-~ -l-- of nL~ scenl analogs involves the use of the "tailing" enzyme, terinal deox)rnucleolide ! . ~ f -- V 'f', to add a 1 - pGI~ or "tail" of n- ul~ nl deoxy analogs to the 3' end of DNA nlignmPrc In practice, the yields obtained in the synthesis of homopolymers when D"h;~ g nuU~ ll analogs for the comml~nly occ~lrring ~m~ lP-$ iS cil,,.;r;-~n~ly less than the yield obtained in the synthesis of he~lupolymers. AlteluaLi~,ly, a single rlbO~III
nllr~ e analog may be added to the 3~ OH of any ~li~mPr using the same en~ne but the dideoxy form of a nuo.esc~l,l analog or a 2'-protected ~uorescent analog, inrllllling the FRET
protected analogs, in exactly the same manner in which, e.g., dideoxy ATP (wl-lc~ l), is used.
A third allci.~Li~e method of Pnlll~hPling hyh. ;.li, .li.~" probes utilizes the action of DNA ligase or RNA ligase, by which non-specific double or single str~ d n..olc~scen~ r~ ,o.. .~. Ir~l i.iP.C can be covalently coupled to either the 3' or 5' end of specific l"iL - ;.1;,,. l ;.-.. probes; the " ~ cs~nl ~lig.l.. l~Lides used in this fashion do not nP~Pc~. ;ly palLi~i~Jale in the Watson-Crick base pairing which ~le~ s~ ;ri~ ;ly of a probe, but may act solely as a generic or universal '' es~l,~ "tag" With each of the ru~CgJiug methnfic, the DNA probes are double stranded and must be denaLulcd to single stranded form using either heat or alkali treatment prior to their use ~WO 95/05391 2 ~ ~ 5 7 5 ~ PCT/US94/09316 (d) an incor~o~ ion method, which can also be used as a standard method of prof~ctinn of fluorescent probes having a ples~"il,ed length and sequence, using standard methods of DNA qmrlifir~q,tion or replication and one of several available DNA
- pol~ scs, inrl-~fiine but not limited to the th.ormnctqhle DNA pol~ll,.,.~es, e.g., Taq polymerase, ~-~lified 17 DNA polyl.. e.ase, Klenow ~r.qgr-ont, and T4 DNA polymerase, but ~,b~ C one of the Lluol~c~ll~ deoxyribonu~lculide analogs, e.g., 2'-deu.~yfull,,y~,u.A-5-triphosphate or 5-amino-deu,~yrulluy. i.- B-5'-l . iplln~l,hqtf- for ATP and GTP, r~ , in the mix of nnrlf otiflf, trirhf-~pl qtf c The lluorc,scent flligf)nn~lf~tides are equivalent in yield and length to the non-rluol~,.L oligomer made with the commnn1y oc~ g nucleotides and h~lidi~ to target t~.l.?l~l-;DNA and display the same thermal stability and capacity to stain with ti~hifli~.. bromide as do the no~uo,c,ce.~L controls once the hybrid duplex has formed. In such qmrliri~ n~, the ~lU ' '- - of rluo..~ L f ligo-m- Ir~Lides can be taken directly as evidence of the ~.~sence of a particular S~L~11Ce, or the identity can be further establishe~l by (i) h~b- i.li,,.l if n with a defined c~ml 1 y probe, and (ii) sf~u.,.._illg to establish the analogous s~~ ; and (e) the use of ~uol~æænt RNA f ligJ~ ~Lides f~m~ y to a specific DNA template which can be srth~ ~i7~ d (i) ~.. 1 . i- ,.lly, by using DNAr;~--.. t~ and, e.g., E. coli RNA pol~ - as illllctrqtf ~ in Figure 12, or (ii) &o~---.--- l~ i- ,.lly, as shown in Figure 13, by . U~Lil g a .~ ~ ...hi'-:~lll plasmid c unLaill llg the promoter for a specific DNAf4~p~
RNApo~ l~ei....... f~ lf~.lyS' to the desired DNA sequence which is used as a t~ mrlqte e.g., a DNA tf~mplqtf~ bearing a T7RNA PO1~ ~~ promoter i"""f~ ly 5' to the fr.qgmPnt of a cloned Chl~znyflia MOMP gene r.,.~;, - ,~1 which has the se.~. which will be used as the target for L~-;-~ l;nn with the probe. For most al,l-li--linl~, ~y~,llic synthesis is the ~l~f~,,lcid method, and the ~.-~,ondillg DNA~lepPn~lPnt RNA pol~ ase will synthesize an RNA
mnlerlllP using ,ibon-~ , e.g., F l~ as a ~ r for ATP and UTP instead of TTP, which is the analogous r~-~r' to one, and only one, of the two strands of the 1. PI~IP The resulting single str~r~lP~ probes can be used directly in a :,ubse~ll Lyl,-i~ linn reaction without a ~lf --~ step.
(2) ~'hPmir~l syntheses. The protected n~o~es~ul deoxyn~lrlPn~ e analog-3'-O-phn~ph~"i----i~lit~, typically those in which Rlo = ~iimpth~ Llilyl~ Rl6 = i~op~uy~l, and RlS =
methyl or beta--y~ hjl, are coupled to the S'-OH of a growing olignnnrlPotide attached to a solid support using standard phn~.h--"....i.~ DNA synthesis te~ h. i~ucs (see ~tkin~on, T., and M. Smith [1984] In O~ ;A~ Synthesis: A Practical Approach, M.J. Gait, ed., IRL Press, Oxford, pp. 35-82). Solid support-bound nlit,.,--... l~Lide, which has already been acid washed to d~1UILCL the 5'-OH group, is reacted with 5'-trityl protected deo~y - ~IP, analog-3'-O-phn~phnrami~itP in anhydrous ~ n~.il ilf in the pl~ e of tf tr~7t)l- under argon, washing away excess reagents, and then nYi/li7ing the rhn~phitP product to the desired rh-~ph~tP with a solution wo gS/05391 2 1 4 ~ 7 5 0 PCT/US94/09316 deprotect the new 5' ~ --i,-..c~, the cycle can be repeated as many times as necessary to achieve the desired length and sequence; a~1itirJn~l nucleotides which are added may be the wmmonly occnrring nucleotides or they may be additional n.lO~ r,~ e analogs. Acwrdingly, one or more fluorophores may be ill~,ul~Juldled within a given probe up to and inrhl~ing w . ' :~

7.,b7~ inll of, e.g., a.l of the A residues in a desired sequence with formycin residues. The wuplings can be pl . r.. -~ manua.ly in a luillil?.,a? Iol vial, utili~ing a 10 minute wupling time, oronaPh,.. -.~ LKl?.GeneAssemblerorsimilari,l,llu~enlutilizingthepro~,.,.. ~r~synthesis protowls. The .~UOlCS~Ilt oligc~n~ P, is then isolated by cleaving the DNA from the porous glass support by irrnh~tion at 55C overnight in NH4O~T Pth~nnl (3:1). The ~uolc~ nt DNA
~ i.. i.. g _ hydroxide solution can then be quickly dried in a Speed-Vac and then separated from failure sequences of a QEAE-HPLC wlumn using a shallow salt and pH gradient.
Yields for the mlrlPn~;~e analog phn~llhn~ p-c are ~f~...l.,..~hle to those obtained with standard amidites based on ,~ ,lilivt; yield r~lr~ tP~l from trityl cation release at the d_~lot~lio-~
step.
To provide specific illn~tr~ti- n~ of how to w ~ ucL and use probe rnr' ~ ' AA ?,., ~
a nuO,~,~I mn 1~ ~Ic analog, fo.lowing are PY~mrl~ . which illn~tr~tP ~.,uculu,es, inrll--ling~ the best mode, for pr~rtirin~ the i~ ion. These ~ I ' should not be ~ lu~l as limiting. Al percentages are by weight and all solvent mixture ~-lupulliûlls are by volume unless olh~"..ii,C
noted.

F~mple 1 - ~'h~mir~ll C~?J.~,.;!,iOI1 of Formvcin A to 2'-Deu"vFol ,-, ?_hl A and Pl~,pdldlio.a of the 5'-Triphosphate and 3'-0-(2-cvanoethyl)-N.N,-Diisû,.,u~,yl Phorphe,d,-,idile Figure 16 depicts the inventiûn scheme used to make the 2r-deoxy-sl-trirhnsph~te or 2'-deoxy-3'-O-pho;~ r,~ itP~ of formycin ~ While the first phase has been pl~;Yio?llsly ac~nmpl -'--~ by the reaction with a-a~clo.~yi,ûl.ulylyl halides as ~1P~rrih~ by De Clerq et a.' ([19871 J. Med Che.~ 30:481), the ~lu~luie prù-luc~ both the 3' and 2' deoxy forms which are difficult to separate and are ~ludu~ ed in low yield. The present ill.~,ulion employs a 3',5'-disila ~lul~lion which has pl~,.;Ou ~ly been applied ~ rully in the ~uv~ ioll of adrnncir~ to 2'-1en~ nncin~. ([1981] J. Am. Chen2. Soc. 103:932). The method appears to be generally applicable to the ~ Jon~li,-g collv~ ion of many Lluolescenl In-- lr~ le analogs.
(I) 7-amino-3-[3'5'-O-(1,L3,3-lelldlaoplù~,yl-1,3--licilnY~n~-dilyl)~-D-liborul~n pyrazolo [4,3d] pvrimirlins 1~3-dichloro-l~l~3~3-leLldisoplulJy~ 3-~licoll - (0.9 g, æ85 mMol) was added to a ~ nl~ of formycin A which had been ~hau~ dch.~ dldlt;d (0.66 g, 2.5 mMol) in anhydrûus pyridine and the reaction was stirred at room t~peldlul~e for 24 hours. The solvent was removed under vacuum at T = 40C and the product ~ ~ ~ . ,-c~ between ethyl acetate and water. The ethyl acetate phase was washed, uz senatzm, with (i) cold 1 N HCl, H20, aqueous _ W0 95/0~391 ~ L PCTrUS94/09316 -- 21~5~50 NaHC03 (c.1 ~ ) and aqueous NaCl (c~q~t~lrqt-f~d) followed by evaporation to a gum. Following flash cl,.u,l.atography on silica gel and stepwise elution with (i) æ5% mf thqnnl-chloroform, and (ii) 5% mf th~nol-chloroform~ the product, which ran as a single spot on silica TLC (RL = 0.80 - in 20% mf t~ ~I-chlo,urulul), was shown to be the 3',5' cyclic protected product by proton NMR
S and f l~mf nt~l analysis.
(II) 7-amino-3-[3 ' ,5 ' -0-(1,1,3,3-tetraisopropvl-1,3-disiloxane-dilvl)-2 '-(phetl(,AyLhio~,l,ul.vl)-~B-D-li1,oru.allosyl] pyrazolo [4,3d] ~v~ i".;fli~ 480 mg of disila protected rul, ~_i,. A (Q93 m Mol) was dii~olvt;d with DMAP (0.9 g, 7.6 mMol) in anhydrous MeCN.
Following dropwise addition of 200 mL of p~ uAylhiO~lL~ l chloride through a dry syringe mnllntf~ in a ground glass joint, the reac~"~ were stirred for 24 hours at room temr~-rqtllre, after which solvent was removed under vacuum and the product again partitioned between ethyl acetate and water. The ethyl acetate phase was washed as above, the solvent evqrnrqtf~A~ and the residue sep~r~qted on flash ~ hl.~lualography and eluted with chloroform-MeCN (50/50). Pooled frqrtinnc of the desired product were ifl-ntifi~ by proton NMR and ~ m~ntql analysis and subjected to a second round of ~lu~ Iinn, as below.
(III) 7-amino-3-(2'-deoxy~-D-ribofulailo~YI) pvrazolo [4,3d] yJtihllidil c (2'-deoxv rullll~_hl A). 240 mg of the product obtained from the plof~lulf d~-crrihed in II, above, were added to 12.5 mg (NH4)2SO4 in a gross excess of ~ ;lq7q~ The reaction mixture was refluxed at >60"C overnight. After e vayolalion under vacuum, the crude trisylyl derivative was l~issolvcd in toluene and reacted with ~;so~ulylullillile and tributyl tin hydride by heating under N2 overnight to attain cnm, ' l~iu~,lion. The product was d~.ut~led in TBAF in THF
at 80C overnight and, after evqrnr.qti~ m fractin-~qted between ethyl acetate and water. The water layer was cr~ d and applied to a Dowex 50W-X8 column equilibrated in water and then eluted with 15% NH40H. The principal product (Rf = 0.3 in 20% ~l-chloroform) wasshown to be i-lPntirql to the purified 2'-deoxy rull-l~. A which had been ~,~,~,ed using the method of De Clerq et aL, supra and by proton NMR and &~ 1 analysis.
(IV) 7-amino-3-~2'-deoxsr~-D-ribofula,lo~vl) p-vrazolo [4~3d] pvrimidine-sl-trirhn~yh~le (2'~eu~yful~ A-5'-~ r) 28 mg (0.11 mMol) of 2'-de~ A was added to a glass ~uppcrcd test tube and mixed with 0.2 mL of reagent grade acetone and 0.1 ml of phncphnroUs oxyrhlnri~l~ The heterogeneous reaction mixture was stored at 4C for 24 hours, during which time the solution turned deep yellow. After cooling and addition of 3 ml cold acetone, 6 mMol of ~ ~ NH40H was added rapidly while mixing. After eval n".l inll of the acetone, and ,~I-.~,lion of the pH to less than 2, the mixture was refluxed for 1.5 hours, then diluted and applied directly to Dowex 1-formate, from which 2'-deu~ fullu~_iu A-MP was eluted with 0.75 M formic acid. 2'-deu,.yfulluy~ A-MP was converted to the triphncph~te by the method of y,~chi~ ~ ~ et aL ([1967] T~tl- ~' e~u,~ Lett. 5095).

WO 95/05391 21 4 5 7 ~ o ` PCT/US94/09316 (V) 7-amino-3-~2'-DEOXY~B-D-lil,vfuldlloavl) pvrazolo [4.3d] l~yl ~-3'-O-phosphoramidite (2'-do~ full.ly.-h, A-3'-O-phosph- r~miAite) 2-deu~rulllly~ Awas treated to attain 5'-O- protection with DMT and b.,~ lalion of the 7-amino group by standard procedures.
To 0.3 mMol of the product and 25 mg of diiavplu~ nnillm t~.tr~7nliAe in 1.5 mL of CH2CI2 S was added a solution ~ nl ~i.. ;--g Q33 mMol of O-cy-~nnethyl-N,N,N',N'-tt;lldiso~lvl~yll~h( spho-u~l; .,"i.ii~. The mixture was mixed for 4 hours and p~lLilioned between CH2CI2 and chilled in ~fllr~3t~.d NaHCO3 sr lntir~n The CH2C12 layer was washed vith ~ lPfl NaCl sohlti~-n, dried (Na2SO4), Itered, and cn~.ff..~ pllrifir~tinn by filtr~tif~n through a 2"
plug of basic alumina in a 25 mm column, eluting with 9:1 CHCl2/ET3N, provided the ph~cph~r~millit~ which could be dried to a foam. Identity of the product was verified by proton NMR, ~l~m.o.nt~l analysis, fluo~ ,~ of the helelo-;y~lc, and use in olig~.n ~ lrotide synthesis.

Example 2 - C nmrlPt~. El~ alic Sub~ n of FTP or 2'dF l~ for ATP or dATP in RNA or DNA Probes A. S~ull~cllic svnthesis of ribose oli,~.... ~ RNA l~lig.. nn. ~ itlf'.C were Sy-nthP~i7tA
from three DNA templates (Figure 12) using (i) ~l~ (F105) as a s ~ lr for ATP, and (ii) a purified E coli RNA pGl~ se as originally A~ s. . il.eA. by Ward et aL ([1969] J. BioL Che~
12:3242), except that synthesis was allowed to run for three hours at 37C before the reaction was stopped; FTP e~f~Lv~l), replaced ATP but not any of the other three normal 111" l~liA~ CTP, UTP, or GTP.
At the end of the synthesis, reaction products were s~-~ from ull~ led reagents by se~ n at 4C on Sepl~ ~ G-50 in normal saline at pH 7. The scheme for s~ of reaction products from unr~ t~l agents is shown as a flow chart in Figure 19.
In the reaction, FTP is an effective ~b ~ n~ for RNA pol~ e.~se with both native and denatured DNA as well as with synthetic d~....... 4~ iAe polymer t~ pl-tr-: In samples f..~ -ining CTP, UTP, GTP, RNA pol~ll,.,.~se, one of the DNA tPmplqtp-c and either ~l~ or ATP, a high m~ r weight product eluted from either sample in the void volume while the amount of --onfl...~ NTP in the retained fraction from either sample was Wllr-~lJo--~
reduced by >70%. No high m~ 1P~-I~r weight fraction other than the small amount of template elutedfromenzyme-freecontrolsandunreactedrNTPswere.. ~Ai,.. i"i~ l similarly,t~ free controls ~."~I~;nPd only unreacted rNTPs which co-eluted in the retained volume with standard 1 iAt~ trirht~ph~tf~ Similar results were o~htqinPA with a variety of DNA tPmplqt~c from natural and synthetic sources, irrlllAing the ~ltt~rn~ting f~JpGI~ ,.a poly d(AC), poly (AG), and poly (ACGT). Moreover, ~ .; hlr. yields of high mt IP~ r weight oligomPr were obtained 3~5 from syntheses in which (i) the N-m~ n~;A- analogs 2~6-diamino-~A~ h~t- 5~ ,h~ h~lr or 2-diamino qA - ^-5~ iph~ h:~t~; were ~ l il ..teA for ATP in the r action mix, or (ii) the C-nllt~l~oncirlp~ r~ll..~ il~ B-5'-i ~ ~Cp~qtP (FbTP) or -amino-formycin B-5'-~ h~ Jh~lp (aFbTP) _WO 95/05391 PCT/US94/09316 -- 21457SO ~ ~

tf --.pl~te No matter what the tennrl~t~P yields obtained by substit~tine several of the deaza- and aza-nnrlPn~i~le analogs for ATP or GTP were ~Ir~ln ~tir~lly lower.
B. Asvmmetric synthesis of RNA or DNA probes. In vitro, DNA ~cpPn~7çnt, RNA
- polymerase lld~ systems for the synthesis of RNAs for use as substr~tpc and h~~ nl~
S probes are a fairly rnmmon tool of molP~ll~r biology. They are uniquely applied here to the development of ~lltnfl~lr~rescent probes and their pro(l~ctinn- The method de._lopf,d is general and applies to any of the phage pol~lu~, ase systems, inr~ ne SP6, T7, and T3. In the present case, the iu~/c;lllioll employs a pair of ~)lu olf;l~ which are sep~r~tply po~;~innf~ on ~ItPrn~tp strands of a duplex plasmid and at opposite ends of a polylin};er as shown in Figure 13. The vectors are used to (i) attach ~ olt;l~ capable of PfflPrtine ~,.l.l.~,llic synthesis through use of a viral polymerase which l~u~,~s one of the pl.,..~nlf -,~, and (ii) replicate multiple copies of a tPmpl~tP. for use in ~ - prodnrtinn of a ~ - w~ probe or of a no~u~ l~nl copy of the probe target. A copy of the DNA target se lL_l.ce is inserted into the polylinker in its duplex form and at a ~ - site adjacent to one of the promoters. RPplir~tinn of the plasmid in cr.. l.e~ -l cells provides large amnllntc of the tPnnpl~tP for l.n.~ .linn. Two separate but parallel methods have been d~._loped for the di~,~ ' synthesis of DNA probes. In the first case, ssDNA probes are srth - ~ from t~ P~ which have primer binding site attached at the S' end of one 1, pl~le strand as shown in Figure 14. In such ~ the primer may be non-~luolw~ll~ or may be srthPAi7P~ using nuolw~.l~ analog phncph-J.,....i~ as shown at the right of the Figure. A ~alialiO~l on this is &.J~.~l.. _llic ~mpliri~-l ;n n and se~ inn in which both strands of a ~ may be replir~tçd by ~plifir~tir n as nuores~nt nliennlPr~, but using a pair of primers in which one, and only one, bears a affinity linker such as biotin which may bsc~lucnlly be used to separate the dcndtuled sense and ~nti~Pn~e strands.
For both RNA and DNA probw, it has proven practical to establish â ,cf~ tPmpl~tPprobe sequence, and target s~ against which all lld~ lions and probe d~ inn se~;liviliw are calibrated. The alpha chain of Xenopus tr~nCl~finn Plnng7tir- factor (Xef-la) servew that purpose and ~.~ iC RNA probe synthesis is used here as l~plwe.lldli~_ of all RNA and DNA synthwis. The Xef-la mRNA is a major I . i..~C~ . il.l ion product of the Xenopus embryo which comprises a large percentage of the non----;lo- l n~lri~l mRNA lldllS~ i that appear j"",.r l;~ y after the rnidblastula tr~ncitinn The gene for the Xef-la was isolated and EcoRI linker sites added at the ends of the clone during co.~llu~ lion of the cDNA library. The 1705 n... lcolide r. ..~ l was inserted into a pSP72 plasmid bearing a T7 pl~ t~r on one strand and an SP6 promoter on the c~mrl Following plasmid rerlir~tinn and template. i, l ;o-~ . . il.lioll with T7 RNA pGl~ -', the rNTPs cytidine, uridine, and ~ o~ P"
together with the ribose trirhncph~tP of either rul.. ~_hl A or ~ PnncinP pl~ ,~d 1749-base-long oli~mPrc ~.".ls.i"i,lg 489 F or A residues, l~ ,e~ ,. T~ c~ .il.l~ less than full length were never obs_ v~d and, in each case, the analogous and control nli~m~o.rc were plO ~ in W O 95/OS391 PCTrUS94/09316 ~
21 4S750 ~

cnmr~r~hle quantities and were generally in.~ i..g~ h~hle in physical bchavior save that the analogous sequence was p~ -.lly fluo.f ,~l.
There are t~,-vo unique features of this novel m~mlf~ctllring system. (1) Synthesis of the c strand, e.g., using SP6 and the r~mmmly OC~,ul ~ g nolLauo-t;sc~.ll rNTPs provides standardi_ed target sequences in high yield. In the corresponding asymmetric synthesis of DNA
probes, distinct primer sites on ~m, ~ t~ry template strands can be used to achieve the same objc~livæ (2) A mixture of plasmids o..li~i"i"g several different plasmids can be used to create a "cocktail" of linP~ri7P~ tPmplqtf~S from which the cu~ po~ g "cocktail" of probes (see FY~mrl~P 7, below), which can bind to multiple sites on a genomic sc.l~c~ce, can be con~ ullC.Illy 0 ~.,.. ~c.~ .etl Example 3 - The Flu~,l~..ce of Nr l~;rlp Analo~ RNA Probes and Proof of Their IIv~lidi~tion in Solution The effective ufili7~ti-~n of FIP in the poly d(AT) directed synthesis in FY~mple 1 produced a polymer appll ely 300 500 bases in length which, when hydroly~ed and/or s~.,.llced, proved to be a p~f~Lly ~ltPrnqting replicate of the DNA tpmr~qtp but with the s~.,e..ce; poly (FU). As pl~li~ d from this Sf~l ~U~, the product could be r - IPA, to like chains by a single thermal cycle, thereby creating the putative product poly (FU):poly (F U); unlike the ~ hly treated poly (FC), which showed no evidence of self-l~b- j,li,"l i- ~- as P~P~tPA the ~nnP~lpd hybrids of poly (FU):poly (FU) stained with 5;~ li.. ,,, bromide in agarose gels and gave a sharp thermal lla~ilion in both ~hc~llb~-^e and nuolcscence, proving that the probes could Ly~lid~ both ~Lf~li~ 1~ and 5pP~ifi~lly~ The absoll,ancc and Pmi~ m spectra of the purified poly (FU), poly (FC), poly (FG), poly (IJFb), poly (CaFb), and poly (FCGU) differ from those of purified poly (AU), poly (AC), poly (AG), poly (TG), and poly (ACGT) controls in four lCSp~l:i; (i) the far UV absoll,au~ ~ .. is shifted slightly for the analogco-~ e , to 265 nm as COlll~àl~ d to 260 nm for the controls; (ii) there is a ~i~nifir~nt~ highly sll..~,luledal)sull,dnce(3peaksatroomtr-.~p~.lllle)between290nmand320nmwithnPgl1;;i~
absoll,dncc at 340 nm; (iii) an ~ -- ;l~lin~ .i-..-.... appears at 303 nm; and (iv) there is a broad P~;~ m band Pytpn~ling into the visible ~..a~_lcu~ , with a peak at 405 nm (Stokes shift = 102 nm). It is an ilu~ UI p~O~"Iy that the n~ .ce is fully ~ d in, e.g, the poly (FU):poly (FU) hybrid, and cannot be detected until the strands are dc~aluled by raising the pH
of the solution to values >pH 10. Once del alult;d~ the rlL~ .cc of the oligomer is fully inte~ t~lhle, with relative rluolc~ lc4 intensity >40% of peak intensity over the range 360 nm to 460 nm.

~Wo 95/05391 PCT/US94/09316 21~;7~o 29 Table l. r.. " li~s of hybrid ~ by poly (AU) and poly (FU) DENATURED HYBRID INTACT HYBRID
RNA RNA
HYBRID WAVELENGTH MAXIMA LENGTH ETHIDIUM MELT
(BASE PAIRS) BROMIDE TEMP
ABSORBANCE EXCITATION EMISSION STAINING
r[AU]:r[AU] 260 nm -- ---- 150-300 yes 32#C
r[E~Ul:r[E~U] 266 nm 303 nm 405 mn 150-300 yes 33#C

FY~mrlP 4 ~ idiLalion of Fluoles~nl Probes to Tar~et RNAs and Tar~et DNAs: Uses of Linkers to AUow Solid Phase Detection The synthetic template poly (TG) was used to produce the C,U1l1P~ ' y RNA probespoly (AC) and poly (FC), neither of which is self ~...~ lc.. ~ .y and in which hybrids could not be ~nnP~lPd or detecte~l of the two only the poly (FC) was fluorescent. In a paraUel .,~c.illlcnl, a poly (AC) template was ~mplifiPd using the bioli~ldled synthetic 22-mer primers, s#BIOTIN-(TG)ll3, together with standard pol), uc~se chain reaction (PCR) methods to produce the DNA
~ having the s~L_ncc~ siyBIoTIN-pol~(TG)3 ~ then sep ~ ed from the ulllcacled primers by gel sizing and/or QEAE ion PYrh~ngP~ ch-u..latû~ hy, after which the pGl~ were r,q.ljna~ labeled using 32P-ATP and the enzyme pGl~llu~lc~lide kinase. When mixed sep~r~tPly, but in equimnlsr ~mountc~ with the biol"l;laled ~mpl s#BIOTIN-poly(TG)3, both of the RNA probes, poly (AC) and poly (FC), formed hybrids which could be ch~r~ctPri7~d by (i) ethi~linm bromide staining, and (ii) melting bChaviOl, as PYpectP~l the ~UVlCScGllcc of the poly (FC) probe was ~ ;hcd by h~b- i.li".~i~)n The hybrids could then be ad~u.bed via the sBIOTIN moiety to ~.~ili~laled beads, washed to remove unL~li.li~d poly (FC), and equal aliquots assayed for ra~lin~ctivity and Quoles~ e. Prior to dP ~ inn of the washed sample, detectable lluu~c~ cc in the solution was nP~gli~hlP~ when d~,llalulcd in high pH buffer, the amount of poly (FC) which had been h~ cd, when Pctimqt~P~ from the nuolesc~,.. ce of dal~ cd ~ ltinnc of the probe, was within 1% of the amount of the target DNA,5#BIOTIN-poly (TG)3, as ~d by the amount of ,,..lin~ label in the sample as culll?alcd to ndàl~liLcd ~lilntin..~

Example S--IIyl,.i~li".~im- of Fluo~ cnl Probes SvnthP-ci7-p~ from N~ 1e Analo~-3'-O-phncrhnr~mi~litPc to Tar~et DNAs In a v~ tinn of the use of the pho~ h~ p-c of the nuo-~7cellt nll~lenC;~e analogs, n-mers which varied in length in mnltir'- of S bases from 25-mers to 60 mers, and having the S~L_nCC (AC)X or (FC)X, where x = 12.5, 15, 17.5, 20, æ.s, 25, 27.5, or 30, were synthP-ci7P~ in parallelusingeitherd~PnncinP-3'-O-rhos~hn.,.. i~lileordF-3'-O-phnc~ho.,~ itetogeth wilh dC-3'-O-phnsphnr~mi~itP in a ph~ LKB Gene Assembler. After cleavage from the solid mJTE SHEE~

WO 9S/05391 PCTtUS94/09316 21~57~0 30`

~mrlimPrc of poly (TG), from F.~ ,1r~ 2 and 3, above, as assessed by DNA melting behavior, ethit~ m bromide staining, and the l`CdlJp~ ''f if 4L_~ched n~ es~e following riP.~ n of the hybrid.

Example 6 - Assay for Chlamydia trachoma~s Usin~ an FTP SnbStitutP~d RNA Probe Chlamydia trachomatis is an obligatory intr~c~ r pathogen which, in its active infP~ctimlc stages, contains from 3x103 to 4x103 copies of rihoso~l RNA (rRNA) and one copy of genomic DNA/l/~c'r~
A primer pair, one of which Co.~ P~d a 5'-biuli~ldled T7 promoter which was 5' to the h~-idi~g primer se4uc ~, was used to ampli~ a 150 base pair DNA segment of the MOMP gene from a stock strain of C trachomatis L2. A~ ~lely 500 ng of the DNA fi~grf nt~ which con~i~l the T7 RNA
pol~.l.c.ase promoter at the 5' end, was I . ;.,-c. ~ ed with T7 RNA polymerase in the ~.es~ e of rCTP, rUTP, rGTP, and with either r~ l~ or rATP (+ control). The reaction was stopped by heat inactivating the enzyme for 3 minutes at 100C. U~u~ ul~Jolaled rNTPs were sep~r~tP~ from the labeled RNA by gel sizing ch.~ r~rhy on a Sephadex G-25 column, after which the probe cnnr~ntr~tinn was P.~ f'~
from its absu.l,allce at 260 nm. Using a simple dual monocLu...alur nuu.~l.ce ~ ~phr~ -f ~, as little as 5 x 10--14 moles of the RNA probe could be detected over l~ac~louL~d when 20 nm slits were used forbothP- ;l~ andf~ r^rmr~ bludl~ . Aphotonf-ountingn~ f-~-ci~n-f~dfors~ ivily (see F~mrl- 9, below) is capable of det~ting between 5 x 1~16 and 5 x 1G17 moles of the same probe, equivalent to the amount of ~ihûsû..lal RNA-f~pect-~ from between 5000 to 50,000 of the bacteria. Two hundred microlites of either (i) C. trachomatis genomic DNA, or (ii) the ~mrlifif~i target DNA were mixed with 200 ,c4L of a 1/200 dilution of the probe in Lyl~ li".l i~n buffer (Q15 M NaCI, 0.02 M sodium citrate, 0.02 M HEP_S, 0.004 M EDTA, pH 7.4) and the mixture boiled for 3 minutes, after which they were allowed to cool slowly to room - ~ ~ over one hour. An aliquot of the genomic DNA sample was eluted into an lllt~filtr~ti~ lUi~ ~ulube or 9~well filter plate (pore size = 0.1 ,um) as ill~lctr~t~ in Figure 17, washed 5 times with Q15 M NaCI, 0.Q2 M sodium citrate, pH 7.4, after which the sample was divided in two, one half denâLul~ in high pH buffer, and both aliquots scanned to measure nLJles~nc~
ba~u~d and the nuO,cs~ cc of L~ li~d probe, ~lJ~livt;ly. Target DNA ~",l,li"-r ~ were treated similarly except that the 5'-b:JIil ylatcd primer end of the target DNA ~O - - were first a~G-l,cd to a~,.liu;laled ~ beads (2.8,um dial--ct~) so that the sample could bewashed without loss of material (Figure 18). With either Ll~ `1, nLOI~I.~e of the probe may be dete ted at ~ ltir)nc of the sample which contain less than 1 x 10--16 moles of target DNA, which is roughly equivalent to the s~,~ilivily required to detect less than 10,000 bacteria if a single siu.ilally sized probe were used to detect rRNA from infectious Chlarrydia. The probe used here is about 150 bases in length, contains a~luA..l.alcl~ 38 rulluy- hl residues per probe, and binds only to a single target site on each copy of the ~ n -~l RNA.
It is an illlpUl l~ feature of this i~. _..lion that h~ lg the number of ~ uphores in a probe, or probe 35 ~cocktail," also h. leases the se.. ,;Livily of detectinn With 13 times as many rul~ residues per probe as the 150 base MOMP gene probe, 1 x 1G18 moles of the Xef-la probe can be detected in a dual ~WO 95/05391 PCT/US94M9316 21~75~

mnnn~ tnr nuolesce.. ,e spectropho~ -,cter whereas lecs than 1 x I~20 mo1es are detected using the photon counting technology tlo~ ed in FYqmr'^ 9.

FYqmr1r 7 - Detection of Mn1tir1O Tar~et Sites An illlpU~ aspect of the a~ luel,ic syntheses to both . ~ - - and thr~ euli~; e.g., ce app1irqtihnc of nucleic acid probes is the capacity for ~ncullent synthesic of probe "cocktails"
which may c~ ""I" i~e probes which differ in length or differ in the 1Or~qtinnc or ~ of the target sitec on RNA or genomic DNA to which they will bind. Uti1i7qtirn of probe corktoi1c to three different types of ~ ;"h~1ir targets i111lctrqte the broad ilupu,~uce of this feature.
A. Single target nucleic acidc precent in multiple copiec. In some species of pqthnge~, multiple copies of rRNA are present in each Oll;aniS~ e.g., each b~-t~ of Chlamydi trf~rhnmA~ contains a~lu~alcl.~ 2 x 104 rRNA r^~ 1 ' per Ol~;alu~ . Since the rRNA of Ch/A-7~iA is typically between 3000 and 5000 nu~,l~Lidcs in length, s~;livily in a ~liq-L - - assay may be il.~,l~scd ci~iri~u~ly by u e of a probe cocktail specific for target s~yu~ s on rRNA and made of as many as 5 to 10 different probe s~l - s, each of which can bind to discrete SJ~ of the target rRNA or target DNA as in~lir.qted with probes (a) to (e) in the lower half of the diagram shown in Figure 20 in which (a), (b), (c), (d), and (e) are analogous c4...p1~ y probes specific for different target sc~ ..ces of a single DNA strand.
There are two disa~anlages in using rRNA s~l as ~ grnctir targets: (i) rRNA s~ - - -are highly col~.v~;d, hence only short variable se~yLe..c~ are useful for the lote~tihn and i~ iri. ~
of i~lious pathogens. One consequence of this to .li g. h~lir s~,~ilivily is that only limited numbers of ,.' labels can be used on each probe, thereby limiting se~;livily; and (ii) only a few I ' ~ ~ carry rRNA in high copy m1n hPrc, and many, such as the DNA viruses, carry no rRNA at all, hence the number of ~liq~nctirc which can employ this strategy is limited.
B. Multiple different tar~et scyl.eac~s on a single strand of DNA. The ge..n---~ of all Ol~;alliSll15 are ~ ri. --lly larger than rRNA and typically carry more ùlls and larger unique sc~ which can serve as target s~y' for nucleic acid probe h~/1-- j.li,"linn For; ,1e, the Cnmp~ ~ genome of Chlamydia ~rachomatis has been isolated and consists of a relative small double strAn~l~ DNA with a m~ Ar weight of >660 2~ 1o6 or slightly more than 1 x 1o6 base pairs. Each bA- t~ also contains a 4.4 x 106 dalton plasmid C~nIA;I~ g >7 kbases. Unlike the rRNA of this species, the plasmid is unique to Ch~A~;~A~ in its entirety--no cross h.~ l in!l can be detected with the DNA from, e.g., Neisse~ia gonorrhea--indeed, no cross-L~- i~ occurs between the different .. ~1 . i. I i-u~ frA~ Ontc of the plasmid itsel~ Even when no other portion of the Chlamydia genomic DNA is chosen for use as L~-iliLalion targets, a cocktail specific for the multiple restriction r.. ~,.. --l~ of the Chlamydi~ plasmid alone is equivalent in length to more than 4 Xef-la probes and can be detected at levels equivalent to betwec,n 35 I00 and I000 bacteria.
C Multiple copies of a sin,ele tar~et sGqu_.l~ on a sin~le strand of DNA. It has only recently been dis~u~,..Gd that flanking S~L_~1CeS on each side of several genes contain -~A-, t~o- to long sl-GI hcs of tandem repeats. Riboso---dl gene repeats are of particular interest in the kinds of DNA based rliAgnncic WO 95/OS391 214 5 7 5 Q PCT/US94/09316 ~

~IP-crrihed in this i..~,..lion. Like the libosoll.al genes, they are present in high copy mlmhP~, which hll~luv~ se~iliviLy of APtP~tinn but, in q~Aitinn, the spacer regions between genes are normally highly variable from species to species, since they are not subject to selective p~ ules. Multiple copies of the same unique se 1~ ~ on a single DNA strand represents a special case in which the L~~ inn targets are a cocktail of loci on each genome; that is, a single probe sequence can probe multiple target sites of the same sequence and on the same DNA strand. They are ideaLly suited as spe ies and genus specific probe targets.
A rt~ livt; example of such probes and targets was created for the different species of the plulo~ud l parasite Eirnena, which causes ~u~ in~;~ in a variety of ~iomP-~tir animals. ~Pnnmir DNA from 0 E. tenella was digested with several different rf~ liol- enzymes, and the r.,.~;,.. , l~ Iigated into a~lup,ialely cut ~.~_I.ic plasmid vectors and were used to ~ r(.. Escherichia coli. Colonies were 5~ ' for repeat se4~ by hyhri~li7qtinn with Eimeria tenella genomic DNA that had been labeled with 35S by random priming. Strongly h~li~li~g clones were picked and subjected to diLre~ ial screening with labeled genomic DNA from E mitis, E. ma~ima, E. acervulina, and E. tenel.'a, as well as DNA from the closely related genera Plasmodi~m, Trypanosoma, and S~ . The majority of clones gave signals of equal intensity with DNA from the other genera. Some clones, however, were r~ o~
sperifirq-lly by the Eimena and one clone was recogni ed only by E. tenella.
The entire s~l - of the ~nsert in the latter clone contains 334 base pairs. Physical rhqrqrtf .i,~inl~ of the l.~ ;n-- f~g ;-~ that the s-P~IuP~re is present in tandemly repeated units of applu ~ ately738 base pairs and that a .. i--i.---.. of 30 genes are tandemly linked and all appear to be on one chromr - - A~ : - probes srthP-~i7-P~ using the tandem repeat as a template contain 179 ru~ ,hl A residues per template S~
Even when no other portion of the Eimeria genomic DNA is chosen for use as a 1-~ li".tinn target, a single sequence probe specific for only the multiple copies of the tandem repeat on the Eime7ia genome is equivalent in length to more than 11 Xef-lcc probes. Since the hlf~Lious particles for Eimeria are oocysts, each of which contains 8g_.1u , such cocktail of targets makes it possible to detect less than 10 oocy-sts. The import of tandem repeat targets extends well beyond se~iLiviLy, however, or simply the ~lPtertinn of this single genus, since tandem repeat sf~ appear in a genomic DNA of a wide variety of species and genera, and are distinct for those species, thereby providing a broad basis for the design of liqglln~tir assays for a wide variety of pathogens, ' - g those for which no rRNA targets exist.

Example 8 - The Use of Non-Specific and Non-H~lidiLill~ Fluorescent Oli~omers as Universal Fl..o-~nl "Ta~s" bv Li~ation or Chemical Linka~ee Simple lnnrljfjrqtinn of the tf~mplqtf to produce a "sticky end" at the 3',5', or both 3' and 5' termini, e.g., to 5 ACGT-polyd(AI~, polyd(AT)-TGCA3, or 5 ACGT-polyd(AT)-TGCA3, l~f~li~
enabled synthesis of nucleic acid probes with all of the above ~lupf;l lies, but which could also be ligated, either (i) to like strands to produce longer nLûl~ scenl probes, or (ii) to other l~h~ ;nn sc~l.,e.~

~WO 95/05391 21 ~ S 7 ~ o PCT/US94/09316 universal label for any cloned DNA r; g,.~ , and allows a given probe to be i-1PntifiP~i by two non-h~~ i,;-,g but highly nuolofsc~ sequences at its termini, without the need to denature the hybrid for rlPtPrtinn as was seen with the simple poly (FU) probe, above. Equivalent non-hyl,-idi~g universal probes can be readily made by I synthesis using, e.g., the etheno analog phngphnr~mirlitPfg, e.g., 1,N6-etheno~lPnnsinP-3'-O-phosphola.l~lilG (eA), to synthesize non-speci_c tags which can ~ul)se lu f.l11y be linked to any L~ l;,f tinn probe. The 3' or 5' termini of such universal probes can also be ~lepàl~ed for r~l, rather than e ~lllaliC sltt~lr' to other olignmr rg or solid phases, through the addition of, e.g., 5'-amino hexyl, S'-~ullh~.llyl hexyl, 3'-aminoheAyl amino, N-Ly-lluA~Io ;Il;~ P esters, and other such linkers. The unique application of this probe technology, which employs the universal end label, is 4~ 1i~tive and works well for routine assays which require high se~ilivily. Another applir~tion of this technology referred to herein as "S.lct~ ~ signal amplifir~tinn" is non-~luanli~livef and can be useful for a ~ l ;n~ where eAtreme sc~ilivily is required to answer "yes" or "no" whether a particular gene marker is at all present, for; r ~"~ where low copy numbers of a target sequence are present. ''sugt5~inpli Signal ~mpliri- ~1 inn" is Ar-~. . ihed in more detail in P~mr1P 8(B), below.

A. The 5' Universal End Label Homopolymers of non-h~l,u~., bonding fluorcscent ~ rlr, analogs, e.g., eth~ rl~PnnginP~
can be used together with ~ fuiC ~..lh~i - of ssDNA and RNA to increase the density of rl~olcs~
labeling on rforLt~ilg of small probes, on small fra" as in sequencing, and to increase se~silivily of labeling of small or low copy number target. The general concept ~u~ -c an nligom~rir probe co.~ ' along a typical phosphodiester b~- Lhn~r" but which can be divided into distinct r _ -I
regions~he 5' nuu.~-.l homopolymer; primer, or promoter cnm,, ' ~; an optional "tether" region, which can connect the homopolymer to the primer, and a target ~u.l-pl~ l. A diagram of this AP-~rrihed 5' universal end label is shown in Figure n.
The filnrt --I regions of the phngrhorliP~trr chain as shown in Figure 22 are:
A=a non-base pairing homopolymer of from 1 to about 50 fluu.~enl nuclcolidc analogs;
B=an optional non-u..~,l~flide ph .~l~hnrlir~ .. "tether" """1" ;~;~ g, e.g., one or more freely rotating alkyl chains inserted as part of the rhngphnr~ipctpr h~rL-honP of the nlig C=an ~,~y ua~ synthesis primer for use in initi~ting e~ alic synthesis of the target-specific 30 D region. R~i~,n~livG pvqmp~^, would be the c~ ~' y se l.,_.. ~ to the T7 RNA pGl~m~ SC
p.~ tcr or to the M13 rO.~ Gd primer as are used in &Jy - RNA or DNA probe systhesis rlr.~. .- il..~ herein;
Regions A, B, and C, typically from 20 to 60 bases in length, can be r~ Ily synth~-ci7P~ The 5~ uui~ al end label c~ c at least regions A and C and, qhPrrqtivley, can also include the optional region B.
D= a target ~ r ~' ' ' I'y sequence of from 40 to 20,000 --,- Iro~ irlPc in length. This sequence may or may not include Çll - ~cenl '- '~ analogs, but fimrtinng primarily as the region which 21~7~0 synthPci7Pd from trmplqtPc adJacent to the promoter or primer site to which region C is c~!mr ' - .-t ~ y.
The entire 5' universal end label can be used as the primer for DNA or RNA replication of the target-specific C.)lllp' Enzymatic synthesis using a 5' universal end label is i~ ctr~tP~ using the M. tuberculosis IS6110 template (a SF~.. _~.~ unique to the ba~ IP. il.. ) which has been inserted into a standard Gemini plasmid to create a synthesis temrl~tP~ Other plasmids can be used as well. This el~ lUaliC synthesis process is shown in Figure Z3. The following advantageous properties of the 5' universal end label have also been di5cu._l~
(1) As shown in Figure 24, the PYrit~tinn sp~llulu of one non-l~JIot,_.l bondingfluorescent analog, et~ ~tiPcncinP (~ip-cigrqt~p~ F185), is cuu,palGd with the romr~r~ble e~ "ir.~
u~ of formycin (F105). The F185 extends further into the UV wa-vrl- ..g~l.c Two ilu~ulL~ t dis uvG.ies have been made about both the ~ l inl~ and Pmiccinn spectra of E185: (i) the wa..~llngtl maxima are the same at both pH 7 and pH 11, and (ii) the 4udllLull, yield is more than 10x that of Flos having values of 0.55 and 0.65 at pH 7 and 11, lwlrG;liv~ . This allows the use of the 5' universal end labelunderawidervarietyofpH~ in,-~andcanresultincignifir~ntlygreaterlu.. i~F-cl~ fromfewer total n~J.upho.G~. It has been shown that an F18s 20-mer which is excited at pH 11 over the range 270nm~s310nm can be equivalent to labeling with between 3 and 10 nuol~s~;u mo'~c~llPc Furthermore, the fluo.w~nce does not quench and can be used with time resolved ~.~,F~Iluseul".
(2) non-base pairing end labels do not interferewith primer .--F~ PA DNA~mplifir~tinn or l~licalion~ are water soluble at c. .i~ tinnc up to 10-3 M, and do not increase the bae~-uund in a binding assay due to non-specific h~ -- to non-target SG~1U~ W.
(3) such probes are beyond the capacity of rhPmir~lly synt~ ' probes because, as is well known in the art, the practical limit of s.yulh~is in l~sonablc yield remains a~lu~LuldlGly 60 basw.
The 5' universal end label can be used to increase sG~ilivily of ~iPte~tirJn by using a cocktail of l~,laliv~
short probw forwhich the length of the "D" region is ap~luAilllàlGly 100 bases. For c rl-~ as illllctrat~P~
in Figure 25, the 1361 bp IS6110 se~ nce of M. tuberculosis has been used as a target for a cocktail of 10 probes, each having a different "D" segment or co.--~ y target sequence. Each probe, however, bears the same 5' universal end label. In the case of M. tuberculosis, there are 16 copies of the IS6110 gene per bacl~ .. By using the end label in a manner shown in Figure 25, each b~rtpnnm has the potential for being labeled by a probe cocktail with pi nuu.uphûres which are equivalent in inct~nt~nP~us ~ --.- to between 480 and 1600 nuOlesCeiu mr'- 1PC
(4) the same labeling device can be used to provide a ~ndd-di~d nuores~nl label in standard DNA sc~, g, but having pre-labeled DNA fr~m~ntc so that s~-._.,ces can be read or recorded directly from the gel. Such a use is depicted in Figure 26.
B. S~Ct~inP~ Si~nal Amplifir~tinn (SSA) The 5' universal end label should prove particularly useful for those situations in which a unique ~WO95/05391 21~57So ~ ~ PCT/US94/09316 For such applir~tinnQ in which eA Iuii,iLely sensitive levels of ~etPrtinn are required, or for which very little target is present, some c~-~-hi~ ;nn of lluo,~escent labeling and signal replication or ~mr1ifir~tinn is required. Hepatitis B presents such a case. The entire genome of I Iepa~ B virus (HBV) is only 3200 bases long and, in the virion, one of the strands is even shorter. The virion contains a DNA pol~l..e,~c which utilizes nucleotide triphosphates from a host cell to complete the short chain as the first step in an i--f~, li The DNA polymerase of the virion utilized together with a novel nuulwcc,.l nllrlPnQi~lP analog ~Pcrrihed herein was cu~ ,c d with a non-PCR type of ~mplifir~tinn which has heletorule been used only for RNA replir~tinn In this scheme, shown in Figure 27, the virion DNA serves as an ut sitz~ template and, in combination with the above~ ;I.c~ asymmetric synthesis method, can be used to ampli~jT the i~llC~i~y of the rluo~escc~ signal. The process, which may be better nn~lFrctood by referring to Figure 27, involves two steps. First~ the sample DNA is combined with (i) deuAynuclP~ide t-iphncFh~tPc (deoAyNTPs) inrl~rling tripho;,phulyldted lluor~s~ nu 1~ P analogs, and, (ii) two primers (shown as A and B in Figure 27), the first of which has at its 5' end a sequence compl~ y to an RNA
pol~ ~ promoter. The primers referred to in this F~mrle are dP ~- ~ ihed as "A" and "B" to indicate the use of two separate primer, These are clP-sr~ihed as such for illustrative ~ulposes and, as such, would be ~ ood by those in the art to refer to any primer which cc~ F~ a sF~~ fe cr r~- Y to a promoter region on the target se IU_liCG and which can be used with a nucleic acid pol~ - In the io-- shown in Figure 27, the T7 RNA pol),~uGlase promoter is lPci~ted by the thicker line at the end of primer A. The sample is first te~ at 37C for 10 minutes to allow the viral DNA polymerase to c~ pl:-lt; the short genomic strand; the sample is then raised to 65C for 1 minute to denature the genome, after which the primers are ~nnr~ P~ at 42C Second, the two enzymes, reverse ~ tase and T7 RNA polymerase, are added, together with the riboseNTPs inf-lo~ling the nuulw~nt lil,~
analogs, and the entire sample is inrl~b~tFd at 42C for 1 hour. This creates a cycling synthesis of DNA
strands and RNA strands as in~lir~tFd in the lower half of Figure 27. The net effect is to produce ~ between 108 and 109 L~OI~CGII~ RNA strands and about 100-fold less n.,J.w~nt DNA
strands. Following a wash of the sample in a cell-free unit to remove the unused m~ m ~ n~,orcsce..l NTP's, the sample can be simply read for IlLorw~ce to det~....i..~- whether any templ~tF in this case II~aLili~ B DNA, was present in the sample.0 T~ '- 9_QI ~"lil; liOI~ of T nminPScPnt Probe Usin~ Time Resolved F1UUIU~_IIV
A novel method for detPrting nLo~ n~rlP,r-Qi~ie analogs, nuulwc~n~ ,r ~ lf ~ti~. C or analogous s~l of the amount of bound nuolw~llt ~lig..l~lf o~ide probe has been developed based on the use of photon ,uun~i. g to measure the amount of a ll-.o-uphore in a sample and is df-~- ihc~
herein below. The method differs from time resolved :,~e,l.osc(j~ in that the method intPgr~tPC all nuulQce..ce ~ n from a nLJIuphore or nucleic acid probe, in-lf-pf~ -1 of the ~ h of the c - and is both a novel ~.. lbiua~ioll of time and spectral integr~tinn and a novel ap~ of ~ $ 7 5 ~ 3 6 photon counting to the ~ r;~ nn~ detection, and ql-Antit^ntifln of nucleic acid target sC~uen~ ~-c to g~ if assays and therapeutic tr~tmf ntc The r"..,i~.... n~ LI~l r^-r,^mPtPr used in any .,.e~U,-,lucnt of l~....i..PCC~fi.^~, is the intensity of the l-....i,..~cc~..f~ l, the units of which are moles of photons per second per liter. Because the nuoresc~llt nuf 1Pr~ei~le analogs used here are, for all practical purposes, pe. ,.. ~nf n lly Lluo.~c~nt and do not photobleachwithin the lifetime of a typical lue~ulcLue-Lt, the lu---i"~s~ e of nuole c~..ce, ..lc-~u-~d in moles of photons emitted per second per mole of fluorophore, can be used as an index of the amount of Lluo.uphore, and hence probe, in a sample. The p.er~;..ed i.~~ .,t~ti~ n for such ul~Ul~,L..~
developed at Ch-o...~.g_n, c~ .~ ~ (i) a 150 watt Hg~Xe CW eylindli~l lamp capable of high intensity excitation over the range 290 nm 5 ~ < 320 nm, (iu) an ultr~hi~h seL~iliviLy photnmllltirliPr in which the photody-node is coated to allow a .~onse only over the range of PmigQi~m 360 nm 5 ~ 5 550 nm, (iii) a ;yli~dli~l cuvette with quartz . -~ l inn windows but glass walls which can serve as the P.mi.Cci~n filter.
The cuvette is mfn~nt~-d so that the entire sample can be rollPctP~ at the face of the photom-lltirlipr tube, and (iv) S ~,uLu~ule-driven photon counting clocks, ronnPrtP~ in senanm, and each capable of .li~. .i.. i,. Ii"g between photons at a frequency of 109 per second.
In ~ with the - ru....... ~.- A and full-length Xef-1~z probe ~v.. l^i"ing 489 Lul~.~_iu residues under ~ of room t- -~l....,.lllle and pH = 10, we have found that (i) the -....i.-Pc~ e of serial tlilntif~nc of the -- and the probe are linearly related to the ~ -and (ii) the l-~ r ~r~ of the probe is e luiv.,l~,L L to the same number of free - ~nn,.. ~ In a typical assay using p- -,.. ~n.-.l Lluo-uphores such a~s those shown in Figures 17 and 18, the amount of target present in a sample is ~ctl - ...i. ^~ by ~l ..-l... i..~ hybrids after u~ou--d probe has been washed away and ~,,r,~ g the amount of probe which wa,c bound. The lluol~s~LIce equivalence of residues in an analogous probe s~, - to the PmiCci~.n of the same number of ,-.~ nh -.^rc, under alkaline r~n-litin-~.
used here, ir~ tPC that there is negligible self-y~ illg in the oligomer and ~ n~ lP~ that the 25 l----.i~ c~ , of the probe can be used directly to q~ ~nli~ lr^. the amount of probe bound by target RNA
or DNA, thereby providing a broad basis for the design of iia,, nct;~ ~Pt~rt~i for a wide variety of nucleic acid assays and ~liAgn~cti.^~i It is an iu.~o.~u- consequence of the invention, that se~i~ivily and signal-to-noise ratios are a function of the number of the photons counted and the number of time periods over which counting is done.
F-~mrle 10 _ ~ttArhm~nt of S' and 3' Linkers for Immobilization of the OliLJnu~lc,otides and Hvbrids or for ~ttArhmPnt of Fluo.~.~l Oli~omers as "Labels"
The ~ h .."i~l i.~.e and ~-ocedu.cs of the iu~ iOIl can be u~sed to create and rhAr~rt~ri7~ any probe srth~-si7Pd using nuOr~nt l n- ~ le analogs, whether the synthesis is e~lic or rhf~.mir~l, for both nuor~cnce and h~ l inn ~l~-iri. ;ly~ Such probes can be used not only in the solution hy~ .tinl-formats ~lP-srrihP~d here, but aLso in the more Ll~u~ y used labol~toly ~lù~edul~;s such as "dot-blot"
detP~ti--n cl~l.u~ho,~i,inagaroseorpolya~;.yL....idegeLs,South~rnblotting,andhyl,li~ lin~onfilter ~WO 95/05391 PCT/US94/09316 21 ~S7So ; ' ` .~

Although linkers are not essential to the solution hyhrj-ii7~ti- n, any ~lu~-iate affinity litlker such as biotin/avidin or homo- or hetelubi~ rn~l linker can be used to capture the probe or hybrid for ~u.~oses of conrt~ntr~tinn, icol~tinn, or detection, as ~ ctr~trd for the PCR amplified DNA fra"
of Figure 18. The present i-.~ ion includes linker derivatized nuo~scent nucleotides, as well as liL -I~ P~~ c~ linker de.;vali~ed prirners for use in ~mrlifir~tinn and subsequent rietertinn with ~uoresce~t oli~nnrl~otide probes, oligonl~r~Poti-le probes, plasmids, and thelapeu~i.s made or otherwise tagged" therefrom, and/or their uses and applir~tinn.c such as are d~.crrih~ herein. Such deriV~ti7~tionc include, but are not limited to, I.,..l~...i..~linns to purine or ~ huidilc nnrl~c;~1~c and/or their ~t~ llu~lulal analogs, amino-thiol, a~ido-, aldehyde, h~d.~ 5' ~minn~lkyl 3' 0-ph~gphnr~mi~iitr. St_thioalkyl_3'_0-rhr, e, 3' ~minnh~.Yyl amino, amino silanes, and aminosilyl d~,~ivalives and other such linkers and groups reactive with linkers or in cnnr1~ linn ~ea~,Liolls such as Schiff base ccmtlf~ngationc of 3' or 5' oxidi_ed cis-diols, as are failiar to one silled in the art. To uctr~t~o, this a specific case is offered:
(i) a set of non-nuo.~n~ ~mrlir~ nll primers for the MOMP gene sequence was chemically synthrci7pll~ at the end of synthesis an ~ litinn~l cycle was used to add 5'-s~minnh~yl 3'_0-phncrhnr~mi-iite to the 5' I~ - ...i...~c of the ~ ~ primer with the addition chemically synthf-ci7~ using standard pho~h~,iQ~er ~h~ ly.
~ii) Following cleavage from the solid phase support in strong eth- lic base, the terminal amino group of each strand was reacted with NHS-biotin ester to provide the 5' biotinylated primers.
(iii) The primers were used for standard qmr!iri~linn~ after which the qmrlimP.nc were captured on ~ ylâlf d 96-well filter plates and washed to remove ullr~ t~ n qtP.riqlc and ~
(iv) The ealJlul~i amplimes were h~-i li~cd with lluu-~,~nl analog labeled oliL,n~ roti-le 25 probes as ~1pcrrihpA above and the amount of target s~ e in the ~.. I.li-.--.~
riPA
Tnrlllrled in the present invention are such q~ -"" -l~ of fluorescent olig.~ oliflPC to other ~luolf~s~nt or non-~ - ~S~..I oligo~m~lrotides to immobilizing beads, filters, or acLivaled plastic plates and done through enzymatic q~ 'l such as ligation, or chP~irql ~I;-- h...f --I through such linkers as are ~lf.C. ' ;hfA herein.

E~ample 11 -Uses of Fluo.~;næ ~PAI -- Ener~v Transfer (E;~ET) to Broaden or Fnhqnee the Uses of Fl~o~ nt Nu~ L d~` Analo~s and Probes Olig....-,- l~Lides can be srthP-ci7p~ or dG~ivaLi~d as dP-crrihed herein which have two or more spectrally distinct, det~bl- labels, either by using two or more ~ "- ;~ analogs with discrete ce,.~ e ,~ or by use of a covalently attached FRET acceptor, such as is f.,. il-ed 1- ~,~bo._. FRET a~l~lol5 can also be used to enhance or broaden the s~ ivily of the WO 95/053917 ~ ' i PCT/US94/09316 2~5~5~) probe f~ ni!ceinn For example, the excitation spectra of such dyes as the cuulua~ s, e.g., 7-amino4-o~3-acetate, 7-methyl-nmhellif~rone, the n~phth"lPnf and ~ h. .- r dyes, etc., overlap the P.micQinn ~e~,LIulu of n~ mf~r!c w..;,llu~ led from the nuoresccnt nnrlPnoirie analogs, e.g., poly (E~U), but not the nlig~mPr~ i~lion s~c~ llulll. Such dyes as 7-amino~-melh~lwulua,in-3-acetate may thus be used either (i) as a covalently attached FRET acceptor, e.g., by reacting the N-lly~llu~ iA-p ester with piesclil)ed amino groups on the oligomer, or (ii) by simply adding the dye to a solution of the probe to act as a FR_T ;--A;.~.,, of probe nuûl~nce. In addition to the obvious advantages of providing a second n~,O~..l label to the hybriAi7~tinn probe, t_is rnPthndnlngy allows ~mrlifir~tinn of the probe signal through more efficient capture of the emitted light, reAu(tinn of ba ~luulld light due to light 0 sr~ttPring from ~.~ ilalio-- sources, and detection at longer visible -a~ k F,~mplP 12 - RNase H Amplification Method First an RNA nuolcsc~ probe is contacted with a DNA sample. The RNA nuolcs~L.t probe h~lidi~ to a target DNA sc~ .ce. RNase H only digests RNA DNA hybrids, not ssRNA probes. The resulting lluu~c~Cenl Illl~nO~ are released into sollltinrl and a second RNA probe can llyl"idi~ to be digested. At the end of the ~ , the are sep~rated from probes on standard --~ ...h~,.nf c, and the amount of mnnnmPr released is lu~ul~,d by simple nl Olull.~ lly. The s~e ;.. ~c with no DNA
for hybrids will show no lluor~

It should be understood that the; , ' and P.mhoAim~.nt!c Ar.~crrjhe~ herein are for ill~lli~tivc p_",oses only and that various ~ r~ in~ or changes in light thereof will be ~ ^~ to persons skilled in the art and are to be included within the spirit and purview of this al)pli;alion and the scope of the appended claims.

WO95/05391 50 PCTrUS94/09316 ~U~:N~ LISTING

( 1 ) ~N~RAT. INFORMATION:
(i) APPLI QNT INFORNATION:
Applicant Name(R): r.~MA~.~N, INC.
Street addre3s: 10441 Ro~elle Street, Suite A
City: San Diego State/Province: California Cuu~lL y: US
Postal code/Zip: 92121 Phone number: (619) 558-1456 Fax number:
Telex number:

(ii) TITLE OF lNv~llON: Applic~ttn~ of Fluo e~_enL N-Nucleosides and Fluorescent Structural AnalogR of N-NucleoRide~
(iii) NUMBER OF S~QU~N~S: 3 (iv) CoT~R~cpoNDENcE AnD~ .eS:
,'A~, AnD~ee~: David R. Saliwanchik ~B STREET: 2421 N.W. 41st Street, Suite A-1 C, CITY: Gainesville D STATE: FL
EI ~UUNlK'Y: USA
~F ZIP: 32606 (V) CO ~!UL~K l~AnART.~ FORM:
'A'I MEDIUM TYPE: Floppy disk B ~O'~u ~: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
~D SOFTWARE: PatentIn Rele~e #1.0, Version ~1.25 (vi) ~uKRh.. APPLICATION DATA:
(A) APPLI QTION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/108,457 (B) FILING DATE: 18-AUG-1993 (C) CLASSIFI QTION:
(vii) PRIOR APPLICATION DATA:
(A) APPLI QTION NUMBER: US 08/021,539 (B) FILING DATE: 12-FEB-1993 (C) CLASSIFI QTION:
(vii) PRIOR APPLI QTION DATA:
(A) APPLICATION NUMBER: US 07/834,456 (B) FILING DATE: 12-FEB-1992 (C) CLASSIFI QTION:
(viii) A LOKN~Y/AGENT INFORMATION:
(A) NAME: Saliwanchik, David R.
(B) REGISTRATION N~IMBER: 31,794 (C) R~K~N~/DOCÆ T NUMBER: Chrom-l.C3 (iX) 'I'~T~ NlQTION lNI"~R~ATION:
(A) TELEPHONE: 904-375-8100 (B) TELEFAX: 904-372-5800 (2) INFORMATION FOR SEQ ID NO:l:

WO95/05391 2 1~ S 7 5 ~ PCTrUS94/09316 ,~A'I LENGTH: 39 ba~e pairs IBI TYPE: nucle;c acid ,C sTR~Nn~nN~s: single ,DJ TOPOLOGY: linear ( ii ) MnT~T!CUT~T' TYPE: DNA (genomic) (iii) ~Y~O.AhllCAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORr-~NT ~M : ~h l: y dia tr~ f ~ i (C) INDIVIDUAL T.COT ~T~: L2/434/Bu (G) OE LL TYPE: Bacterium (vii) IMMEDIATE SOURCE:
(A) T-TRRARY: lambda 1059 recombinant (B) CLONE: lamdba gtll/L2/33 (viii) POSITION IN GENOME:
(A) ~RnMnSnMT/SEGMENT: ompll2 ORF
(Xi) ~'yU~N~ D~CrRTpTIoN: SEQ ID NO:l:
AACGTTCGAG ACGr-~r-cc cTTAr-r-~rr-~ L~G 39 (2) INFORMATION FOR SEQ ID NO:2:
( i ) ~LyUL . _~ CHARACTERISTICS:
~Al LENGTH: 39 base pair~
~'B~ TYPE: nucleic acid ,'C, STR~Nn~nN~CS: ~ingle D,I TOPOLOGY: linear ( ii ) MnnT~CTTT~ TYPE: tran~cribed DNA or RNA
(iii) ~Y~lAhllCAL: NO
(iv) ANTI-SENSE: YES
(ix) FEATURE:
(A) NAME/KEY: Compl~ ~ry probe (C) ~ l~lCATION METHOD: Hybri~ tion to SEQ ID NO. l (D) OTHER INFORMATION: Control for SEQ ID NO. 3 (Xi) ~ yuL~ c DT!.C:r.RTPTION SEQ ID NO:2:
TTGCAAGCTC TGC~l~GG GAATCCTGCT r-~r~r-c 39 (2) INFORMATION FOR SEQ ID NO:3:
( i ) ~yU~N~ CHARACTERISTICS:
/A1 LENGTH: 39 ba~e pairs BI TYPE: nucleic acid ~CI sTR~N~n~s single ~D~ TOPOLOGY: linear (ii) MnT.T~'CUT.T~ TYPE: tran~cribed DNA or RNA
(iii) ~Y~LlCAL: NO
(iv) ANTI-SENSE: YES
(ix) FEATu-RE:
(A) NAME/KEY: Analogou~ c ~ ry probe ~ WO95/05391 2 14 5 7 ~ O PCTrUS94/09316 (D) OTHER INFORMATION: Analog to SEQ ID NO. 2 (xi) ~QU~N~ D~TPTION: SEQ ID NO:3:
TTGCNNGCTC TGCCTGTGGG ~N~ ~ C~ ~ GCT ~N~C~NN~C 39

Claims (11)

Claims

1. A fluorescent nucleoside, or structural analog thereof, having the following structure:

II
I

wherein X1,X2,X3,X4,X5,and X6 = N,O,C,S,or Si, wherein at least one of X1,X2, X3,X4,X5, orX6=N;
R4 is a reactive group derivatizable with a detectable label;
R5 is H or part of an etheno linkage with R4;
R6 is H, NH2, SH, or =O;
R8 and R9 can be hydrogen, methyl, bromine, fluorine, or iodine; alkyl or aromatic substituent, or optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof such as R1-(CH2)x-R2 wherein x is an integer in from 1 to 25 inclusive, and R1 and R2 are H, OH, alkyl, acyl, amide, thioether, or disulfide;
R10 is hydrogen, an acid-sensitive/base-stable blocking group, or a phosphorous derivative;
R11 = R13 = H;
R12 is hydrogen, OH, 3' amino, 3'-azido, 3'-thiol, 3'-unsaturated or a 3'-phosphorous derivative; and R14 is H, OH, or OR3 where R3 is a reactive group, protecting group, or additional fluorophore.

2. The use of a compound of claim 1 as a substitute for any of the six commonlyoccurring non-fluorescent N-nucleotides in the synthesis, amplification, enzymatic digestion, base-pairing, labeling, sequencing, replication transcription, location, detection, or identification of (c) hybridizing a fluorescent nucleoside analog probe comprising a sequence complementary to the promoter of the inserted target sequence; and (d) extending the probe sequence from the hybridized promoter region, using a nueleic acid polymerase, to synthesize a specific probe complementary to the inserted target sequence.

12. A method for increased sensitivity of detection of a target nucleotide sequence, said method comprising (a) chemically synthesizing a 5' universal end label;
(b) enzymatically synthesizing a plurality of nucleic acid sequences complementary to a discreet segment of said target sequence, wherein each of said plurality ofnucleic acid sequences comprises an end label of step (a);
(c) providing as a cocktail a mixture of the plurality of 5' end label nucleic acid sequences, and (d) hybridizing the labeled nucleic acid sequences to a target nucleic acid sequence.

13. A method for determining the sequence of bases in a polynucleotide, said method comprising (a) synthesizing a plurality of nucleic acid fragments each having different lengths, wherein each of the nucleic acid fragments has attached thereto a 5' universal end label;
(b) separating on a gel each of the different lenths of said nucleic acid fragments;
and (e) detecting directly on the gel, each of said fragment lengths.

14. A method for sustained signal amplification, said method comprising (a) extending a first strand of a double stranded nucleic acid to form a complementary double-stranded nucleic acid sequence;
(b) separating the two strands and hybridizing a separate primer sequence complementary to each of the nucleic acid sequences;
(e) extending with a nucleic acid polymerase the first strand of nucleic acid; and (d) providing a reverse transcriptase and a ribonucleic acid polymerase to cyclically synthesize nucleic acid strands.

DNA or RNA oligonucleotides.

3. The use, according to claim 2, wherein said amplification, synthesis, labeling, detection, or identification of DNA or RNA oligonucleotides is by (i) chemical synthesis, polymerization, or linking methods; or (ii) enzymatic methods of amplification, replication, endonuclease DNA
or RNA digestion, transcription, terminal labeling, filling in, or nick translation.

4. A polynucleotide probe for the detection or amplification of target polynucleotide sequence, said probe comprising a fluorescent nucleoside.

5. A method for detecting a target polynucleotide sequence, said method comprising contacting a sample suspected of having said target sequence with an effective amount of a composition comprising, a probe of claim 2 under conditions which permit hybridization; and detecting any hybridization by observing fluorescence or changes in fluorescence.

6. The method, according to claim 5, which comprises the simultaneous detection of multiple sites in a genome.

7. A method for producing a 2'-deoxy-form of a fluorescent nucleoside analog for use in oligonucleotide synthesis, wherein said method comprises (1) conversion of said analog to a 3',5' disila protected analog, and (2) deprotection to produce the 2'-deoxy-5'-triphosphate or the 2'-deoxy-3'-O-phosphoramidite form of the nucleoside.

8. A 5' universal end label for a nucleic acid sequence, wherein said label comprises a 5' homopolymer, said homopolymer comprising a non-hybridizing fluorescent nucleoside analog, and a sequence complementary to a promoter region on a nucleic acid sequence.

9. The universal end label, according to claim 8, further having a nucleic acid sequence disposed between and connected to said homopolymer sequence and said promoter complement.

10. The universal end label, according to claim 8, wherein said universal end label further comprises a nucleic acid sequence complementary to a unique target sequence.

11. A method for synthesizing a fluorescent 5' end label probe comprising (a) restricting, with a specific restriction enzyme, a sequence having a knownpromoter site and known restriction site downstream from the known promoter site;