EP0472648A1

EP0472648A1 - Crosslinking oligonucleotides

Info

Publication number: EP0472648A1
Application number: EP90908844A
Authority: EP
Inventors: Rich B. Meyer; John C. Tabone; Gerald D. Hurst
Original assignee: MicroProbe Corp
Current assignee: Nanogen Inc
Priority date: 1989-05-18
Filing date: 1990-05-15
Publication date: 1992-03-04
Also published as: JPH04507402A; EP0472648A4; WO1990014353A1

Abstract

Cette invention concerne de nouveaux oligonucléotides comprenant au moins un agent de réticulation et l'utilisation de ces nouveaux oligonucléotides à des fins diagnostiques et thérapeutiques. Les agents de réticulation de l'invention ont la formule (I'): R1-B-(CH2)q-(Y)r-(CH2)mA', dans laquelle R1 représente l'hydrogène, ou une fraction sucre ou un analogue de celle-ci éventuellement substitué dans sa position 3' ou 5' par un dérivé de phosphore fixé par l'intermédiaire d'oxygène à la fraction sucre par un atome d'oxygène et comprenant les groupes Q1, Q2 et Q3, ou avec un précursseur réactif de ceux-ci appropriés à la formation de liaisons nucléotides; Q1 représente hydroxy, phosphate ou diphosphate; Q2 représente =O ou =S; Q3 représente CH2-R', S-R', O-R', ou N-R'R''; chacun des R' and R'' représente indépendamment l'hydrogène ou un alkyle de C1 à C6; B est une base d'acide nucléique ou un analogue de celle-ci qui est un composant d'un oligonucléotide; Y est un groupe de liaisons fonctionnelles; chacun des m et q représente indépendamment un chiffre de 0 à 8; r est égal à 0 ou 1; et A' est un groupe sortant.The present invention relates to novel oligonucleotides comprising at least one crosslinking agent and the use of these novel oligonucleotides for diagnostic and therapeutic purposes. The crosslinking agents of the invention have the formula (I '): R1-B- (CH2) q- (Y) r- (CH2) mA', in which R1 represents hydrogen, or a sugar fraction or a analog thereof optionally substituted in its 3 ′ or 5 ′ position by a phosphorus derivative attached via oxygen to the sugar moiety by an oxygen atom and comprising the groups Q1, Q2 and Q3, or with a reactive precursor thereof suitable for the formation of nucleotide bonds; Q1 represents hydroxy, phosphate or diphosphate; Q2 represents = O or = S; Q3 represents CH2-R ', S-R', O-R ', or N-R'R' '; each of R 'and R' 'independently represents hydrogen or C1 to C6 alkyl; B is a nucleic acid base or an analog thereof which is a component of an oligonucleotide; Y is a group of functional bonds; each of m and q independently represents a number from 0 to 8; r is 0 or 1; and A 'is an outgoing group.

Description

CROSSLINKING OLIGONUCLEOTIDES

This application is a continuation-in-part of application Serial No. 250,474, filed on September 28, 1988.

BACKGROUND OF THE INVENTION

This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides.

The concept of crosslinkable nucleotide probes for use in therapeutic and diagnostic applications is related to the pioneering work of B.R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors," Wiley, New York, (1967) , who used what was termed "active-site-directed enzyme inhibitors" in chemotherapeutic applications.

In recent years, the concept of incorporating a crosslink in an oligonucieotide has been sporadically discussed in efforts to develop superior sequence probes. Knorre and Vlassov, Proσ. Nucl. Acid Res. Mol. Biol.. 32:291 (1985) , have discussed sequence-directed crosslinking ("complementary addressed modification") using an N-(2-chloroethyl)-_7-methylaniline group attached to either the 3'- or 5'-terminus of oligonucleotides. Sum erton and Bartlett, J. Mol. Biol.. 122.:145 (1978) have shown that an 8-atom chain, attached to a cytosine residue at its C-4 position and terminating in the highly reactive bromomethyl ketone group, can crosslink to the W-7 of guanosine.

Webb and Matteucci, Nucleic Acids Res.. 14:7661 (1986) , have prepared oligonucleotides containing a 5-methyl-tf,tf-ethanocytosine base which is capable of slow crosslinking with a complementary strand. In a conceptually related alkylation via a linker arm within a DNA hybrid, Iverson and Dervan, Proc. Natl-. Acad. Sci. USA. 85:4615 (1988) , have shown opposite strand methylation, triggered by BrCN activation of a methylthio ether, predominately on a guanine base located two pairs from the base bearing the linker.

Oligonucleotides may be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus, a parasite or a bacterium. In nature, some RNA expression in bacteria is controlled by "antisense" RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity. A variety of recent studies using plasmid vectors for the introduction of antisense RNAs into eukaryotic cells have shown that they effectively inhibit expression of mRNA targets in vivo (reviewed in Green, et al., Ann. Rev. Bioche . 55: 569-597 (1986)). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment, which binds to the mRNA and prevents its translation into protein on ribosomes (Paterson, et al., Proc. Natl. Acad. Sci 74: 4370-4374

(1977); Hastie et al. , Proc. Natl. Acad. Sci. 75: 1217-1221 (1978)) .

In the first demonstration of the concept of using sequence-specific, antisense oligonucleotides as regulators of gene expression and as chemotherapeutic agents, Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA. 25:280 (1978), showed that a small antisense oligodeoxynucleotide probe can inhibit replication of Rous Sarcoma Virus in cell culture, and that RSV viral RNA translation is inhibited under these conditions (Stephenson et al., Proc. Natl. Acad. Sci. USA 25:285 (1978)). Zamecnik et al., Proc. Natl. Acad. Sci. USA. .83.:4143 (1986) , have also shown that oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucieotide concentrations of about 70 μM. Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intact and are reasonably stable to metabolism.

Uncharged methylphosphonate oligodeoxynucleotides with a sequence complementary to the initiation codon regions of rabbit globin mRNA inhibited the translation of the mRNA in both cell-free systems and in rabbit reticulocytes (Blake et al., Biochemistry 4:6139 (1985)). Another uncharged methylphosphonate oligonucieotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of Herpes simplex virus, Type 1, can inhibit virus replication in intact Vero cells. However, fairly high concentrations (>25 mM) of this nonionic probe were required for this inhibition. Although the impact of crosslinking oligonucleotides in the chemotherapeutic field might be of great significance, their impact in DNA probe-based diagnostics is of equally great importance. The ability to covalently crosslink probe-target hybrids has the potential to dramatically improve background and sensitivity limits in diagnostic assays as well as permit novel assay formats.

Specific innovations (discussed previously by Gamper et al_.. , Nucl. Acids Res.. 14, 9943 (1988)) include:

(a) incorporation of a denaturing wash step to remove background; (b) use of the crosslink as an additional tier of discrimination;

(c) crosslinking occurring at or near the melting temperature of the expected hybrid to insure exquisite specificity and to substantially reduce secondary structure in the target, thereby increasing the efficiency of hybrid formation; and

(d) novel solution hybridization formats as exemplified by the Reverse Southern protocol.

The concept of crosslinking, however, suggests potential problems that must be circumvented. For instance, the oligonucieotide containing a crosslinking arm might covalently bond to the target sequence so readily that mismatching of sequences will occur, possibly resulting in host toxicity. On the other hand, the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.

This issue can be addressed by constructing an oligonucieotide that, upon hybridization, results in a duplex whose T_m is just above the physiological temperature of 37'C. Thus, even a single mismatched base will prevent hybrid formation and therefore crosslinkage. The optimization can be accomplished by judicious choice of oligonucieotide length and base composition, as well as position of the modified base within the probe. The probe must be long enough, however, to insure specific targeting of a unique site.

European Patent Application No. 86309090.8 describes the formation of chemically modified DNA probes such as 5-substituted uridinyl in which the substituent does not crosslink but contains a chemical or physical reporter group. WO8707611 describes a process for labeling DNA fragments such as by chemically modifying the fragment followed by reaction with a fluorescent dye. Yabusaki et al. in U.S. Patent No. 4,599,303 disclose a scheme for covalently crosslinking oligonucleotides such as by formation of furocoumarin monoadducts of thymidine which are made to covalently bond to other nucleotides upon photoexcitation. EP 0259186 describes adducts of macromolecules and biotin which can be used as crosslinking nucleic acid hybridization probes. W08503075 describes crosslinking disulfonic esters useful as nucleic acid fragmentation agents. DE3310337 describes the covalent crosslinking of single-stranded polynucleotides to such macromolecules as proteins with the resulting complex subsequently used as a marker in hybridization experiments in the search for complementary sequences in foreign polynucleotides. A need exists for probe oligonucleotides, consisting of sufficient base sequences to identify target sequences with high specificity, that are provided with one or more crosslinking arms which readily form covalent bonds with specific complementary bases. Such oligonucleotides may be used as highly selective probes in hybridization assays. The oligonucleotides may also be used as antisensing agents of RNAs, e.g., in chemotherapy.

SUMMARY OF THE INVENTION This invention is directed to crosslinking agents which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides. The crosslinking reaction observed is of excellent specificity. The invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes.

More particularly, the crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (I'):

R_χ - B - (CH₂)_q - (Y)_r - (CH₂)_m - A' (I>)

wherein,

R-L is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached via oxygen to the sugar moiety by an oxygen and including groups 0-, Q- and Q~, or with a reactive precursor thereof suitable for nucleotide bond formation;

Q₁ is hydroxy, phosphate or diphosphate; Q₂ is =0 or =S;

Q₃ is CH₂-R', S-R¹, O-R', or N-R'R"; each of R¹ and R" is independently hydrogen or C^gal yl; B is a nucleic acid base or analog thereof that is a component of an oligonucieotide;

Y is a functional linking group; each of m and q is independently 0 to 8, inclusive; r is 0 or 1; and

A* is a leaving group.

The invention also provides novel oligonucleotides comprising at least one of the above nucleotide base derivatives of formula I' . Nucleotides of this invention and oligonucleotides into which the nucleotides have been incorporated may be used as probes. Since probe hybridization is reversible, albeit slow, it is desirable to ensure that each time a probe hybridizes with the correct target sequence, the probe is irreversibly attached to that sequence. The covalent crosslinking arm of the nucleotide bases of the present invention will permanently modify the target strand, or cause depurination. As such, the oligonucleotides of this invention are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free and cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucieotide probe comprising at least one of a labeled nucleotide base of the present invention.

Unless otherwise indicated, in the above description and throughout this document, the notation C, ₆ is inclusive, such that it indicates compounds having one, two, three, four, five, and six carbons and their isomeric forms. A further understanding of the nature and advantages of the invention may be realized by reference to the remaining portions of the specifications and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a modified deoxyuridine residue of an oligodeoxynucleotide crosslinked via an acetamidopropyl sidearm to a deoxyguanosine residue located two sites away from the complementary base along the 5' direction;

Figure 2 depicts an autoradiogram of P labeled HPV target and crosslinked product following cleavage at the 3 ' side of the crosslinked guanosme. Lane 1: 32P-labeled

15-mer size marker. Lane 2: 24 hour reaction at 20 "C. Lane 3: 72 hour reaction at 20^βC. Lane 4: 24 hour reaction at 30^βC. Lane 5: 72 hour reaction at 30^βC. Reactions were quenched with 2-aminoethanothiol and treated with piperidine solution to effect cleavage; and

Figure 3 depicts an autoradiogram of 32P labeled HPV target and crosslinked product showing hybrid separation by denaturing polyacrylamide gel electrophoresis. Lane l:

Control 32P-labeled CMV target. Lane 2: 24 hour reaction at 20^βC. Lane 3: 72 hour reaction at 20^βC. Lane 4: 24 hour reaction at 30^βC. Lane 5: 72 hour reaction at 30^βC. Reaction solutions were treated with 2-aminoethanothiol, which quenches the iodoacetamido group.

DESCRIPTION OF SPECIFIC EMBODIMENTS This invention provides novel substituted nucleotide bases with a crosslinking arm which are useful in preparing nucleosides and nucleotides and are useful as crosslinking agents. The substituted bases are of the following formula (I'):

R_χ - B - (CH₂)_g - (Y)_r - (CH₂)_m - A^» (I')

wherein,

R₁ is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached via oxygen to the sugar moiety by an oxygen and including groups Q^ , Q₂ and Q_, or with a reactive precursor thereof suitable for nucleotide bond formation;

Q, is hydroxy, phosphate or diphosphate;

Q₂ is =0 or =S; Q₃ is CH₂-R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C-L_₆alkyl;

B is a nucleic acid base or analog thereof that is a component of an oligonucieotide;

A' is a leaving group. In the practice of the present invention, the sugar moiety or analog thereof is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, ribose, deoxyribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl. The sugar moiety is preferably ribose, deoxyribose, arabinose or 2-'0-methylribose and embraces either ano er, or β .

The phosphorus derivative attached via oxygen to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate, and the like.

A reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucieotide. Reactive groups particularly useful in the present invention are those containing phosphorus. Phosphorus-containing groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose.

The nucleic acid base or analog thereof (B) may be chosen from the purines, the pyrimidines, and the deazapurines. It is preferably selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, where the purines are attached to the sugar moiety of the oligonucleotides via the 9-position, the pyrimidines via the 1-position, and the pyrrolopyrimidines via the 7-position. The functional linking group Y may be chosen from nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof, for example trifluoroacetamido, phthalimido, CONR' , NR'CO, and S0₂NR', where R' = H or C₁__galkyl. such functionalities, including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the -(CH₂) -A' group. Amino groups and blocked derivatives thereof are preferred.

The leaving group A' may be chosen from, for example, such groups as chloro, bro o, iodo, S0₂R"^!, or S- R'"R"", where each of R*" and R"" is independently C₁_₆ alkyl or aryl or R"' and R"" together form a C-__₆ alkylene bridge. Chloro, bromo and iodo are preferred. The leaving group will be altered by its leaving ability. Depending on the nature and reactivity of the particular leaving group, the group to be used is chosen in each case to give the desired specificity of the irreversibly binding probes.

Examination of double-stranded DNA by ball-and-stick models and high resolution computer graphics indicates that the 7-position of the purines and the 5-position of the pyrimidines lie in the major groove of the B-form duplex of double-stranded nucleic acids. These positions can be substituted with side chains of considerable bulk without interfering with the hybridization properties of the bases. These side arms may be introduced either by derivatization of dThd or dCyd, or by straightforward total synthesis of the heterocyclic base, followed by glycosylation. These modified nucleosides may be converted into the appropriate activated nucleotides for incorporation into oligonucleotides with an automated DNA synthesizer. Crosslinking Side Chains The crosslinking side chain should be of sufficient length to reach across the major groove from a purine 7- or 8-position, pyrimidine 5-position, pyrrolopyrimidine 5-position and reacting with the N-7 of a purine (preferably guanine) located above (on the oligomer 3'-side) the base pair containing the modified analog. Thus, the side chain should be of at least three atoms, preferably of at least five atoms and more preferably of at least six atoms in length. A generally preferred length of the side chain is from about 5 to about 9 carbon atoms.

To optimize strand crosslinking, it would be desirable to have the target strand base which is being attacked paired to the first or second base which is on the 3' side of the modified base in the oligonucieotide containing the crosslinking arm. For example, in the case where the target strand base under attack is a guanine, the target sequence for a probe containing a modified uracil should contain the complement GZA (preferably GGA) , where Z is any base, with the probe oligonucieotide containing UZC (preferably UCC) , where TJ is dUrd 5-substituted with the crosslinking arm. In oligonucleotides containing crosslinking adenine derivatives, for example, the adenme-modified AZ C triplet would target GZ T, where Z is any base. It has been found that when the modified base containing the crosslinking arm is a uracil and the target sequence is GGA, alkylation of the second guanine on the target's 5' side of the crosslinker-modified base pair is the exclusive action observed (as shown in Figure 1) . The crosslinking reaction seems to be very specific for the

"best fit" of electrophile to nucleophile, i.e., two or more guanine residues may need to neighbor the complement of the modified base to discover the site of alkylation. Crosslinking Acrents - Preparation of Nucleosides One class of modified 2'-deoxynucleosides have demonstrated particular usefulness in the present invention for incorporation into oligonucleotides as sequence-directed crosslinking agents. This is the

5-substituted-2'-deoxyuridines whose general structure is presented below:

(CH₂)_q __(γ)r __(CH_2in __A,

The 5-(substituted)-2'-deoxyuridines may be prepared by the routes shown in Schemes 1 and 2. The moiety Y' in Schemes 1 and 2 refers to -(Y)_r-(CH₂)_m-A' .

Scheme 1 :

(XX)

Pd(0)

deoxyribose

(xxπ)

deoxyribose (XXIII)

For example, the general procedure of Robins et al. (J. Can. J. Chem.. .60:554 (1982); J. Org. Che .. 48:1854 (1983)) may be adapted, as shown in Scheme 1, to the palladium-mediated coupling of a substituted 1-alkyne (XXI) to 5-iodo-2'-deoxyuridine (XX) to give the acetylene-coupled product (XXII) . The acetylenic dϋrd analog XXII is reduced, with Raney nickel for example, to give the saturated compound (XXIII) , which is then used for direct conversion to a reagent for use on an automated DNA synthesizer, as described below.

Scheme 2 :

4- HCrCH (CH₂) -__2 Y" deoxyribose (XXV)

deoxyribose (XXVI)

TFA/TFAA

— Y'

deoxyribose (XXVII)

(XXIII) When 5-chloromercurio-2'-deoxyuridine (XXIV) is used as a starting compound, it cannot be directly coupled to an olefin group to give the olefinic compound (XXVII) by palladium-catalyzed coupling with functionalized olefins. Instead, as shown in Scheme 2, a substituted alkene (XXV) and 5-chloromercurio-2'-deoxyuridine (XXIV) are reacted together with methanol to give the alpha- ethoxy adduct (XXVI) , which is converted to the olefinic compound XXVII by trifluoroacetic acid and trifluoroacetic anhydride. Reduction gives the saturated compound (XXIII) , to be converted to the DNA synthesizer-ready reagent as described below.

In the practice of the present invention, the sugar moiety or its analog is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl. The sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, a or β .

A reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucieotide. Reactive groups particularly useful in the present invention are those containing phosphorus. Phosphorus-containing groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose. Oligonucleotides - Utility and Preparation

Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valuable in chemotherapy because they increase the efficiency of inhibition of mRNA translation or gene expression control by covalent attachment of the oligonucieotide to the target sequence. This can be accomplished by crosslinking agents being covalently attached to the oligonucieotide, which can then be chemically activated to form crosslinkages which can then induce chain breaks in the target complementary sequence, thus inducing irreversible damage in the sequence. Examples of electrophilic crosslinking moieties include alpha-halocarbonyl compounds, 2-chloroethylamines and epoxides. When oligonucleotides of the invention are utilized as a probe in nucleic acid assays, a label is attached to detect the presence of hybrid polynucleotides. Such labels act as reporter groups and act as means for detecting duplex formation between the target nucleotides and their complementary oligonucieotide probes.

A reporter group as used herein is a group which has a physical or chemical characteristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.

Oligonucleotides of the present invention may comprise at least one and up to all of their nucleotides from the substituted nucleotide bases of formula I• . To prepare oligonucleotides, protective groups are introduced onto the nucleosides of formula I' and the nucleosides are activated for use in the synthesis of oligonucleotides. The conversion to protected, activated forms follows the procedures as described for 2*-deoxynucleosides in detail in several reviews. See,

Sonveaux, Bioorganic Chemistry. 14.:274-325 (1986) ; Jones, in "Oligonucieotide Synthesis, a Practical Approach", M.J. Gait, Ed., IRL Press, p. 23-34 (1984).

The activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA and RNA nucleotides, in that the correct nucleotides will be sequentially linked to form a chain of nucleotides which is complementary to a sequence of nucleotides in target DNA or RNA. The nucleotides may be incorporated either enzy atically or via chemical synthesis. The nucleotides may be converted to their 5•-O-dimethoxytrityl-3 ' - (N,N- diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in "Oligonucieotide Synthesis: A Practical Approach", supra . The W-protecting groups are then removed, along with the other oligonucieotide blocking groups, by post-synthesis aminolysis, by procedures generally known in the art.

In a preferred embodiment, the activated nucleotides may be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed. The oligonucleotides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.

The leaving group, such as a haloacyl group, may be added to the aminoalkyl tails (-CH₂) -Y) following incorporation into oligonucleotides and removal of any blocking groups. For example, addition of an α-haloacetamide may be verified by a changed mobility of the modified compound on HPLC, corresponding to the removal of the positive charge of the amino group, and by subsequent readdition of a positive charge by reaction with 2-amino- ethanethiol to give a derivative with reverse phase HPLC mobility similar to the original aminoalkyloligonucleotide. In specific embodiments, each of the following electrophilic leaving groups were attached to an aminopropyl group on human papillomavirus (HPV) probes: bromoacetyl, iodoacetyl and the less reactive but conformationally more flexible 4-bromobutyryl. Bromoacetyl and iodoacetyl were found to be of equal reactivity in crosslinking. Oligonucieotide Probe Labelling

An oligonucieotide probe according to the invention includes at least one labeled substituted nucleotide base of formula I' .

Probes may be labeled by any one of several methods typically used in the art. A common method of detection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P labeled probes or the like. Other reporter groups include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates. Alternatively, the same components may be indirectly bonded through a ligand-antiligand complex, such as antibodies reactive with a ligand conjugated with label. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.

The choice of label dictates the manner in which the label is incorporated into the probe. Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope. The radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3' end of probes with terminal transferase, by copying M13 plasmids having specific inserts with the Klenow fragment of DNA poiymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA poiymerase in the presence of radioactive rNTP's. Non-radioactive probes can be labeled directly with a signal (e.g., fluorophore, chemiluminescent agent or enzyme) or labeled indirectly by conjugation with a ligand. For example, a ligand molecule is covalently bound to the probe. This ligand then binds to a receptor molecule which is either inherently detectable or covalently bound to a detectable signal, such as an enzyme or photoreactive compound. Ligands and antiligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring antiligand. Alternatively, any haptenic or antigenic compound can be used in combination with a suitably labeled antibody. A preferred labeling method utilizes biotin-labeled analogs of oligonucleotides, as disclosed in Langer et al., Proc. Natl. Acad. Sci. USA. 78:6633-6637 (1981) , which is incorporated herein by reference. Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, rare earths, etc. Chemiluminescers include luciferin, acridiniu esters and 2,3-dihydrophthalazinediones, e.g., luminol. Hybridization and Crosslinking of Probe and Target - Assays The specific hybridization and crosslinking conditions are not critical and will vary in accordance with the investigator's preferences and needs. Various hybridi¬ zation solutions may be employed, comprising from about 20% to about 60% volume, preferably about 30%, of a polar organic solvent. A common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HC1, PIPES or HEPES, about 0.05% to 0.5% detergent, such as sodium dodecylsulfate, and between 1-10 mM EDTA, 0.01% to 5% ficoll (about 300-500 kDal), 0.1% to 5% polyvinylpyrrolidone (about 250-500 kDal), and 0.01% to 10% bovine serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/ml, e.g., partially fragmented calf thymus or salmon sperm, DNA, and/or partially fragmented yeast RNA and optionally from about 0.5% to 2% wt./vol. glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anioniσ polyacrylate or polymethylacrylate, and charged saccharidic polymers, such as dextran sulfate. The particular hybridization technique is not essential to the invention. Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach", Hames and Higgins, Eds. , IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. Sci.. U.S.A.. €>3_:378-383 (1969); and John et al.. Nature. 22J3:582-587 (1969) . As improvements are made in hybridization techniques, they can readily be applied.

The amount of labeled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.

Various degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex. The degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20^β to 80°C, usually 25^β to 75°C. For probes of 15-50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65*C. With routine experimentation, one can define conditions which permit satisfactory hybridization at room temperature. The stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of about 20% to about 50%. Treatment with ultrasound by immersion of the reaction vessel into commercially available sonication baths can oftentimes accelerate the hybridization rates. After hybridization and crosslinking at a temperature and time period appropriate for the particular hybridization solution used, the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar reagents (e.g., sodium chloride, buffers, organic solvents and detergent) , as provided in the hybridization solution. These reagents may be at similar concentrations as the hybridization medium, but often they are at lower concentrations when more stringent washing conditions are desired. The time period for which the support is maintained in the wash solutions may vary from minutes to several hours or more.

Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.

The probe may be conjugated directly with the label. For example, where the label is radioactive, the support surface with associated hybridization complex substrate is exposed to X-ray film. Where the label is fluorescent, the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector ("Physical Biochemistry", Freifelder, D. , W.H. Freeman & Co., 1982, pp. 537-542). Where the label is an enzyme, the sample is detected by incubation with an appropriate substrate for the enzyme. The signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence. The preferred label for dipstick assays generates a colored precipitate to indicate a positive reading. For example, alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly colored and insoluble formazans. Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and antiligand interactions as between a ligand-conjugated probe and an antiligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.

The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label. (Tijssen, P., "Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology", Burdon, R.H. , van Knippenberg, P.H., Eds., Elsevier, 1985, pp. 9-20).

The amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybridization medium and/or wash medium. Generally, substantial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids. Method of Identifying Target Seguences

The invention is also directed to a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucieotide probe including at least one labeled substituted nucleotide moiety of formula I'.

In one embodiment, the method comprises the steps of:

(a) denaturing nucleic acids in the sample to be tested; (b) hybridizing to the target nucleic acids an oligonucieotide probe including at least one labeled substituted nucleotide moiety of formula I• , wherein the probe comprises a sequence complementary to that of the target nucleic acids, under conditions which permit crosslinking of probe and target;

(c) washing the sample to remove unbound probe;

(d) incubating the sample with detection agents; and (e) inspecting the sample.

The above method may be conducted following procedures well known in the art.

An assay for identifying target nucleic acid sequences utilizing an oligonucieotide probe including at least one labeled substituted nucleotide moiety of formula I' and comprising the above method is contemplated for carrying out the invention. Such an assay may be provided in kit form. For example, a typical kit will include a probe reagent component comprising an oligonucieotide including at least one labeled nucleotide moiety of formula I', the oligonucieotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single-stranded nucleic acid; and a hybridization reaction mixture. The kit can also include a signal-generating system, such as an enzyme for example, and a substrate for the system.

The following examples are provided to illustrate the present invention without limiting same. General Thin layer chromatography was performed on silica gel 60 F 254 plates (Analtech) using the following solvent mixtures: A- 90% methylene chloride:10% methanol; B- 50% ethyl acetate:50% hexanes; C- 70% ethyl acetate: 10% methanol:10% water:10% acetone; D- 50% ether:50% hexanes. Flash chromatography was performed using 60 F 254 silica (Merck) . Oligonucleotides were synthesized on an Applied Biosystems Model 380B Synthesizer. Oligonucleotides were isotopically labeled using T4 Polynucleotide kinase (BRL) and r-³²P-ATP (New England Nuclear) . RT refers to room temperature.

EXAMPLE 1: 5-(4-Phthalimidobut-l-yn-l-yl)-2'-deoxyuridine.

5-Iodo-2*-deoxyuridine (354 mg, 1 mmol) was dissolved in 10 L of dimethylformamide. Cuprous iodide (76 mg, 0.4 mmol), tetrakis(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol), and triethylamine (200 mg, 2.0 mmol) were added. 4-Phthalimidobut-l-yne (300 mg, 1.5 mmol) was added all at once and the reaction kept at 60^βC for three hours. The clear yellow reaction was then evaporated and methylene chloride was added. Scratching of the flask induced crystallization of nearly all of the product which was filtered and recrystallized from 95% ethanoi to give 335 mg (78%) of title compound as fine, feathery needles.

EXAMPLE 2:

5-(4-Phthalimidobut-l-yl)-2'-deoxyuridine.

1.00 Gram of deoxyuridine from Example 1 was dissolved in 95% EtOH and about 3 g of neutral Raney nickel was added. After 48 hours, the catalyst was removed by cautious filtration and the filtrate was evaporated to a solid which was recrystallized from methanol-water to give 960 mg (97%) of the title compound.

EXAMPLE 3:

5-(3-Iodoacetamidopropyl)-2*-deoxyuridine.

5-(3-Trifluoroacetamidoprop-1-yl)-2'-deoxyuridine (0.3 mmol) is treated with ammonia and then with N-hydroxy- succinimidyl ce-iodoacetate (0.5 mmol) . The reaction mixture is evaporated to dryness and purified by chromatography to give 5-(3-iodoacetamidopropyl)-2'-deoxyuridine. EXAMPLE 4 :

5-(4-(4-Bromobutyramido)butyl)-2'-deoxyuridine.

Following the procedure of Example 3, 5-(4-phthalimidobut-l-yl)-2'-deoxyuridine, from Example 2, is treated with ammonia and then with N-hydroxysuccinimidyl 4-bromobutyrate to give 5-(4-(4-bromobutyramido)butyl)- 2*-deoxyuridine.

Preparation of Synthetic Oligonucleotides

EXAMPLE 5: Phosphoramidite Preparation and DNA Synthesis.

Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3•-phosphoramidite was made using diisopropylamino /3-cyanoethylchlorophosphite (as described in "Oligonucieotide Synthesis: A Practical Approach", supra) with diisopropylethylamine in ethylene chloride. The phosphoramidite was made into a 0.2N solution in acetonitrile and placed on the automated DNA synthesizer. Incorporation of these new and modified phosphoramidites gave incorporation similar to ordinary phosphoramidites

(97-99% as judged by assay of the trityl color released by UV.)

Oligonucleotides were removed from the DNA synthesizer in tritylated form and deblocked using 30% ammonia at 55^βC for 6 hours. Ten μL of 0.5M sodium bicarbonate was added to prevent acidification during concentration. The oligonucieotide was evaporated to dryness under vacuum and redissolved in 1.0 L water. The oligonucleotides were purified by HPLC using 15-55% acetonitrile in 0.1N triethylammonium acetate over 20 minutes. Unsubstituted oligonucleotides came off at 10 minutes; amino derivatives took 11-12 minutes. The desired oligonucieotide was collected and evaporated to dryness, then it was redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group. Desalting was accomplished with a G25 Sephadex column and appropriate fractions were taken. The fractions were concentrated, brought to a specific volume, dilution reading taken to ascertain overall yield and an analytical HPLC done to assure purity. Oligonucleotides were frozen at -20^βC until use. Following the above procedures, the nucleoside

5-(3-trifluoroacetamidoprop-1-yl)-2•-deoxyuridine was converted to the 5'-O-dimethoxytrityl-3• (N,N-diisopropyl)- phosphoramidite cyanoethyl ester derivative. This was added to a DNA synthesizer and the following 14-mer oligonucieotide was prepared:

3'-cτ Tec U-HΓG TAG GTC-5' where U is 5-(3-amιnoprop-l-yl)-2'-deoxyuridine (oligo A).

In the same manner, 5-(4-phthalimidobut-l-yl)- 2'-deoxyuridine was converted to the 5'-O-dimethoxytrityl- 3'-(N,N-diisopropyl)phosphoramidite cyanoethyl ester derivative and added to a DNA synthesizer to prepare the above 14-mer oligonucieotide sequence where U is 5-(4-aminobut-l-yl)-2'-deoxyuridine (oligo C) .

A corresponding 14-mer oligonucieotide was also prepared where U is the unmodified deoxyuridine.

EXAMPLE 6:

Derivatization of Oligonucleotides.

In general, to add the crosslinking arm to an aminoalkyloligonucleotide, a solution of 10 μg of the aminoalkyloligonucleotide and a 100X molar excess of n-hydroxysuccinimide haloacylate such as α-haloacetate or 4-halobutyrate in 10 μL of 0.1 M borate buffer, pH 8.5, was incubated at ambient temperature for 30 min. in the dark. The entire reaction was passed over a NAP-10 column equilibrated with and eluted with distilled water. Appropriate fractions based on UV absorbance were combined and the concentration was determined spectrophotometrically.

Introduction of the haloacyl moiety was examined by HPLC. A Zorbax® oligonucieotide column (Dupont) eluted with a 20 minute gradient of 60% to 80% B composed of: A (20% acetonitrile:80% 0.02 N NaH₂P0₄) and B (1.2 N NaCl in 20% acetonitrile:80% 0.02 N NaH₂P0₄) . The presence of a reactive α-haloacyl moiety was indicated by return of the retention time of the α-haloacylamidoalkyl oligonucieotide to the corresponding aminoalkyl oligonucieotide after exposure to IN cysteamine. Introduction of cysteamine created equivalent charge patterns between the aminoalkyl oligonucieotide and the α-haloacylamido oligonucieotide. Following the above procedure, the 14-mer oligonucieotide:

3'-CT TCC υ TG TAG GTC-5' where U 1 i.s 5-(3-amιnoprop-1-yl)-2'-deoxyuridine (oligo A,

Example 5) , was reacted with n-hydroxysuccinimide α-iodoacetate to give the above 14-mer oligonucieotide where Uι is 5-(3-ιodoacetamιdoprop-l-yl)-2'-deoxyuridine

(oligo B) .

Oligo A and oligo B, as well as the above 14-mer where Uι i.s the unmodi.fied deoxyuridine were resolved in the

Zorbax column, all of identical sequence, with the following retention times: unmodified 14-mer, 9.31 min; aminopropyl

14-mer (oligo A), 7.36 min; and iodoacetamidopropyl 14-mer

(oligo B) , 10.09 min.

In the same manner, the aminopropyl 14-mer (oligo A) was reacted with N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U is 5-(3-(4-bromobutyramido) prop-1-yl)-2'-deoxyuridine.

Also, the aminobutyl 14-mer (oligo C, Example 5) was reacted with either N-hydroxysuccinimide α-iodoacetate or

N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U is 5-(4-iodoacetamidobut-l-yl)-2'-deoxyuridine or

5-(4-(4-bromobutyramido)but-l-yl)-2'-deoxyuridine, respectively. EXAMPLE 7 :

Evaluation of Crosslinking Potential of Modified Oligonucleotides.

The reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 μg of haloacylamidoalkyl probe and 10 ng of 32P-labeled cordycepm-tailed target in

200 μL of 0.1 M Tris, pH 8.0, and 0.9 M NaCl incubated at

20° or 30^βC. Aliquots were removed at 24- or 72-hour intervals and diluted in 20 μL of 10 mM cysteamine to quench the haloacylamido group. These solutions were stored at RT, and 1 μL was used for analysis by denaturing polyacrylamide gel electrophoresis (PAGE) .

Following the above procedure, two model oligonucleotides were utilized to evaluate the crosslinkage potential of the modified probe to its complement. The sequences, derived from human papillomavirus (HPV) or human cytomegalovirus (CMV) , are shown below:

HPV System:

5 10 15 20 25 30 I I I I I I

Target: 5'-AGA CAG CAC AGA ATT CGA AGG AAC ATC CAG-3' Probe: 3'-CT TCC UTG TAG GTC-5•

CMV System:

5 10 15 20

I I

Target: 5'-ACC GTC CTT GAC ACG ATG GAC TCC-3^» Probe: 3'-GAA CTG TGC UAC CTC-5•

U = 5-[3-(α-iodoacetamido)- or 3-

(4-bromobutyramido)-propyl]-2'-deoxyuridine, or

U = 5-[3-(α-iodoacetamido)- or 4-

(4-bromobutyramido)-butyl]-2'-deoxyuridine. The target for HPV is a 30-mer, and for CMV it is a 24-mer. The crosslinking probes were a 14-mer for HPV and two 15-mers for CMV. Each probe contained a single modified deoxyuridine designated as U in the sequences above. Results of the reaction of HPV target with a limiting amount of crosslinking probe containing a 5-(3-iodoacetamidopropyl) sidearm are shown in Figure 2. Analysis of the cleavage pattern on a denaturing PAGE gel showed the loss of the crosslinked hybrid with the concomitant appearance of a discrete low molecular weight band. The intensity of this band was dependent upon the extent of crosslinkage in the initial reaction. The localization of signal into two discrete bands on the gel strongly argues that no non-sequence-directed alkylation of either target or probe strands had occurred (including intramolecular probe alkylation) . Comparison to an authentic 15-mer run in an adjacent lane suggested that the major cleaved fragment is a 9-mer. Upon close examination of the original autoradiogram, a slower moving band of very weak intensity was visible. This pattern would be consistent with major alkylation at G-21 and minor alkylation at G-20. An examination of a Dreiding model of the crosslinkable HPV hybrid shows that the 5-(3- iodoaceta idopropyl) sidearm can contact the G-21 residue of the target strand with only minor distortion of the helix.

If alkylation occurs predominately at a guanosine on the target strand located two units on the 5' side of the modified-deoxyuridine base pair, the CMV sequence should not react with the probe. This result was in fact observed. The absence of reaction of probe with CMV further supports the specificity of crosslinking scheme of the invention.

EXAMPLE 8:

Time and Temperature Dependence.

Time and temperature dependence studies were carried out with the HPV system of Example 7 where U is 5-(3-iodoacetamidoprop-l-yl)-2'-deoxyuridine. The target was ³²P-labeled by cordycepin tailing with terminal transferase (Maniatis et al. , "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, 1982, p. 239) and incubated with excess probe in a pH 8.0 Tris buffer at either 20" or 30^βC. Aliquots were removed after 0, 24, or 72 hours incubation, quenched with an equivalent volume of 10 mM mercaptoethylamine (which reacts with the iodoacetamide) , and stored at RT for subsequent analysis by denaturing or non-denaturing PAGE.

Crosslinkage of the hybrid, which was monitored by denaturing PAGE, was evident for the 24 and 72 hour time points at both temperatures (see Figure 3) . The amount of crosslinked hybrid increased with both temperature and time. Approximately 20% of the hybrid was crosslinked after 72 hours incubation at 30^βC. Separate experiments at a range of temperatures indicated that the half-life for crosslinking at 37^βC is approximately 2 days, and that the reaction is complete after 24 hours at 58^βC. This time-dependent reaction implies that the iodoacetamido moiety does not hydrolyze or react with the buffer. The increased reaction rate at higher temperature indicates that the hybrid is maintained, and subsequently the rate of alkylation shows the expected increase with temperature.

EXAMPLE 9: Site Specificity of Alkylation.

To elucidate the site specificity of alkylation, the crosslinked HPV hybrid of Example 7 (where U is 5- (3-iodoacetamidoprop-l-yl)-2'-deoxyuridine) was subjected to a 10% piperidine solution at 90"C for 60 minutes. As shown by Maxam et al. (Proc. Natl. Acad. Sci. USA_f 24:560 (1977), this treatment quantitatively cleaves the target strand 3'- to the site of alkylation. The resulting data indicated that the alkylation of the second guanine above the crosslinker-modified base pair (i.e., the guanine above the target base) was the exclusive action observed, indicating that the crosslinking reaction in the HPV model system is remarkably specific.

Claims

WHAT IS CLAIMED IS:

1. A compound having the following formula (I¹):

R_± - B - (CH₂)_g - (Y)_r - (CH₂)_m - A^« (I')

wherein, R, is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached via oxygen to the sugar moiety by an oxygen and including groups Q₁, Q₂ and Q₃, or with a reactive precursor thereof suitable for nucleotide bond formation;

Q₁ is hydroxy, phosphate or diphosphate; Q₂ is =0 or =S;

Q₃ is CH₂-R', S-R^«, 0-R^», or N-R'R"; each of R' and R" is independently hydrogen or C^galkyl;

A' is a leaving group.

2. A compound according to Claim 1 wherein B is selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3- d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]- pyrimidin-5-yl.

3. A compound according to Claim 2 wherein R-, is a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate or a reactive precursor suitable for nucleotide bond formation.

4. A compound according to Claim 3 wherein m is 0, 1, 2 or 3; q is 2, 3 or 4; and r is 1.

5. A compound according to Claim 3 wherein B is uracil-5-yl.

6. A compound according to Claim 3 wherein the sugar moiety is ribose, deoxyribose, arabinose or 2'-O-methylribose.

7. A compound according to Claim 6 wherein the sugar moiety is deoxyribose.

8. A compound according to Claim 6 wherein the group -(CH₂) -(Y)_r-(CH₂)_m-A' is 3-iodoacetamidopropyl, 3-(4-bromobutyramido)propyl, 4-iodoacetamidobutyl, or 4-(4-bromobutyramido)butyl.

9. A compound according to Claim 8 wherein B is uracil-5-yl.

10. A compound according to Claim 9 wherein the sugar moiety is deoxyribose.

11. An oligonucieotide which comprises at least one of the following:

R - B - (CH₂)_g - (Y)_r - (CH₂)_m - A' (I^«)

wherein,

R, is a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a monophosphate derivative attached to the sugar moiety by an oxygen;

B is a nucleic acid base or analog thereof that is a component of an oligonucieotide; Y is a functional linking group; each of m and q is independently 0 to 8, inclusive; r is 0 or 1; and

A* is a leaving group.

12. An oligonucieotide according to Claim 11 wherein B is selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl.

13. An oligonucieotide according to Claim 12 wherein m is O, 1, 2 or 3; q is 2, 3 or 4; and r is l.

14. An oligonucieotide according to Claim 12 wherein B is uracil-5-yl.

15. An oligonucieotide according to Claim 12 wherein the sugar moiety is ribose, deoxyribose, arabinose or 2^«-O-methylribose.

16. An oligonucieotide according to Claim 15 wherein the sugar moiety is deoxyribose.

17. An oligonucieotide according to Claim 15 wherein the group -(CH₂) -(Y)_r-(CH₂)_m-A' is 3- iodoacetamidopropy1, 3-(4-bromobutyramido)propyl, 4-iodoacetamidobutyl, or 4-(4-bromobutyramido)butyl.

18. An oligonucieotide according to Claim 17 wherein B is uracil-5-yl.

19. An oligonucieotide according to Claim 18 wherein the sugar moiety is deoxyribose.

20. A method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucieotide probe including at least one labeled compound of formula (I¹) as defined in Claim 1.

21. A method according to Claim 20 which comprises the steps of: (a) denaturing nucleic acids in the sample to be tested;

(b) hybridizing to the target nucleic acids an oligonucieotide probe including at least one labeled substituted compound of formula (I'), wherein the probe comprises a sequence complementary to that of the target nucleic acids, under conditions which permit crosslinking of probe and target;

(c) washing the sample to remove unbound probe; and

(d) detecting duplex formation between the target and probe nucleic acids.

22. An assay for identifying target nucleic acid sequences, which assay comprises utilization of an oligonucieotide probe including at least one labeled substituted compound of formula (I') as defined in Claim 1.

23. A kit for identifying target nucleic acid sequences, which kit comprises a probe reagent component comprising an oligonucieotide including at least one labeled substituted compound of formula (I¹) as defined in Claim 1, the oligonucieotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single-stranded nucleic acid; and a hybridization and crosslinking reaction mixture.