Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reissue Patent
2000-10-19
2004-02-03
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026700, C536S026800, C536S026120, C536S026130, C536S026140, C536S027600, C536S027800, C536S027810, C536S028500, C536S028540, C536S024500
Reissue Patent
active
RE038416
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides. It also relates to derivatives of pyrazolo [3,4-d] pyrimidine which are useful as nucleic acid bases for the preparation of oligonucleotides.
Oligonucleotides are useful as diagnostic probes for the detection of “target” DNA or RNA sequences. In the past, such probes were made up of sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine nucleotide bases (U.S. Pat No. 4,711,955; Robin et al., J. Can. J. Chem, 60:554 (1982); Robins et al., J. Org. Chem., 48:1854 (1983)). The method for attaching chemical moieties to these bases has been via an acetoxy-mercuration reaction. which introduce, covalently bound mercury atoms into the 5-position of the pyrimidine ring, the C-8 position of the purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., Proc. Natl. Acad. Sci. USA, 70:2238 (1973); Dale et al., Biochemistry, 14:2447 (1975)), or by the reaction of organomercurial compounds with olefinic compounds in the presence of palladium catalysts (Ruth et al., J. Org. Chem., 43:2870 (1978); Bergstrom et al., J. Am. Chem. Soc., 100:8106 (1978); Bigge et al., J. Am. Chem. Soc., 102:2033 (1980)).
The sugar component of oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural &bgr;-arabinose (patent publication EP 227,459).
A novel class of nucleotide base, the 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-
d
]-pyrimidines, has now been found which offers several advantages over the prior art. The de novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties. Also, adenine, guanine and hypoxanthine analogs we obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts. Similar pyrazolo [3,4-d] pyrimidines are known (Kobayashi. Chem. Pharm. Bull., 21:941 (1973)); however, the substituents on the group are different from those of the present invention and their only use is as xanthine oxidase inhibitors. The concept of crosslinkable nucleoside 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, N.Y., (1967), who used who was termed “active-site-directed enzyme inhibitors” in chemotherapeutic applications.
In recent years, the concept of incorporating a crosslink in an oligonucleotide has been sporadically discussed in efforts to develop superior sequence probes. Knorre and Vlassov, Prog. Nucl. Acid Res. Mol. Biol., 32:291 (1985), have discussed sequence-directed cross-linking (“complementary addressed modification”) using an N-(2-chloroethyl)-N-methylaniline group attached to either the 3′- or 5′-terminus of oligonucleotides. Summerton 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 laminating in the highly reactive bromomethyl ketone group, can crosslink to the N-7 of guanosine.
Webb and Matteucci, Nucleic Acids Res., 14:7661(1986). have prepared oligonucleotides containing a 5-methyl-N,N-ethanocytosine base which is capable of slow cross linking 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 “antionse” 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 land they effectively inhibit expression of MRNA targets in vivo (reviewed in Green, et at., Ann. Rev. Biochem., 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. Zameonik and Stephenson, Proc. Natl. Acad. Sci. USA, 75:280 (1978), showed the 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., Pro. Natl. Acad. Sci. USA 75:285 (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 oligonucleotide concentration of about 70 &mgr;M. Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intend and are reasonably stable to metabolism.
Uncharged methylphosphonate oligodeoxynucleotides with a sequence complementary to the initiation colon regions of rabbit globin mRNA inhibited the translation of the mRNA in both cell-free systems and in rabbit reticulocytes (Blake et al., Biochemistry 24:6139 (1985)). Another unchanged methylophosphonate oligonucleotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of Herpes simply 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 in covalently crosslink probe-target hybrids has the potential to dramatically improve background and sensitivity limits in diagnostic assays as well as permit novel assay funnels. 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 oligonucleotide 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 oligonucleotide 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 ther
Hurst Gerald D.
Meyer Rich B.
Petrie Charles R.
Tabone John C.
Epoch Biosciences, Inc.
Owens, Jr. Howard V.
Townsend and Townsend / and Crew LLP
Wilson James O.
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