Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-23
2003-12-09
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S024300, C536S025300, C435S006120
Reexamination Certificate
active
06660845
ABSTRACT:
TECHNICAL FIELD
The disclosure concerns the use of nucleotide analogues to provide improved properties to hybridization probes, including DNA and RNA probes and modified nucleic acid probes, such as peptide nucleic acids (PNAs), and to chimeric probes containing two or more types of nucleic acid and/or modified nucleic acid.
BACKGROUND
Hybridization analysis is central to a variety of techniques in molecular biology and diagnostics, including gene cloning, gene identification, forensic analysis, pharmacogenomics and identification of genetic polymorphisms. Hybridization can be used as an endpoint of an assay, whereby the presence of hybridized probe constitutes the readout for the assay; or hybridization can be used as an initial step in an assay, wherein an event subsequent to hybridization (such as, for example, extension of a hybridized primer or hydrolysis of a hybridized probe) is used as the readout.
Traditionally, hybridization probes and primers have been DNA molecules; however, there are certain disadvantages to the use of DNA as a probe or primer. For example, the base composition of a DNA molecule can affect its effectiveness as a probe or primer in several ways. A DNA molecule with a high concentration of G residues is often difficult to handle (e.g., problems with aggregation and poor solubility) and can yield high background in hybridization reactions. It is also well-known that G-rich DNA molecules are prone to the production of artifacts in the analysis of DNA sequences by gel electrophoresis, presumably due to the adoption of secondary structure by such molecules, despite the denaturing conditions under which such analyses are conducted.
Various modified forms of DNA and DNA analogues have been used in attempts to overcome some of the disadvantages of the use of DNA molecules as probes and primers. Among these are peptide nucleic acids (PNAs, also known as polyamide nucleic acids). Nielsen et al. (1991)
Science
254:1497-1500. PNAs contain heterocyclic base units, as found in DNA and RNA, that are linked by a polyamide backbone, instead of the sugar-phosphate backbone characteristic of DNA and RNA. PNAs are capable of hybridization to complementary DNA and RNA target sequences and, in fact, hybridize more strongly than a corresponding nucleic acid probe. Furthermore, PNAs are resistant to many types of nuclease which attack the sugar-phosphate DNA and RNA backbones. Additional advantages of PNAs include the ability of specifically modified PNAs to cross the blood-brain-barrier and the observation that PNAs injected intrathecally can mediate antisense affects in vivo. During et al. (1999)
Nature Biotechnol.
17:753-754.
The synthesis of PNA oligomers and reactive monomers used in the synthesis of PNA oligomers have been described in U.S. Pat. Nos. 5,539,082; 5,714,331; 5,773,571; 5,736,336 and 5,766,855. Alternate approaches to PNA synthesis and monomers for PNA synthesis have been summarized. Uhlmann et al. (1998)
Angew. Chem. Int. Ed.
37:2796-2823.
However, as they become more widely used, disadvantages of PNAs are also becoming apparent. For example, long PNA oligomers, depending on their sequence, are prone to aggregation, difficult to purify and difficult to characterize. In addition, purine-rich PNA oligomers tend to aggregate and are poorly soluble in aqueous media. Gangamani et al. (1997)
Biochem. Biophys. Res. Comm.
240:778-782; Egholm,
Cambridge Healthtech Institute's Seventh Annual Nucleic Acid-Based Technologies,
Jun. 21-23, 1999, Washington, D.C.; Uhlmann,
Cambridge Healthtech Institute's Seventh Annual Nucleic Acid-Based Technologies,
Jun. 21-23, 1999, Washington, D.C. Consequently, effective use of PNAs in hybridization is limited to sequences in which there are no more than 4-5 consecutive purines, no more than 6 purines in any 10-base portion of the sequence, and/or no more than 3 consecutive G residues. See, for example, http://www.resgen.com/perseptivedesign.html. Furthermore, since PNA-PNA interactions are even stronger than PNA-DNA interactions, PNA-containing probes and primers containing self-complementary sequences cannot generally be used for hybridization to a target sequence. Another consequence of the very strong interaction between PNAs and complementary DNA and/or RNA molecules is that it is difficult to obtain single nucleotide mismatch discrimination using PNA probes. Demidov et al. (1995)
Proc. Natl. Acad. Sci. USA
92:2637-2641.
Uhlmann et al., supra reviewed approaches for increasing the solubility of PNAs, including synthesis of PNA/DNA chimeras and addition of terminal lysine residues to a PNA oligomer. They did not disclose the use of nucleotide analogues to increase solubility and improve hybridization properties of PNA oligomers.
Similar design constraints are required in the synthesis of non-PNA-containing oligonucleotide probes and primers. See, for example, the publication entitled “Sequence Detection Systems Quantitative Assay Design and Optimization,” PE Biosystems, Stock No. 117MI02-01. In these cases, the G/C content of an oligomer must be kept within the range of 20-80% and runs of an identical nucleotide, particularly guanine (G), should be avoided. In particular, the aforementioned publication advises against stretches of four or more G residues and against the presence of a G residue at the 5′ end of a 5′-fluorescently labeled probe. In the case of primers, the five nucleotides at the 3′ end should comprise no more than two G and/or C residues.
The synthesis of pyrazolo[3,4-d]pyrimidine and 7-deazapurine nucleosides, as well as their phosphoramidite monomers for use in oligomer synthesis, have been described. Seela et al (1985)
Nucl. Acids Res.
13:911-926; Seela et al. (1988a)
Helv. Chim. Acta
71:1191-1198; Seela et al. (1988b)
Helv. Chim. Acta
71:1813-1823; and Seela et al. (1987)
Biochem.
26:2232-2238. Pyrazolo[3,4-d]pyrimidine and 7-deazapurine nucleosides for use in DNA sequencing and as antiviral agents are disclosed in EP 286 028. Co-owned PCT publication WO 99/51775 discloses the use of pyrazolo[3,4-d]pyrimidine containing oligonucleotides for hybridization and mismatch discrimination. It has been reported that incorporation of 2′-deoxy-7-deazaguanosine into DNA eliminates band compression in GC-rich stretches during DNA sequence analysis by gel electrophoresis (U.S. Pat. No. 5,844,106), decreases tetraplex formation by G-rich sequences (Murchie et al. (1994)
EMBO J.
13:993-1001) and reduces formation of aggregates characteristic of DNA molecules containing 2′-deoxyguanosine (U.S. Pat. No. 5,480,980). However, substitution of oligonucleotides with either 7-deazaadenosine (in place of A) or 7-deazaguanosine (in place of G) lowers the T
m
of hybrids formed by such substituted oligonucleotides, with greater than one degree reduction in T
m
per substituted base. Seela et al. (1987) supra; and Seela et al. (1986)
Nucl. Acids Res.
14:2319-2332.
On the other hand, stabilization of duplexes by pyrazolopyrimidine base analogues has been reported. Seela et al. (1988)
Helv. Chim. Acta.
71:1191-1198; Seela et al. (1988)
Helv. Chim. Acta.
71:1813-1823; and Seela et al. (1989)
Nucleic Acids Res.
17:901-910. Oligonucleotides in which one or more purine residues have been substituted by pyrazolo[3,4-d]pyrimidines display enhanced duplex- and triplex-forming ability, as disclosed, for example, in Belousov et al. (1998)
Nucleic Acids Res.
26:1324-1328; U.S. Pat. No. 5,594,121 and co-owned PCT publication WO 98/49180. Pyrazolo[3,4-d]pyrimidine residues in oligonucleotides are also useful as sites for attachment of various pendant groups to oligonucleotides. See co-owned PCT Publication WO 90/14353, Nov. 29, 1990 and U.S. Pat. No. 5,824,796. None of these references disclose the use of pyrazolopyrimidines or any other type of base analogue for reducing aggregation and/or increasing solubility of an oligomer, or for decreasing quenching of a fluorophore conjugated to an oligomer.
Conjugates comprising a
Dempcy Robert O.
Gall Alexander A.
Kutyavin Igor V.
Vermeulen Nicolaas M. J.
Epoch Biosciences, Inc.
Riley Jezia
Townsend and Townsend / and Crew LLP
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