Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-05
2001-04-03
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025310, C536S025320, C536S025330, C536S025340, C536S025400
Reexamination Certificate
active
06211354
ABSTRACT:
The present invention relates to a DNA probe having an optically active phosphonic diester linkage and a method of its preparation. The present invention also relates to a method of selective cleavage of a trialkylsilyl ether linkage used for preparing the DNA probe, particularly to a method of selective elimination of a trialkylsilyl group as a hydroxyl-protecting group. The oligonucleotide obtained according to the present invention can be used for identification, extraction and control of expression of a target gene in the fields such as genetic engineering, clinical diagnosis and medical treatment.
In detection and quantification of a target nucleic acid, the ability of the target nucleic acid to hybridize with a nucleic acid probe having a base sequence complementary to a specific nucleotide sequence, namely a specific base sequence in the target nucleic acid is utilized.
On the other hand, intercalative dyes such as oxazole yellow, thiazole orange and ethidium bromide are known to remarkably enhance the fluorescence upon binding to a double-stranded nucleic acid.
Using this property, the present inventors invented a method of assay of a nucleic acid containing a specific nucleic acid sequence (a target nucleic acid) by detecting the specific nucleic acid sequence, which comprises a step of adding a single-stranded oligonucleotide containing a nucleic acid sequence complementary to the specific nucleic acid sequence in the target nucleic acid as a probe to a sample and hybridizing the probe with the target nucleic acid, wherein the probe is a single-stranded oligonucleotide labeled with a fluorescent intercalative dye which intercalates into the hybrid of the target nucleic acid and the single-stranded oligonucleotide (JP-A-8-211050, U.S. Pat. No. 5,814,447 and EP 714986).
The above-mentioned invention provided a labeled nucleic acid probe for homogeneous and simple one-step detection of a nucleic acid containing a specific nucleic acid sequence which enabled detection of hybridization or quantification of the resultant hybrid without the necessity of a step of separating the unhybridized excess probe in detection of a target nucleic acid.
When an intercalator as a label was attached in the middle of a nucleic acid probe via an aminoalkylphosphonate linker having no influence on the hybridization with the target nucleic acid, it was possible to detect even one base substitution in a target nucleic acid, though the method of labeling the nucleic acid probe is not limited thereto.
A phosphonic diester linkage as an inter-nucleotide linkage has a chiral center on the phosphorus atom in it. Namely, a DNA probe obtained by introducing phosphonic acid in the middle of an oligonucleotide has two stereoisomers having R- and S-absolute configurations.
Antisense DNA containing methylphosphoric acid under extensive research as a gene drug is known to vary in stability of its hybridization with a complementary strand (Tm), conformation and reactivity with nucleases, depending on the absolute configuration of the methylphosphonic acid.
The object of the present invention is to provide a nucleic acid probe having an intercalator as a label attaching in the middle of the nucleic acid sequence via an aminoalkylphosphonate linker, which has a configurationally different phosphorus atom.
As a result of extensive research with a view to attaining this object, the present inventors have accomplished the present invention. Namely, the present invention provides an optically active DNA probe and a method of its preparation, wherein the nucleic acid probe has an intercalator as a label attaching in the middle of the nucleic acid sequence via a chiral aminoalkylphosphonate linker.
The present invention also provides an optically active phosphonic dinucleotide useful as a precursor for preparation of the optically active DNA probe and a method of its preparation. The present inventors have found that a phosphonic dinucleotide obtained in the form of a racemate can show an Rf difference between its stereoisomers depending on the type of the protecting group in it. On the basis of this discovery, they resolved the stereoisomers easily and established a method of preparing an optically active phosphonic dinucleotide.
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R. Horie et al; “Intercalator-linked DNA Probe Having Stereoregular 2-Aminoethyl-phosphonate Diester Linkage.” Nucleic Acids Symposium Series. vol. 39. 1998, pp. 39-49, Xp002116549.
Database WPI, Section Ch, Week 8615, Derwent Publications Ltd., London, GB; Class B04, AN 86-098371, XP002116550. & JP 61 04353 A (Yuki Gosei Yakuhin Kogyo KK), Mar. 4, 1986 (1986-03-0) *Abstract*.
Horie Ryuichi
Ishiguro Takahiko
Horlick Kenneth R.
Sughrue Mion Zinn Macpeak & Seas, PLLC
Tosch Corporation
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