DK2365078T3 - Fremgangsmåder under anvendelse af oligonucleotid med dobbelt specificitet og oligonucleotid med dobbelt specificitet - Google Patents
Fremgangsmåder under anvendelse af oligonucleotid med dobbelt specificitet og oligonucleotid med dobbelt specificitet Download PDFInfo
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- DK2365078T3 DK2365078T3 DK11168429.6T DK11168429T DK2365078T3 DK 2365078 T3 DK2365078 T3 DK 2365078T3 DK 11168429 T DK11168429 T DK 11168429T DK 2365078 T3 DK2365078 T3 DK 2365078T3
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- oligonucleotide
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Claims (21)
1. Oligonucleotid med dobbelt specificitet og med forøget annealing-specificitet, der er repræsenteret ved følgende almene formel: 5’-Xp-Yq-Zr-3’ hvor Xp repræsenterer et 5’-specificitetsparti med høj Tm, der har en hybridise-ringsnucleotidsekvens, der i det væsentlige er komplementær med et sted på en initial målskabelonnucleinsyre til hybridisering dermed, Yq repræsenterer et separeringsparti omfattende mindst tre på hinanden følgende universelle baser, Zr repræsenterer et 3’-specificitetsparti med lav Tm, der har en hybridise-ringsnucleotidsekvens, der i det væsentlige er komplementær med et sted på den initiale målskabelonnucleinsyre til hybridisering dermed; p, q og r repræsenterer antallet af nucleotider, og p repræsenterer et helt tal på mindst 15, q repræsenterer et helt tal på mindst 3, og r repræsenterer et helt tal på mindst 3; og X, Y og Z er deoxyribonucleotid eller ribonucleotid; Tm af 5’-specificitetspartiet med høj Tm er mindst 20 °C højere end den af 3’-specificitetspartiet med lav Tm, separeringspartiet har den laveste Tm i de tre partier; 5’-specificitetspartiet med høj Tm er længere end 3’-specificitetspartiet med lav Tm; separeringspartiet danner en ikke-baseparring under betingelser, hvor 5’-specificitetspartiet med høj Tm og 3’-specificitetspartiet med lav Tm anneales til den initiale målskabelonnucleinsyre, hvilket gør det muligt, at 5’-specificitetspartiet med høj Tm kan separeres fra 3’-specificitetspartiet med lav Tm med hensyn til annealing-specificitet til den initiale målskabelonnucleinsyre, hvorved annealing-specificiteten af oligonucleotidet til den initiale målskabelonnucleinsyre bestemmes dobbelt af 5’-specificitetspar-tiet med høj Tm og 3’-specificitetspartiet med lav Tm således, at den overordnede annealing-specificitet af oligonucleotidet forøges.
2. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor den universelle base i separeringspartiet er valgt blandt gruppen bestående af deoxyinosin, ino- sin, 7-deaza-2'-deoxyinosin, 2-aza-2'-deoxyinosin, 2'-OMe-inosin, 2'-F-inosin, deoxy-3-nitropyrrol, 3-nitropyrrol, 2'-OMe-3-nitropyrrol, 2'-F-3-nitropyrrol, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrol, deoxy-5-nitroindol, 5-nitroindol, 2'-OMe-5-nitroindol, 2'-F-5-nitroindol, deoxy-4-nitrobenzimidazol, 4-nitrobenzimida-zol, deoxy-4-aminobenzimidazol, 4-aminobenzimidazol, deoxy-nebularin, 2'-F-nebularin, 2'-F-4-nitrobenzimidazol, PNA-5-introindol, PNA-nebularin, PNA-ino-sin, PNA-4-nitrobenzimidazol, PNA-3-nitropyrrol, morpholino-5-nitroindol, morpholino-nebularin, morpholino-inosin, morpholino-4-nitrobenzimidazol, morpholino-3-nitropyrrol, phosphoramidat-5-nitroindol, phosphoramidat-nebula-rin, phosphoramidat-inosin, phosphoramidat-4-nitrobenzimidazol, phosphorami-dat-3-nitropyrrol, 2'-0-methoxyethyl-inosin, 2'0-methoxyethyl-nebularin, 2'-0-methoxyethyl-5-nitroindol, 2'-0-methoxyethyl-4-nitro-benzimidazol, 2'-0-metho-xyethyl 3-nitropyrrol og kombinationer heraf.
3. Oligonucleotid med dobbelt specificitet ifølge krav 2, hvor den universelle base er deoxyinosin, 1-(2’-deoxy-beta-D-ribofuranosyl)-3-nitropyrrol eller 5-nitroindol.
4. Oligonucleotid med dobbelt specificitet ifølge krav 3, hvor den universelle base er deoxyinosin.
5. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor p repræsenterer et helt tal på 15 til 40, fortrinsvis 15 til 25.
6. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor q repræsenterer et helt tal på 3 til 10.
7. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor r repræsenterer et helt tal på 3 til 15.
8. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor Tm af 5’-specifici- tetspartiet med høj Tm er i området fra 40 °C til 80 °C.
9. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor Tm af 3’-specifici-tetspartiet med lav Tm er i området fra 10 °C til 40 °C.
10. Oligonucleotid med dobbelt specificitet ifølge krav 1, hvor Tm af separeringspartiet er i området fra 3 °C til 15 °C.
11. Fremgangsmåde til syntetisering af et nucleinsyremolekyle under anvendelse af et oligonucleotid med dobbelt specificitet ved en skabelonafhængig forlængelsesreaktion, der omfatter trinene: (a) at anneale oligonucleotidet med dobbelt specificitet til et skabelonnucleinsy-remolekyle, hvor oligonucleotidet med dobbelt specificitet er repræsenteret ved følgende almene formel: 5’-Xp-Yq-Zr-3’ hvor Xp repræsenterer et 5’-specificitetsparti med høj Tm, der har en hybridise-ringsnucleotidsekvens, der i det væsentlige er komplementær med et sted på en skabelonnucleinsyre til hybridisering dermed, Yq repræsenterer et separeringsparti omfattende mindst tre på hinanden følgende universelle baser, Zr repræsenterer et 3’-specificitetsparti med lav Tm, der har en hybridiseringsnucleotidse-kvens, der i det væsentlige er komplementær med et sted på skabelonnucleinsy-ren til hybridisering dermed; p, q og r repræsenterer antallet af nucleotider, og p repræsenterer et helt tal på mindst 15, q repræsenterer et helt tal på mindst 3, og r repræsenterer et helt tal på mindst 3; og X, Y og Z er deoxyribonucleotid eller ribonucleotid; Tm af 5’-specificitetspartiet med høj Tm er mindst 20 °C højere end den af 3’-specificitetspartiet med lav Tm, separeringspartiet har den laveste Tm i de tre partier; 5’-specificitetspartiet med høj Tm er længere end 3’-specificitets-partiet med lav Tm; separeringspartiet danner en ikke-baseparring, der ikke tjener som hybridiseringssted og ikke interagerer med skabelonen til baseparring, under betingelser, hvor 5’-specificitetspartiet med høj Tm og 3’-specificitetspartiet med lav Tm anneales til skabelonnucleinsyren, hvilket gør det muligt, at 5’-speci-ficitetspartiet med høj Tm kan separeres fra 3’-specificitetspartiet med lav Tm med hensyn til annealing-specificitet til skabelonnucleinsyren, hvorved annealing-specificiteten af oligonucleotidet bestemmes dobbelt af 5’-specificitetspartiet med høj Tm og 3’-specificitetspartiet med lav Tm således, at den overordnede annealing-specificitet af oligonucleotidet forøges; og (b) at forlænge oligonucleotidet med dobbelt specificitet til syntetisering af nucleinsyremolekylet, der er komplementært med skabelonnucleinsyren.
12. Fremgangsmåde ifølge krav 11, hvor syntesen af nucleinsyremolekylet opnås ved gentagelse af processen med den skabelonafhængige forlængelsesreaktion, hvor trinene med annealing og forlængelse af oligonucleotidet med dobbelt specificitet følges af et denatureringstrin.
13. Fremgangsmåde ifølge krav 11, hvor oligonucleotidet med dobbelt specificitet er en probe, der er immobiliseret på et substrat.
14. Fremgangsmåde ifølge krav 11, hvor annealingen udføres ved en temperatur i området fra 45 °C til 68 °C.
15. Fremgangsmåde ifølge krav 11, hvor p repræsenterer et helt tal på 15 til 40, fortrinsvis 15 til 25.
16. Fremgangsmåde ifølge krav 11, hvor q repræsenterer et helt tal på 3 til 10.
17. Fremgangsmåde ifølge krav 11, hvor r repræsenterer et helt tal på 3 til 15.
18. Fremgangsmåde ifølge krav 11, hvor Tm af 5’-specificitetspartiet med høj Tm er i området fra 40 °C til 80 °C.
19. Fremgangsmåde ifølge krav 11, hvor Tm af 3’-specificitetspartiet med lav Tm er i området fra 10 °C til 40 °C.
20. Fremgangsmåde ifølge krav 11, hvor Tm af separeringspartiet er i området fra 3 °C til 15 °C.
21. Anvendelse af universelle baser til fremstilling af et oligonucleotid med dobbelt specificitet og med forøget annealing-specificitet for at gøre det muligt, at en annealing-specificitet af oligonucleotidet med dobbelt specificitet kan bestemmes dobbelt via en struktur af oligonucleotidet med dobbelt specificitet, hvor fremstillingen omfatter trinene: (a) at vælge en målnucleinsyresekvens; (b) at designe en sekvens af et oligonucleotid med (i) en hybridiseringssekvens, der i det væsentlige er komplementær med målnucleinsyren, og (ii) et separeringsparti omfattende mindst tre på hinanden følgende universelle baser, så at separeringspartiet griber ind i hybridiseringssekvensen til dannelse af tre partier i oligonucleotidet; og (c) at bestemme positionen af separeringspartiet i oligonucleotidet, så at et parti ved 5’-retningen af separeringspartiet kan have en højere Tm end et parti ved 3’-retningen af separeringspartiet, og så at separeringspartiet kan have den laveste Tm i de tre partier, hvorved der tilvejebringes et oligonucleotid med tre distinkte partier med forskellige Tm-værdier fra hinanden, hvor (i) et 5’-specifici-tetsparti med høj Tm af oligonucleotidet har en hybridiseringsnucleotidsekvens, der i det væsentlige er komplementær med målnucleinsyren, (ii) et 3’-specifici-tetsparti med lav Tm af oligonucleotidet har en hybridiseringsnucleotidsekvens, der i det væsentlige er komplementær med målnucleinsyren; og (iii) separeringspartiet af oligonucleotidet mellem 5’-specificitetspartiet med høj Tm og 3’-specifi-citetspartiet med lav Tm omfatter mindst tre på hinanden følgende universelle baser; 5’-specificitetspartiet med høj Tm har en længde på mindst 15 nucleotider, separeringspartiet har en længde på mindst 3 nucleotider, og 3’-specificitetspar-tiet med lav Tm har en længde på mindst 3 nucleotider; Tm af 5’-specificitetspar- tiet med høj Tm er mindst 20 °C højere end den af 3’-specificitetspartiet med lav Tm, separeringspartiet har den laveste Tm i de tre partier; 5’-specificitetspartiet med høj Tm er længere end 3’-specificitetspartiet med lav Tm, hvorved annealing-specificiteten af oligonucleotidet til målnucleinsyren bestemmes dobbelt af både 5’-specificitetspartiet med høj Tm og 3’-specificitetspartiet med lav Tm.
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