Modified polynucleotides and methods of preparing same
DC CAFCFirst Claim
1. An oligo- or polynucleotide containing a nucleotide having the structure:
- ##STR26## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C1 '"'"'-position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is a pyrimidine, the sugar moiety is attached at the N1 -position of the pyrimidine;
wherein A comprises at least three carbon atoms and represents at least one component of a signaling moiety capable of producing a detectable signal;
wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine;
wherein one of x and y represents ##STR27## and the other of x and y is absent or represents --OH or --H; and
wherein z represents H-- or HO--.
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Abstract
Compounds having the structure: ##STR1## wherein B represents 7-deazapurine, or pyrimidine moiety covalently bonded to the C1 '"'"'-position of the sugar moiety, provided that when B is 7-deazapurine, it is attached at the N9 -position of the 7-deazapurine and when B is pyrimidine, it is attached at the N1 -position;
wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, or DNA-RNA hybrid;
wherein the dotted line represents a chemical linkage joining B and A, provided that if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and
wherein each of x, y and z represents ##STR2## either directly, or when incorporated into oligo- and polynucleotides, provide probes which are widely useful.
306 Citations
71 Claims
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1. An oligo- or polynucleotide containing a nucleotide having the structure:
- ##STR26## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C1 '"'"'-position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is a pyrimidine, the sugar moiety is attached at the N1 -position of the pyrimidine;
wherein A comprises at least three carbon atoms and represents at least one component of a signaling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine; wherein one of x and y represents ##STR27## and the other of x and y is absent or represents --OH or --H; and
wherein z represents H-- or HO--. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)
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23. A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is ##STR33##
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24. A method in accordance with claim 19 wherein said aqueous solvents include a buffer.
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25. A method in accordance with claim 24 wherein said buffer is selected from the group consisting of sodium acetate, potassium acetate, sodium citrate, potassium citrate, potassium citrate-phosphate, tris-acetate, and borate-sodium hydroxide.
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26. A method in accordance with claim 25 wherein the concentration of said buffer is less than about 2.0 molar.
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27. A method in accordance with claim 19 wherein in step (b) the aqueous solvent additionally includes an organic solvent.
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28. A method in accordance with claim 27 wherein said organic solvent is water-miscible.
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29. A method in accordance with claim 27 wherein said organic solvent is selected from the group consisting of alcohols, ketones, and amides.
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30. A method in accordance with claim 29 wherein said organic solvent is selected from the group consisting of methanol, ethanol, propanol, glycerin, dioxane, acetone, pyridine, and dimethylformamide.
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31. A method in accordance with claim 29 wherein A comprises biotin or iminibiotin, and M is selected from the group consisting of N-hydroxy-succinimide ester, imidate, anhydride, isothiocyanate, and epoxide.
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32. A method in accordance with claim 19 wherein M is an N-hydroxysuccinimide ester.
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33. A method in accordance with claim 19 wherein M is selected from the group consisting of imidate, anhydride, isothiocyanate, and epoxide.
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34. A method in accordance with claim 19 wherein . . . N is selected from the group consisting of thiol, carboxylic acid, epoxide, and amine.
- 35. A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is
- space="preserve" listing-type="equation">--CH═
CH--CH.sub.2 --NH-biotin.
- space="preserve" listing-type="equation">--CH═
- ##STR26## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C1 '"'"'-position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is a pyrimidine, the sugar moiety is attached at the N1 -position of the pyrimidine;
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36. A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is ##STR34##
- 37. A method in accordance with claim 19 wherein said chemical moiety represented by the formula . . . N is
- space="preserve" listing-type="equation">--CH═
CH--CH.sub.2 --NH-iminobiotin.
- space="preserve" listing-type="equation">--CH═
- -galactosidase.
- -galactosidase.
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42. An oligo- or polynucleotide sequence comprising at least one of a moiety having the structure:
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space="preserve" listing-type="equation">--BAwherein B represents a 7-deazapurine or a pyrimidine moiety suitable for incorporation into a polynucleotide and; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the sequence to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine. - View Dependent Claims (43, 44, 46, 47)
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45. An oligo- or polynucleotide sequence wherein A is fluorescent, electron dense, or is an enzyme capable of reacting with a substrate to form a detectable reaction product.
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48. An oligo- or polynucleotide containing therein a sugar moiety having the structure:
- ##STR36## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is a pyrimidin, the sugar moiety is attached at the N1 -position of the pyrimidine;
wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; and wherein B and A are covalently attached directly or through a linkage group that does not substantially interfere with the characteristic ability of the oligo- or polynucleotide to hybridize with a nucleic acid and does not substantially interfere with formation of the signalling moiety or detection of the detectable signal, provided also that if B is 7-deazapurine, A or the linkage group is attached to the 7-position of the deazapurine, and if B is pyrimidine, A or the linkage group is attached to the 5-position of the pyrimidine. - View Dependent Claims (49, 50, 51, 53)
- ##STR36## wherein B represents a 7-deazapurine or a pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that whenever B is a 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is a pyrimidin, the sugar moiety is attached at the N1 -position of the pyrimidine;
Specification