Cationic oligonucleotides, and related methods of synthesis and use
First Claim
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1. A composition comprising molecules of an oligonucleotide having at least one cationic internucleoside linkage having the structure (I) ##STR10## wherein:
- W is selected from the group consisting of O, S and Se;
X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected from the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl;
Z is selected from the group consisting of O, S, C 1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O, S or NR7 ;
R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond;
R2 and R3 are independently selected from the group consisting of H, C1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or R2 and R3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and
wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations,and further wherein the linkage is stereouniform among the molecules of the oligonucleotide.
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Abstract
Novel oligonucleotides are provided having cationic internucleoside linkages. The cationic internucleoside linkage has the structure ##STR1## wherein W, X, Y, Z, R1, R2, and R3 are as defined herein, P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the internucleoside linkage is stereouniform. Certain of these oligonucleotides may have alternating anionic and cationic internucleoside linkages, which are not necessarily stereouniform. A method for synthesizing the compounds are provided as well, as are methods for using the compounds, e.g., as antisense molecules and in nucleic acid hybridization assays.
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Citations
36 Claims
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1. A composition comprising molecules of an oligonucleotide having at least one cationic internucleoside linkage having the structure (I) ##STR10## wherein:
- W is selected from the group consisting of O, S and Se;
X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected from the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl; Z is selected from the group consisting of O, S, C 1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O, S or NR7 ; R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond; R2 and R3 are independently selected from the group consisting of H, C1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or R2 and R3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage is stereouniform among the molecules of the oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
- W is selected from the group consisting of O, S and Se;
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18. A composition comprising molecules of an oligonucleotide having alternating cationic and anionic internucleoside linkages wherein the cationic internucleoside linkages have the structure (II) ##STR11## wherein:
- W is selected from the group consisting of O, S and Se;
X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected from the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl; Z is selected from the group consisting of O, S, C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O or S; R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond; R2 and R3 are independently selected from the group consisting of H, C1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or wherein R2 and R3 are linked to form a five- or six-membered alkyl or aryl ring or N-, O- or S-containing heterocycle; and wherein P is a phosphorus atom that may or may not be capable of existing in two distinct stereoisomeric configurations and further wherein the linkage may or may not be stereouniform among the molecules of the oligonucleotide. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
- W is selected from the group consisting of O, S and Se;
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19. A method for making a composition comprising molecules of an oligonucleotide containing at least one cationic internucleoside linkage having the structure (I) ##STR12## wherein:
- W is selected from the group consisting of O, S and Se;
X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected from the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl; Z is selected from the group consisting of O, S, C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O, S or NR7 ; R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond; R2 and R3 are independently selected from the group consisting of H, C 1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or R2 and R3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage is stereouniform among the molecules of the oligonucleotide, said method comprising; (a) synthesizing a point racemic mixture of protected cationic nucleotide dimers comprising the cationic internucleoside linkage and a 3'"'"'--O--t-butyldimethyldisilyl protecting group; (b) optionally resolving the stereoisomers in the mixture; (c) deprotecting the cationic nucleotide dimer isolated in step (b); (d) converting the deprotected cationic nucleotide dimer provided in step (c) into the corresponding 3'"'"'--O--CH2 CH2 CN phosphoramidite derivative by reaction with Cl--P(N(iPr)2)--O--(β
-cyanoethyl); and(e) coupling the 3'"'"'--O--CH2 CH2 CN phosphoramidite derivative to an unprotected hydroxyl-containing terminal unit of an oligonucleotide chain.
- W is selected from the group consisting of O, S and Se;
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32. A composition comprising molecules of an oligonucleotide having at least one cationic internucleoside linkage having the structure (I) ##STR13## wherein:
- W is S;
X and Y are O; Z is NH; R1 is (CH2)3 ; R2 and R3 are CH3 ; and wherein P* represents an asymmetric phosphorus atom, such that the linkage exists in stereoisomeric configuration that corresponds to the configuration of the first-eluted stereoisomer when a point racemic mixture of a nucleotide dimer containing the internucleoside linkage is resolved using silica gel column chromatography.
- W is S;
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33. A nucleic acid hybridization assay comprising:
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(a) providing a labeled oligonucleotide probe containing at least one cationic stereouniform internucleoside linkage, wherein the cationic intemucleoside linkage has the structure (I) ##STR14## wherein W is selected from the group consisting of O, S and Se, X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected for the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl, Z is selected from the group consisting of O, S, C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O, S or NR7, R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond, R2 and R3 are independently selected from the group consisting of H, C1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or R2 and R3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S- containing heterocycle, and wherein P* represents and asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations; (b) hybridizing the probe to a single-stranded analyte nucleic acid to produce a labeled duplex; and (c) detecting the labeled duplex. - View Dependent Claims (35, 36)
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34. A nucleic acid hybridization assay comprising:
(a) providing a labeled oligonucleotide probe containing at least one cationic internucleoside linkage, wherein the cationic internucleoside linkage has the structure (II) ##STR15## wherein;
W is selected from the group consisting of O, S and Se;X and Y are independently selected from the group consisting of (i) O, (ii) S, (iii) C(R4)R5 where R4 and R5 are independently selected from the group consisting of H and C1 -C6 alkyl, and (iv) NR6 where R6 is H or C1 -C6 alkyl; Z is selected from the group consisting of O, S, C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene and NR7 where R7 is H or C1 -C6 alkyl, with the proviso that when W, X and Y are O, Z is O or S; R1 is selected from the group consisting of C1 -C6 alkylene, C2 -C6 alkenylene, C2 -C6 alkynylene, monocyclic arylene and a bond; and R2 and R3 are independently selected from the group consisting of H, C1 -C6 alkyl, C1 -C6 alkyl substituted with 1 to 4 NH2 groups, and monocyclic aryl, or wherein R2 and R3 are linked to form a five- or six-membered alkyl or aryl ring or N-, O- or S-containing heterocycle; (b) hybridizing the probe to a single-stranded analyte nucleic acid to produce a labeled duplex; and (c) detecting the labeled duplex.
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Current AssigneeNorthwestern University, Siemens Healthcare Diagnostics Incorporated (Siemens AG)
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Original AssigneeBayer Corporation (Bayer AG)
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InventorsBalasubramaniam, Tanjore N., Horn, Thomas, Letsinger, Robert L.
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Primary Examiner(s)Marschel, Ardin H.
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Assistant Examiner(s)Riley, Jezia
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Application NumberUS08/693,831Time in Patent Office1,273 DaysField of Search435/6, 536/22.1, 536/23.1, 536/24.3, 536/25.33, 536/25.4US Class Current435/6.14CPC Class CodesC07H 21/00 Compounds containing two or...C12Q 1/68 involving nucleic acidsC12Q 1/6839 Triple helix formation or o...
