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US5808036A
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Stem-loop oligonucleotides containing parallel and antiparallel binding domains
Assignee: University of Rochester
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Filing: 1995-06-06
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Granted: 1998-09-15
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Expires: 2015-06-06
Abstract
Abstract The present invention provides stem-loop oligonucleotides containing a double-stranded stem domain of at least about 2 base pairs and a single-stranded loop domain. The loop domains of the present oligonucleotides include at least one parallel binding (P) domain separated by at least about 3 nucleotides from a corresponding anti-parallel binding (AP) domain. Each P and corresponding AP domain of the present oligonucleotides can bind detectably to one strand of a defined nucleic acid target wherein the P domain binds in a parallel manner to the target and the corresponding AP domain binds in an anti-parallel manner to the target. The present stem-loop oligonucleotides can bind to both single-stranded and double-stranded target nucleic acids. The present invention also provides methods of using these oligonucleotides as well as kits and pharmaceutical compositions containing these oligonucleotides.
Inventors
- 👤 Eric T. Kool
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 21:22
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6823; C12Q1/6839
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
88 words
A stem-loop oligonucleotide comprising a double-stranded stem domain of at least about 2 base pairs and a single-stranded loop domain comprising at least one parallel binding (P) domain separated by at least about 3 nucleotides from a corresponding anti-parallel binding (AP) domain; wherein each P and corresponding AP domain simultaneously and detectably binds to one strand of a defined nucleic acid target; and wherein said P domain binds in a parallel manner to said target and said corresponding AP domain binds in an anti-parallel manner to said target.
Claim 2
Dependent Claim
88 words
A stem-loop oligonucleotide comprising a double-stranded stem domain of at least about 2 base pairs and a single-stranded loop domain comprising at least one parallel binding (P) domain separated by at least about 3 nucleotides from a corresponding anti-parallel binding (AP) domain; wherein each P and corresponding AP domain simultaneously and detectably binds to one strand of a defined nucleic acid target; and wherein said P domain binds in a parallel manner to said target and said corresponding AP domain binds in an anti-parallel manner to said target.
Claim 3
Dependent Claim
24 words
The oligonucleotide of claim 1 wherein said target, said P domain and said AP domain independently comprise from about 2 to about 200 nucleotides.
Claim 4
Dependent Claim
24 words
The oligonucleotide of claim 2 wherein said target, said P domain and said AP domain independently comprise from about 6 to about 36 nucleotides.
Claim 5
Dependent Claim
23 words
The oligonucleotide of claim 1 wherein each P domain is separated from a corresponding AP domain by about 3 to about 2000 nucleotides.
Claim 6
Dependent Claim
23 words
The oligonucleotide of claim 4 wherein each P domain is separated from a corresponding AP domain by about 4 to about 8 nucleotides.
Claim 7
Dependent Claim
12 words
The oligonucleotide of claim 1 wherein said target is single-stranded or double-stranded.
Claim 8
Dependent Claim
12 words
The oligonucleotide of claim 1 wherein said target is RNA or DNA.
Claim 9
Dependent Claim
21 words
The oligonucleotide of claim 1 wherein said target consists of a domain located within the sequence of a nucleic acid template.
Claim 10
Dependent Claim
65 words
The oligonucleotide of claim 1 wherein said P domain and said AP domain bind to said target in a staggered binding arrangement resulting in a target:stem-loop oligonucleotide complex which is partially two stranded and partially three-stranded and wherein said two stranded portion of said complex comprises at least one of a P:target duplex, without bound AP nucleotides, or an AP:target duplex, without bound P nucleotides.
Claim 11
Dependent Claim
21 words
The oligonucleotide of claim 1 wherein said P domain or said AP domain have less than 100% complementarity to said target.
Claim 12
Dependent Claim
23 words
The oligonucleotide of claim 10 wherein said P domain or said AP domain have about 30% to about 40% complementarity to said target.
Claim 13
Dependent Claim
12 words
The oligonucleotide of claim 1 wherein said oligonucleotide is DNA or RNA.
Claim 14
Dependent Claim
14 words
The oligonucleotide of claim 1 wherein said oligonucleotide is taken up in a cell.
Claim 15
Dependent Claim
32 words
The oligonucleotide of claim 1 wherein said oligonucleotide is a conjugated oligonucleotide further comprising at least one of a cellular receptor, cholesterol group, an aryl group, a steroid group or a polycation.
Claim 16
Dependent Claim
17 words
The oligonucleotide of claim 1 wherein said P and AP domains are separated by a non-nucleotide spacer.
Claim 17
Dependent Claim
12 words
The oligonucleotide of claim 15 wherein said non-nucleotide spacer is polyethylene glycol.
Claim 18
Dependent Claim
17 words
The oligonucleotide of claim 16 wherein said polyethylene glycol is pentaethylene glycol, hexaethylene glycol or heptaethylene glycol.
Claim 19
Dependent Claim
24 words
The oligonucleotide of claim 1 wherein said oligonucleotide further comprises at least one methylphosphonate, phosphorothioate, phosphorodithioate, phosphotriester, siloxane, carbonate, acetamidate, thioether or phosphorus-boron linkage.
Claim 20
Dependent Claim
17 words
The oligonucleotide of claim 1 wherein said oligonucleotide is a conjugated oligonucleotide further comprising a reporter molecule.
Claim 21
Dependent Claim
17 words
The oligonucleotide of claim 1 wherein two nucleic acid strands of said stem domain are covalently cross-linked.
Claim 22
Dependent Claim
47 words
A kit for the isolation, detection or diagnosis of a target nucleic acid, comprising: at least one first container providing a stem-loop oligonucleotide of claim 1 wherein at least one P domain and corresponding AP domain of said oligonucleotide can selectively bind to said target nucleic acid.
Claim 23
Dependent Claim
13 words
The kit of claim 21 wherein said target is poly (A) + mRNA.
Claim 24
Dependent Claim
30 words
An expression vector comprising a sequence encoding the oligonucleotide of claim 1 operably linked to a segment of said vector which effects expression of an RNA copy of said oligonucleotide.
Claim 25
Dependent Claim
14 words
The expression vector of claim 23 wherein said vector is a eukaryotic expression vector.
Claim 26
Dependent Claim
12 words
A complex formed between the oligonucleotide of claim 1 and a target.
Claim 27
Dependent Claim
116 words
The oligonucleotides of claim 1 wherein said oligonucleotide comprises a first pair of corresponding P and AP binding domains and a second pair of corresponding P and AP binding domains; wherein the corresponding P and AP binding domains of said first pair each constitute at least part of a spacer separating the P binding domain and the AP binding domain of said second pair, when said second pair binds to a second target; wherein the corresponding P and AP binding domains of said second pair each constitute at least part of a spacer separating the P binding domain and the AP binding domain of said first pair, when said first pair binds to a first target.
Claim 28
Dependent Claim
35 words
The oligonucleotide of claim 26 wherein said first pair of P and AP domains each comprise 2-20 cytosines while said second pair of P and AP binding domains each comprise no more than 1 cytosine.
References Cited (Backward Citations)
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