Replicative RNA reporter systems
First Claim
1. A method of determining the presence of a biopolymer analyte in a sample, which method comprises(i) exposing the sample to an affinity molecule for said analyte under conditions whereby binding occurs between the affinity molecule and the analyte;
- (ii) if said affinity molecule is not itself a replicative RNA, joining, either before or after step (i), a replicative RNA to the affinity molecule employed in step (i);
(iii) employing a RNA-dependent RNA polymerase to catalyze replication of replicative RNA that is or had been joined to affinity molecule bound to analyte or that is affinity molecule that had been bound to analyte; and
(iv) detecting RNA made by the reaction of step (iii).
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Abstract
Highly sensitive methods for assaying for biopolymers, such as by immunoassay or nucleic acid probe hybridization assay, and compositions for carrying out the methods, are provided. The methods employ as reporter group a RNA capable of being autocatalytically replicated by an RNA-dependent RNA polymerase, such as the replicase of bacteriophage Qβ. The high sensitivity of the assay methods is due to the rapid, exponential increase in concentration of such a replicative RNA, associated specifically with biopolymer analyte, that can be achieved by autocatalytic RNA replication.
43 Citations
53 Claims
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1. A method of determining the presence of a biopolymer analyte in a sample, which method comprises
(i) exposing the sample to an affinity molecule for said analyte under conditions whereby binding occurs between the affinity molecule and the analyte; -
(ii) if said affinity molecule is not itself a replicative RNA, joining, either before or after step (i), a replicative RNA to the affinity molecule employed in step (i); (iii) employing a RNA-dependent RNA polymerase to catalyze replication of replicative RNA that is or had been joined to affinity molecule bound to analyte or that is affinity molecule that had been bound to analyte; and (iv) detecting RNA made by the reaction of step (iii). - 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, 51, 52, 53)
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- 27. An affinity molecule-replicative RNA hybrid wherein the affinity molecule is joined to a replicative RNA through a first linking moiety, joined to the replicative RNA without eliminating the replicability of the replicative RNA by a RNA-dependent RNA polymerase, and a second linking moiety, joined to the affinity molecule without eliminating the specificity of binding between affinity molecule and its analyte, said first and second linking moieties being covalently joined to each other or being a specific binding pair.
- 38. A replicative RNA joined to a linking moiety, without eliminating the replicability in vitro of the replicative RNA by an RNA-dependent RNA polymerase, said linking moiety being capable of effecting a linkage between the replicative RNA and an affinity molecule through covalent linkage to, or being one member of a specific binding pair with, a linking moiety which is joined to the affinity molecule.
- 42. A smart probe comprising a replicative RNA covalently joined to a nucleic acid affinity molecule, wherein the affinity molecule is a nucleic acid which comprises a sequence which is capable of hybridizing with a complementary sequence of the replicative RNA and wherein the affinity molecule portion and the replicative RNA portion are associated so that the replicative RNA portion is inactive for replication by the RNA-dependent RNA polymerase unless the affinity molecule portion is associated with its analyte.
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46. An affinity molecule-replicative RNA hybrid wherein the replicative RNA is a template for replication in vitro by Qβ
- replicase and the affinity molecule is joined to the replicative RNA by hybridization of the replicative RNA to a first segment of nucleic acid at least 10 bases in length joined to or included in the affinity molecule, provided that, if the affinity molecule is a nucleic acid, the affinity molecule comprises a second segment which is not hybridized to the replicative RNA in the affinity molecule-replicative RNA hybrid.
- View Dependent Claims (47, 48, 49, 50)
Specification