Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1988-04-16
1990-09-18
Yarbrough, Amelia Burgess
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 7, 435 91, 435188, 436501, 436547, 536 27, 530387, 935 78, C12Q 168
Patent
active
049578588
DESCRIPTION:
BRIEF SUMMARY
RNA can be joined to the affinity molecule before or after the affinity molecule has complexed with any of its biopolymer analyte present in an assay system.
Because, after replicative RNA is joined to an affinity molecule, replication of the RNA is required to render detectable affinity molecule that has bound to analyte, either (a) the RNA is joined to the affinity molecule in a manner that allows the RNA, while joined, to be replicated by a RNA-dependent RNA polymerase or (b) the RNA is joined to the affinity molecule in a manner that allows the RNA to be severed from the affinity molecule in a form whereby the severed RNA can be replicated by a RNA-dependent RNA polymerase.
The detection of the RNA replicated from the replicative RNA bound to affinity molecule, that in turn had bound to analyte in an assay system, is accomplished by any of numerous known techniques. For example, the replication of the RNA can be carried out with radioactively labeled ribonucleoside-5'-triphosphates, and detection is then of radioactive RNA resulting from the replication. Alternatively, a biotinylated ribonucleoside-5'-triphosphate can be employed as a substrate in the replication process and any resulting biotinylated RNA can be detected using an enzyme-avidin adduct as disclosed by, for example, Leary et al., supra. Also, replicated RNA can be detected directly by its ultraviolet absorbance or by staining.
DETAILED DESCRIPTION OF THE INVENTION
In one of its aspects, the present invention is a method of determining the presence of an analyte in a sample, which method comprises conditions whereby binding occurs between the affinity molecule and the analyte; either before or after step (i), a replicative RNA to the affinity molecule employed in step (i); replicative RNA that is or had been joined to affinity molecule that bound to analyte or that is affinity molecule that had been bound to analyte; and
In another of its aspects, the invention entails an affinity molecule-replicative RNA hybrid molecule, i.e., and affinity molecule joined to a replicative RNA. The affinity molecule can be joined to the replicative RNA through a first linking moiety, covalently 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, being a specific binding pair, or forming simultaneously, with a common third linking moiety, specific binding pairs.
The invention also entails "smart probes," which are compounds in which an affinity molecule, which is a nucleic acid, is covalently joined to a replicative RNA and in which the affinity molecule portion is associated with the replicative RNA portion so that the replicative RNA is inactive as a template for replication by an RNA-dependent RNA polymerase unless the affinity molecule is associated with analyte of affinity molecule.
The invention entails further an affinity molecule non-covalently joined to a replicative RNA through base-pairing.
In yet a further aspect, the invention involves a replicative RNA joined to a linking moiety, without eliminating the replicability of the replicative RNA by an RNA-dependent RNA polymerase, said linking moiety being one of a pair of linking moieties whereby linkage between the replicative RNA and an affinity molecule, joined to the other linking moiety of the pair, can be effected by covalent joining of the linking moieties or by interaction of the linking moieties as a specific binding pair.
Such a replicative RNA joined, in accordance with the invention, to one of a pair of linking moieties, is a universal reporter group for any affinity molecule joined, in a manner that does not eliminate the specificity of its binding to its analyte, to the other linking moiety of the pair.
"Analyte" means a substance whose presence, concentration or amount in a sample is
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Chu Barbara
Kramer Fred R.
Lizardi Paul
Orgel Leslie E.
Burgess Yarbrough Amelia
The Salk Instute for Biological Studies
The Trustees of Columbia University
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