Replicative RNA reporter systems - Patent Review 4957858

What is claimed is:

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, which is a template for replication in vitro by Q.beta. replicase, joining, either before or after step (i), a replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to the affinity molecule employed in step (i);

(iii) employing Q.beta. replicase to catalyze replication of replicative RNA, which (a) is a template for replication in vitro by Q.beta. replicase and (b) or had been joined to affinity molecule bound to analyte or is affinity molecule that had been bound to analyte; and

(iv) detecting RNA made by the reaction of step(iii).

2. A method according to claim 1 wherein the analyte is a nucleic acid, the affinity molecule is a replicative RNA which (a) is a template for replication in vitro by Q.beta. replicase and (b) includes a segment with a sequence of about 20 to about 4000 bases complementary to the sequence of a segment of the analyte, and the replication of said replicative RNA is after dissociation thereof from analyte.

3. A method according to claim 1 wherein the joining of replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to affinity molecule is effected through a first linking moiety, joined to said replicative RNA without eliminating the replicability thereof by Q.beta. replicase, and a second linking moiety, joined to the affinity molecule without eliminating the specificity of binding between affinity molecule and analyte, said first and second linking moieties either being covalently joined to each other or being a specific binding pair.

4. A method according to claim 1 wherein the joining of replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to affinity molecule is effected by hybridization of said replicative RNA to a segment of nucleic acid at least 10 bases in length joined to or included in affinity molecule.

5. A method according to claim 2 wherein the replication of replicative RNA is carried out with a radioactively labeled ribonucleoside-5'-triphosphate and the resulting replicated RNA is radioactively labeled.

6. A method according to claim 3 wherein the replication of replicative RNA is carried out with a radioactively labeled ribonucleoside-5'-triphosphate and the resulting replicated RNA is radioactively labeled.

7. A method according to claim 6 wherein, prior to exposing the affinity molecule to the sample, the first linking moiety and second linking moiety are covalently joined to each other by a disulphide moiety.

8. A method according to claim 7 wherein the first linking moiety is of formula --O--PO.sub.2 -NH-(CH.sub.2).sub.n --S--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide of the replicative RNA, which is a template for replication in vitro by Q.beta. replicase, and n is 2 to 8, and the second linking moiety is of formula --O--PO.sub.2 --NH--CH.sub.2).sub.m --S--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide of a nucleic acid affinity molecule and m is the same as or different from n and is 2 to 8.

9. A method according to claim 6 wherein the affinity molecule is a nucleic acid with a segment of at least 1 purine residue, which segment is at the 3'-terminus of the affinity molecule and outside the analyte-binding segment of the affinity molecule, said 3'-terminus of the affinity molecule bonded through a phosphodiester to the 5'-carbon of the 5'-nucleotide of the replicative RNA, which is a template for replication in vitro by Q.beta. replicase.

10. A method according to claim 7 wherein affinity molecule and replicative RNA, which is a template for replication in vitro by Q.beta. replicase, are combined in a smart probe.

11. A method according to claim 6 wherein the first and second linking moieties are a specific binding pair.

12. A method according to claim 11 wherein one of the first and second linking moieties is biotinyl and the other is avidin joined to affinity molecule or replicative RNA, which is a template for replication in vitro by Q.beta. replicase, through complexing to biotinyl.

13. A method according to claim 12 wherein a biotinyl moiety is linked to the 5'-nucleotide of the replicative RNA, which is a template for replication in vitro by Q.beta. replicase, by a spacer arm of formula --O--OP.sub.2 --NH--(CH.sub.2).sub.p (SS).sub.q (CH.sub.2).sub.r NH--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide, wherein p and r are the same or different and are each 2 to 8, and wherein q is 0 or 1.

14. A method according to claim 13 wherein the affinity molecule is a biotinylated antibody and avidin is complexed to the biotinyl joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase.

15. A method according to claim 13 wherein the affinity molecule is a biotinylated lectin and avidin is complexed to the biotinyl joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase.

16. A method according to claim 13 wherein the affinity molecule is a nucleic acid biotinylated photochemically with photobiotin, enzymatically with dUTP or UTP that is linked to biotinyl through C-5 of the uracil moiety or dATP that is linked to biotinyl through C-6 or C-8 of the adenine moiety, or chemically at the 5'-carbon of the 5'-nucleotide through a spacer arm of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.s (SS).sub.t (CH.sub.2).sub.u NH--, wherein the phosphocamidate moiety is bonded to the 5'-carbon of the 5'-nucleotide, wherein s and u are the same or different and are each 2 to 8, and wherein t is 0 to 1; and wherein avidin is complexed to biotinyl joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase.

17. A method according to claim 16 the affinity molecule is biotinylated at the 5'-carbon of the 5'-nucleotide.

18. A method according to claim 7 wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte, and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide covalently joining the first and second linking moieties.

19. A method according to claim 8 wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide covalently joining the first and second linking moieties.

20. A method according to claim 9 wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by acid depurination followed by .beta.-elimination to sever the phosphodiester bond.

21. A method according to claim 10 wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reducing of the disulfide covalently joining the first and second linking moieties.

22. A method according to claim 13 wherein q in the spacer group is 1; and wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide of the spacer arm joining said replicative RNA to biotinyl.

23. A method according to claim 14 wherein q in the spacer group is 1; and wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide of the spacer arm joining said replicative RNA to biotinyl.

24. A method according to claim 15 wherein q in the spacer group is 1; and wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide of the spacer arm joining said replicative RNA to biotinyl.

25. A method according to claim 17 wherein q in the spacer group is 1; and wherein, after binding of affinity molecule, joined to replicative RNA, which is a template for replication in vitro by Q.beta. replicase, to analyte and prior to replication of said replicative RNA, said replicative RNA is severed from affinity molecule by reduction of the disulfide of the spacer arm joining said replicative RNA to biotinyl.

26. A method according to claim 4 wherein the affinity molecule is a nucleic acid.

27. An affinity molecule-replicative RNA hybrid wherein the affinity molecule is joined to a replicative RNA, which is a template for replication in vitro by Q.beta. replicase, through a first linking moiety, joined to said replicative RNA without eliminating the replicability thereof by Q.beta. replicase, 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.

28. An affinity molecule-replicative RNA hybrid, in accordance with claim 27, wherein the second linking moiety and first linking moiety are covalently joined to the affinity molecule and the replicative RNA, respectively, and are covalently joined to each other by a disulfide moiety.

29. An affinity molecule-replicative RNA hybrid, in accordance with claim 28, wherein the affinity molecule is a nucleic acid, the first linking moiety is of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.n --S--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide of the replicative RNA and n is 2 to 8, and the second linking moiety is of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.m --S--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide of the affinity molecule, and m is the same as or different from n and is 2 to 8.

30. An affinity molecule-replicative RNA hybrid, in accordance with claim 27, wherein the affinity molecule is a nucleic acid with a segment of at least one purine residue which is at the 3'-terminus of the affinity molecule and outside the segment of affinity molecule with sequence complementary to that of target segment of analyte, said 3'-terminus of the affinity molecule bonded through a phosphodiester to the 5'-carbon of the 5'-nucleotide of the replicative RNA.

31. An affinity molecule-replicative RNA hybrid, in accordance with claim 27, wherein the first and second linking moieties are a specific binding pair.

32. An affinity molecule-replicative RNA hybrid, in accordance with claim 31, wherein one of the linking moieties is biotinyl and the other is avidin, joined to the affinity molecule or the replicative RNA through complexing to biotinyl.

33. An affinity molecule-replicative RNA hybrid, in accordance with claim 32, wherein a biotinyl moiety is linked to the 5'-nucleotide of the replicative RNA by a spacer arm of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.p (SS).sub.q (CH.sub.2 .sub.r NH--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide, wherein p and r are the same or different and are each 2 to 8, and wherein q is 0 or 1.

34. An affinity molecule-replicative RNA hybrid, in accordance with claim 33, wherein the affinity molecule is a biotinylated antibody.

35. An affinity molecule-replicative RNA hybrid, in accordance with claim 33, wherein the affinity molecule is a biotinylated lectin.

36. An affinity molecule-replicative RNA hybrid, in accordance with claim 33, wherein the affinity molecule is a nucleic acid biotinylated photochemically with photobiotin, enzymatically with dUTP or UTP, that is linked to biotinyl through C-5 of the uracil moiety, or dATP, that is linked to biotinyl through C-6 or C-8 of the adenine moiety, or chemically at the 5'-carbon of the 5'-nucleotide through a spacer arm of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.s (SS).sub.t (CH.sub.2).sub.u NH--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide, wherein s and u are the same or different and are each 2 to 8, and wherein t is 0 or 1.

37. An affinity molecule-replicative RNA hybrid, in accordance with claim 36, wherein the affinity molecule is biotinylated at the 5'-carbon of the 5'-nucleotide.

38. A replicative RNA, which is a template for replication in vitro by Q.beta. replicase, joined to a linking moiety, without eliminating the replicability in vitro of the replicative RNA by Q.beta. replicase, 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.

39. A replicative RNA in accordance with claim 38 wherein the linking moiety to which the replicative RNA is joined is sulfur, biotinyl, or avidin which is joined through a complex with biotinyl which, in turn, is joined to the replicative RNA.

40. A replicative RNA in accordance with claim 39 wherein the linking moiety is biotinyl or avidin, and, with either, biotinyl is linked to the 5'-nucleotide of the replicative RNA by a spacer group of formula --O--PO.sub.2 --NH--(CH.sub.2).sub.p (SS).sub.q (CH.sub.2).sub.r NH--, wherein the phosphoramidate moiety is bonded to the 5'-carbon of the 5'-nucleotide, wherein p and r are the same or different and are each 2 to 8, and wherein q is 0 or 1.

41. A replicative RNA in accordance with claim 39 wherein the linking moiety is sulfur and is joined to the 5'-nucleotide of replicative RNA by a spacer group of formula --O(PO.sub.2)NH(CH.sub.2).sub.p --, wherein the phosphoramidate group is joined to the 5'-carbon of the 5'-nucleotide and wherein p is 2 to 8.

42. A smart probe comprising a replicative RNA, which is a template for replication in vitro by Q.beta. replicase, covalently joined to a nuclei acid affinity molecule.

43. A smart probe according to claim 42 wherein the 5'-carbon of the 5'-nucleotide of the replicative RNA and the 5'-carbon of the 5'-nucleotide of the affinity molecule are joined by a moiety of formula --O(PO.sub.2)NH(CH.sub.2).sub.y (SS)(CH.sub.2).sub.z NH(PO.sub.2)O--, wherein y and z are the same or different and are each from 2 to 8.

44. A smart probe according to claim 43 wherein the affinity molecule has both a 5'-clamp segment and a 3'-clamp segment.

45. A smart probe according to claim 43 wherein the replicative RNA is a recombinant replicative RNA with a segment with a sequence complementary to that of a segment at the 3'-end of the affinity molecule.

Description Submit all comments and votes

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

(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 that 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).

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 being determined in an assay. An analyte is sometimes referred to as a target substance or a target segment of an assay. With assays according to the present invention, the analyte is usually a biopolymer or a segment of a biopolymer. Analytes include, for example, proteins, including glycoproteins and lipoproteins, enzymes, hormones, receptors, antigens, and antibodies; nucleic acids (DNAs and RNAs); segments of nucleic acids; and polysaccharides.

With assays of the present invention, an analyte is often associated with a biological entity which is present in a sample if and only if the analyte is present. Such biological entities include viroids (analyte is the nucleic acid or a segment thereof); viruses (analyte is, e.g., a viral coat protein, viral genome, or segment of viral genome, or antibody against the virus); other microorganisms (analyte is, e.g., a segment of the genome or the RNA of the microorganism, a toxin produced by the microorganism, or an heterologous protein made by the microorganism if it is genetically engineered) (with reference to protozoan parasites, see Lizardi and Noguiera, European Patent Application Publication No. 0 135 108); abnormal cells, such as cancer cells (analyte is, e.g., a cell surface antigen of the abnormal cell); or an abnormal gene (analyte is, e.g., a gene segment which includes the altered bases which render the gene abnormal, a messenger RNA segment which includes altered bases as a result of having been transcribed from the abnormal gene, or an abnormal protein product expressed from the abnormal gene). An analyte may also be a particular protein, such as, for example, a hormone, whose presence or concentration in serum or other body fluid is to be ascertained in an assay. In the case of immunoassays which entail the use of two antibodies, analyte may be antigen bound to first antibody (in the case of a sandwich assay) or first antibody bound to antigen (in the case of an immunosorbent assay). Many other types of analyte will be apparent to the skilled.

From the description of analyte, it is apparent that the present invention has widespread applicability, including in applications in which immunoassays or nucleic acid probe hybridization assays are employed. Thus, among other applications, the invention is useful in diagnosing diseases in plants and animals, including humans; and in testing products, such as food, blood, and tissue cultures, for contaminants.

An "affinity molecule" for an analyte is a molecule of an affinity substance (or, by a different name, a specific binding substance) for the analyte. Specific binding substances for particular analytes and methods of preparing them are well known in the art.

For an antigen analyte (which itself may be an antibody), antibodies, including monoclonal antibodies, are available as specific binding substances. For certain antibody analytes in samples which include only one antibody, an antibody binding protein such as Staphylococcus aureus Protein A can be employed as specific binding substance.

For an analyte which is a nucleic acid (DNA or RNA), or a segment thereof, oligonucleotides or polynucleotides (both also either DNA or RNA) which include a segment with a sequence complementary to that of a segment of the analyte are available as specific binding substances. Such affinity molecules can be made by any of numerous known in vivo or in vitro techniques, including automated synthesis techniques. As understood in the art, the length that a DNA or RNA affinity molecule must have to provide a pre-determined specificity in an assay will depend in part on the amount and complexity of nucleic acid in the sample being assayed. Such an affinity molecule will usually require at least 10 nucleotides.

For an analyte, such as a glycoprotein or class of glycoproteins, or a polysaccharide or class of polysaccharides, which is distinguished from other substances in a sample by having a carbohydrate moiety which is bound specifically by a lectin, a suitable specific binding substance is the lectin.

For an analyte which is a hormone, a receptor for the hormone can be employed as a specific binding substance. Conversely, for an analyte which is a receptor for a hormone, the hormone can be employed as specific binding substance.

For an analyte which is an enzyme, an inhibitor of the enzyme can be employed as a specific binding substance. For an analyte which is an inhibitor of an enzyme, the enzyme can be employed as a specific binding substance.

Usually, an analyte molecule and an affinity molecule for the analyte molecule are related as a specific binding pair, i.e., their interaction is only through non-covalent bonds (e.g., salt-bridges, hydrogen-bonding, hydrophobic interactions).

The skilled can easily determine conditions whereby, in a sample, binding occurs between affinity molecule and analyte that may be present. In particular, the skilled can easily determine conditions whereby binding between affinity molecule and analyte, that would be considered in the art to be "specific binding," can be made to occur. As understood in the art, such specificity is usually due to the higher affinity of affinity molecule for analyte than for other substances and components (e.g., vessel walls, solid supports) in a sample. In certain cases, the specificity might also involve, or might be due to, a significantly more rapid association of affinity molecule with analyte than with other substances and components in a sample.

A sample on which the assay method of the invention is carried out can be a raw specimen of biological material, such as serum or other body fluid, tissue culture medium or food material. More typically, the method is carried out on a sample which is a processed specimen, derived from a raw specimen by various treatments to remove materials that would interfere with detection of analyte, such as by causing non-specific binding of affinity molecules. Methods of processing raw samples to obtain a sample more suitable for the assay methods of the invention are well known in the art.

Thus, the method can be carried out on nucleic acid from cells following the colony hybridization method of Grunstein and Hogness, Proc. Natl. Acad. Sci. (U.S.A.) 72, 3961-3965 (1975) (see also, e.g., Falkow and Moseley, U.S. Pat. No. 4,358,535; and Shafritz, U.S. Pat. No. 4,562,159) or the plaque lift method of Benton and Davis, Science 196, 180-182 (1977). It can also be carried out on nucleic acids isolated from viroids, viruses or cells of a specimen and deposited onto solid supports (Gillespie and Spiegelman, J. Mol. Biol. 12, 829-842 (1965)); including solid supports on dipsticks and the inside walls of microtiter plate wells. The method can also be carried out with nucleic acid isolated from specimens and deposited on solid support by "dot" blotting (Kafatos et al., Nucl. Acids Res. 7, 1541-1552 (1979); White and Bancroft, J. Biol. Chem. 257, 8569-8572 (1982); Southern blotting (Southern, J. Mol. Biol. 98, 503-517 (1975); "northern" blotting (Thomas, Proc. Natl. Acad. Sci. (U.S.A.) 77, 5201-5205 (1980); and electroblotting (Stellwag and Dahlberg; Nucl. Acids Res. 8, 299-317 (1980)). Nucleic acid of specimens can also be assayed by the method of the present invention applied to water phase hybridization (Britten and Kohne, Science 161, 527-540 (1968)) and water/organic interphase hybridizations (Kohne et al., Biochemistry 16, 5329-5341 (1977). Water/organic interphase hybridizations have the advantage of proceeding with very rapid kinetics but are not suitable when an organic phase-soluble linking moiety, such as biotin, is joined to the nucleic acid affinity molecule.

The assay method of the invention can also be carried out on proteins or polysaccharides isolated from specimens and deposited onto solid supports by dot-blotting, by "Western" blotting (see, e.g., Example XII), or by adsorption onto walls of microtiter plate wells or solid support materials on dipsticks.

Still further, the method of the invention is applicable to detecting cellular proteins or polysaccharides on the surfaces of whole cells (see, e.g., Example XI) from a specimen or proteins or polysaccharides from microorganisms immobilized on a solid support, such as replica-plated bacteria or yeast.

Either before or after affinity molecule is bound to analyte that might be present in a sample being assayed, a replicative RNA must be joined to the affinity molecule.

A "replicative RNA" can be any RNA capable of being autocatalytically replicated in vitro, i.e., replicated in vitro in a reaction catalyzed by an RNA-dependent RNA polymerase. Suitable RNA polymerases and suitable replicative RNAs for practice of the instant invention are described in Example I below.

In this connection, it is to be understood that reference herein to bacteriophage Q.beta. is not limited to any particular variant or mutant or population thereof. Such reference, unless otherwise specifically limited, is to any variant, mutant or population which, upon infection therewith of E. coli susceptible to bacteriophage Q.beta. infection, is capable of causing production of an RNA-dependent RNA-polymerase.

For other phages which, upon infection of bacteria susceptible to infection therewith, produce RNA-dependent RNA polymerases, and associated replicative RNAs capable of being autocatalytically replicated in vitro, which can be employed in the present invention, see, e.g., Miyake et al., Proc. Natl. Acad. Sci. (U.S.A.) 68, 2022-2024 (1971).

Replicative RNA can be joined to affinity molecule in numerous different ways, some of which are described in the Examples.

If replicative RNA is joined to affinity molecule prior to binding affinity molecule to analyte, it is, as the skilled will understand, essential that the specificity of the affinity molecule for analyte not be eliminated, i.e., the affinity molecule, joined to replicative RNA, must retain capability to bind with some specificity to the analyte to be tested for in an assay.

If replicative RNA is joined to affinity molecule after affinity molecule binds to analyte, it is essential, as the skilled will also understand, that affinity molecule with replicative RNA joined be capable of being separated from replicative RNA that has not joined to affinity molecule. This is not a significant problem. In the usual case, with affinity molecule bound to analyte bound in turn to a solid support, such separation is readily accomplished by simple washing, because joining of replicative RNA to bound affinity molecule does not significantly disrupt the connection of affinity molecule to solid support. If the usual case does not obtain, such separation can be readily accomplished by any of several well known chromatographic and electrophoretic techniques.

Finally, the joining of replicative RNA to affinity molecule must be such that the replicability of the RNA by a RNA-dependent RNA polymerase is not eliminated, i.e., either the replicative RNA, as joined to affinity molecule, is a template for replication by an RNA-dependent RNA polymerase or is capable of being severed from the affinity molecule to a form which is a template for replication by an RNA-dependent RNA polymerase.

In one type of connection between replicative RNA and affinity molecule, the affinity molecule itself is a replicative RNA, namely a recombinant RNA which includes a segment with an appropriate sequence. Such an affinity molecule will be for an analyte which is a nucleic acid or segment thereof. The affinity molecule will be a recombinant RNA prepared from a replicative RNA, preferably by the procedure of Miele et al., supra, and Kramer et al., U.S. patent Application Ser. No. 614,350, supra, to include a segment with a sequence complementary to that of a segment of the analyte. This segment of complementary sequence will be at least 10 ribonucleotides in length, to provide specificity, and can be up to about 4500 nucleotides in length without eliminating replicability. Example IX illustrates use of a recombinant replicative RNA as an affinity molecule.

The connection between replicative RNA and affinity molecule can be non-covalent or covalent.

A non-covalent connection between affinity molecule and replicative RNA can be effected by joining to or including in affinity molecule a nucleic acid segment of sequence complementary to that of a segment of replicative RNA and hybridizing the replicative RNA to the segment joined to or included in affinity molecule. For joining such a nucleic acid segment to an affinity molecule which is a protein, see, e.g., Dattagupta et al., European Patent Application Publication No. 0 154 884. For an affinity molecule which is a nucleic acid, methods of joining or including such a segment are well known in the art. With such a connection between affinity molecule and replicative RNA, separation of replicative RNA from affinity molecule bound to analyte, to enable the replicative RNA to be replicated for detection, is accomplished by heating above the melting temperature of the complex between replicative RNA segment and nucleic acid segment joined to or included in affinity molecule.

A non-covalent connection between replicative RNA and affinity molecule can also be effected through binding of a first linking moiety, joined to replicative RNA, and a second linking moiety, joined to affinity molecule, said linking moieties being related as a specific binding pair.

A covalent connection between replicative RNA and affinity molecule is through bonds between the two which (but for bonds arising from secondary or tertiary structure in the complex) are only covalent. Usually a covalent connection involves a first linking moiety, which is covalently joined to replicative RNA; a second linking moiety, which is covalently joined to affinity molecule; and a covalent connection between the first and the second linking moiety.

Reference herein to "covalent joining" or "covalent linkage" or "covalent connection" of a linking moiety to a replicative RNA or an affinity molecule means that all bonds between the linking moiety and the replicative RNA or affinity molecule, respectively, other than bonds arising from secondary or tertiary structure, are covalent. Reference herein to "joining" or "linkage" or "connection" of a linking moiety to a replicative RNA or an affinity molecule, without qualification, means that the linking moiety is either "covalently" or "non-covalently" joined or linked to the replicative RNA or affinity molecule, respectively. "Non-covalent" joining or linkage or connection means that at least some bonds, other than bonds due to secondary or tertiary structure, between linking moiety and replicative RNA or affinity molecule are non-covalent.

Examples of covalent linkages between linking moiety and replicative RNA, whereby the replicability of the RNA is not eliminated, include, among others, the following:

(a) Linking moiety is a phosphate group and linkage is directly between the phosphate and the 5'-carbon of the 5'-nucleotide of replicative RNA. The phosphate linking moiety, bonded to the 5'-carbon of the 5'-nucleotide of replicative RNA, will usually be involved in covalently joining a replicative RNA directly to the 3'-carbon of the 3'-nucleotide of a nucleic acid affinity molecule or to the 3'-carbon of the 3'-nucleotide of a segment of nucleotides which is a linking moiety considered to be bonded to the 3'-end of a nucleic acid affinity molecule and which is covalently joined, through a phosphte at the 5'-carbon of its 5'-nucleotide, to the 3'-carbon of the 3'-nucleotide of the affinity molecule. The 5'-terminal nucleotide of a replicative RNA can be phosphorylated at the 5'-carbon with T4 polynucleotide kinase by methods known in the art. See also Example I. Affinity molecule, or nucleic acid linking moiety of affinity molecule, can then be connected to the 5'-phosphate of the 5'-nucleotide of replicative RNA by known methods employing T4 RNA ligase. This latter reaction