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WO1991018117A1 - Sondes d'arn recombine hybridable replicable et test d'hybridation - Google Patents

Sondes d'arn recombine hybridable replicable et test d'hybridation Download PDF

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Publication number
WO1991018117A1
WO1991018117A1 PCT/US1991/003634 US9103634W WO9118117A1 WO 1991018117 A1 WO1991018117 A1 WO 1991018117A1 US 9103634 W US9103634 W US 9103634W WO 9118117 A1 WO9118117 A1 WO 9118117A1
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rna
recombinant
oligo
interest
polynucleotide
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PCT/US1991/003634
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English (en)
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Fred R. Kramer
Paul M. Lizardi
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The Trustees Of Columbia University In The City Of New York
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Publication of WO1991018117A1 publication Critical patent/WO1991018117A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • RNA synthesis by QB replicase is that a small number of template strands can initiate the synthesis of a large number of product strands (Haruna and Spiegleman, 1965b). Million-fold increases in the amount of RNA routinely occur in vitro (Kramer, et al. 1974) as a result of an autocatalytic reaction mechanism (Weissman, et al. 1986; Spiegelman, et al. 1968): single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products; after the completion of product strand elongation, both the product and the template are released from the replication complex (Dobkin, et al.
  • Qß replicase was first isolated from bacteriophage Qß- infected Escherichia coli by Haruna and Spiegelman (1965a). It is composed of four polypeptides, only one of which is specified by the viral RNA. The other three polypeptides are E. coli proteins, and have been identified as the protein synthesis elongation factors Tu and Ts and the ribosomal protein SI. When provided with the single- stranded RNA from Qß, the replicase mediates the exponential synthesis of infectious viral RNA (Spiegelman et al., 1965). The enzyme is highly template selective. No other viral RNA, nor any E. coli RNA, will serve as a template (Haruna and Spiegelman, 1965c).
  • RNA from a temperaturesensitive mutant of Qß was used as a template with wild-type replicase, mutant RNA was synthesized, demonstrating that the template is the instructive agent (Pace and Spiegelman, 1966).
  • the replicative process (Spiegelman et al., 1969; Weissmann et al., 1968) proceeds in the following manner:
  • the replicase uses the viral (+) strand as a template to direct the synthesis of a complementary (-) strand. Both of these strands serve as templates for the synthesis of additional (+) and (-) strands; and exponential increase is observed in the number of RNA strands present.
  • there are enough strands to saturate the available enzyme molecules, after which the number of strands increases linearly with time.
  • Qß replicase Because of the complementary nature of this process, it is often referred to as "self-replication". There are a number of advantages to using the amplification of RNA by Qß replicase as the basis of a signal-generating system: Qß replicase is highly specific for its own template RNAs (Haruna and Spiegelman, 1965c); as little as one molecule of template RNA can, in principle, initiate replication (Levisohn and Spiegelman, 1968); and the amount of RNA synthesized (typically, 200 ng in 50 ⁇ l in 15 minutes) is so large that it can be measured with the aid of simple colorimetic techniques. There are a number of naturally occurring Qß replicase templates that are much smaller than Qß RNA.
  • RNAs have been isolated from in vitro Qß replicase reactions that were incubated in the absence of exogenous template RNA. They include: MDV-1 RNA (Kacian et al., 1972), microvariant RNA (Mills et al., 1975), the nanovariant RNAs (Schaffner et al., 1977), RQ120 RNA (Munishkin et al., 1989), and cordycepin-tolerant RNA (Priano et al., 1989). Although the origin and biological role of these RNAs is not known, they have been extensively characterized and are all excellent templates for Qß replicase.
  • MDV-1 RNA serves as an excellent exogenous template. It is bound by Qß replicase and replicated in a manner similar to Qß RNA (Kacian et al., 1972). MDV-1 RNA is much smaller (221 nucleotides) than Qß RNA (4,220 nucleotides), which led to the determination of its complete nucleotide sequence (Mills et al., 1973; Kramer and Mills, 1978). Two striking aspects of the MDV-1 sequence are its unusually high proportion of guanosine and cytidine residues and the occurrence of many intrastrand complements capable of forming hairpin structures.
  • MDV-1 has been directly visualized (Klotz et al., 1980), utilizing hollow-cone, dark-field electron microscopy. Observations made with native, partially denatured, and fully denatured molecules indicate that native single-stranded MDV-1 RNA is a highly condensed molecule, possessing substantial tertiary structure. Specific secondary structures were identified by reacting MDV-1 RNA with chemical agents that modify single- stranded regions (Mills et al., 1980). The location of the altered nucleotides was determined by sequencing the modified RNA.
  • MDV-1 RNA The tertiary structure of MDV-1 RNA was probed by subjecting it to mild cleavage with ribonuclease T 1 (Kramer et al., 1989), which only cleaves single-stranded regions. Because of the extensive secondary and tertiary structure present in MDV-1 RNA, combined with the macromolecular dimensions of ribonuclease T 1 , the initial sites of attack were limited to those on the exterior of the molecule. The few guanosines in each strand that were hypersusceptible to ribonuclease T 1 were located in hairpin loops.
  • MDV-1 RNA synthesis was facilitated by the development of an electrophoretic technique for separating the complementary strands (Mills et al., 1978). An excess of pure MDV-1 (-) RNA was used as template in the presence of a small amount of Qß replicase, in a series of experiments designed to elucidate the synthetic cycle. Mutant MDV-1 (-) RNA (Kramer et al., 1974) was added to these reactions after the initiation of chain elongation to see whether replicase molecules retain the same template through many rounds of synthesis. It was shown that a single replicase molecule bound to a single template strand is sufficient to carry out a complete synthetic cycle.
  • a particular sequence at 3' end of the template strand is required for the initiation of product strand synthesis.
  • MDV-1 RNA fragments lacking nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments lacking nucleotides at their 3' end were unable to serve as templates, even though they were able to form complexes with the replicase (Nishihara et al., 1983).
  • the sequence required for initiation to occur includes at least some of the 3' terminal cytidines, since the conversion of these cytidines to uridines by sodium bisulfite treatment was accompanied by a concomitant loss in the ability of the RNA to initiate synthesis (Mills et al., 1980).
  • MDV-1 (+) RNA was completely modified by bisulfite treatment, except for the 3'-terminal cytidines, which were protected by a complementary oligonucleotide mask.
  • the resulting RNA was able to initiate synthesis.
  • the 5' end of the template need not be present for the synthesis of multiple copies of complementary RNA (Bausch et al., 1983). However, it must be present if exponential synthesis is to occur, because the 5' terminal sequence serves as the template for the required 3' initiation sequence in the product strand.
  • An exhaustive comparison of the nucleotide sequence of MDV-1 RNA and Qß RNA found only two significant homologies. These regions encompass the internal replicase binding site required for template recognition and the cytidine-rich 3'-terminal sequence required for product strand initiation.
  • the exponential synthesis of heterologous RNAs was achieved by constructing recombinant RNAs by the insertion of heterologous sequences at an appropriate site within MDV-1 RNA (Miele et al., 1983).
  • the insertion site chosen (between nucleotides 63 and 64 of MDV-1 (+) RNA) was located away from the regions that are required for template recognition and product strand initiation.
  • the site was in hairpin loop, where it was least likely to disturb structure; and it was in a loop where viable mutations were known to occur, indicating that the sequence in that region was not essential for replication.
  • this site was located on the exterior of the molecule in a hairpin loop that was hypersusceptible to cleavage by ribonuclease T 1 .
  • the first recombinant RNA constructed was prepared by cleaving MDV-1 (+) RNA at the selected site with ribonuclease T 1 and then inserting decaadenylic acid there by direct ligation with the aid of bacteriophage T4 RNA ligase.
  • the resulting 231-nucleotide recombinant was an excellent template for Qß replicase, demonstrating that the inserted sequence did not interfere with replication.
  • the products consisted of full-length copies of the recombinant RNA, and both complementary strands were synthesized.
  • An analysis of the kinetics of recombinant RNA synthesis demonstrated that the amount of recombinant RNA increased exponentially at a rate that was indistinguishable from that of MDV-1 RNA.
  • a plasmid that contains a strong bacteriophage T7 promoter (Lizardi et al., Mills et al., 1990) directed towards the MDV-1 cDNA sequence is a good template for MDV-1 RNA synthesis (Axelrod and Kramer, 1985). Bacteriophage RNA polymerases are highly specific for their own promoters (Chamberlin and Ring, 1973). Incubation with a bacteriophage RNA polymerase results in the exclusive transcription of the nucleotide sequence immediately downstream from its homologous promoter (McAllister et al., 1981; Melton et al., 1984). Moreover, the transcripts are virtually homogenous; and as many as 300 transcripts can be synthesized from each promoter in a 30 minute incubation.
  • RNA segments can be inserted within the sequence of a small, naturally occurring template for Qß replicase, MDV-1 RNA (Kacian, et al. 1972), without affecting its replicability Miele, 1983 (Miele, et al. 1983); and the construction of a plasmid that serves as a template for the synthesis of MDV-1 (+) RNA when the plasmid is incubated in vitro with bacteriophage T7 RNA polymerase (Mills, et al. 1990).
  • This plasmid has been modified in the present invention by inserting a polylinker within the MDV-1 cDNA sequence, and then inserting synthetic hybridization probe sequences within the polylinker.
  • the resulting plasmids served as templates for the synthesis of "recombinant RNAs," which consist of a probe sequence embedded within the sequence of MDV-1 (+) RNA.
  • the site for inserting the polylinker and probe into MDV-1 RNA is on the exterior of the molecule to reduce any possible interference with replication and hybridization of the probe sequence to its target.
  • the probe sequences employed in this invention and which will be further described in the Experimental Details section to follow, are known to hybridize specifically to the repetitive DNA of Plasmodium falciparum (Franzen, et al.
  • RNA molecules are bifunctional, in that they are able to hybridize specifically to complementary DNA targets and they are also able to serve as templates for exponential amplification by Qß replicase.
  • HIV-1 human immunodeficiency virus type 1
  • Suitable assays would make use of a macromolecular probe having extremely high affinity for a particular component of the infections agent and very low affinity for all the other components of the sample.
  • oligonucleotide probes can seek out and bind to the integrated HIV-1 DNA, or the retroviral messenger RNA, present in a single infected cell.
  • oligonucleotide probes are not sufficient to ensure detection.
  • An infected cell contains only about 6000 molecules of retroviral messenger RNA (Pelligrino, et al. 1987), so the problem becomes how to detect the probes once they are bound to such a small number of targets.
  • the classic detection strategy is to attach reporter groups to the probes, such as fluorescent organic molecules or radioactive phosphate groups. More recently, biotin groups have been incorporated into probes (Langer, et al. 1981).
  • enzymes such as peroxidase or phosphatase are linked to the biotin, then incubated with a colorless substrate, leading to the accumulation of a large number of colored product molecules for each enzyme-probe adduct (Leary, et al. 1983).
  • the practical limit of detection of these schemes is about 10 6 target molecules.
  • they cannot be used to detect a single cell in a sample that contains only 6000 retroviral messenger RNAs.
  • a particularly attractive strategy for detecting rare targets is to link each probe to a replicatable reporter, which can be exponentially amplified after hybridization to reveal the presence of the probe (Chu, et al. 1986).
  • the present invention concerns a novel version of this approach, in which a probe sequence is embedded within the sequence of a replicatable RNA (Lizardi, et al. 1988).
  • the resulting recombinant RNAs hybridize to their target sequences as ordinary hybridization probes do and, as in a classical hybridization assay, nonhybridized probes are then washed away.
  • the hybridized probes are then freed from their targets and released into solution.
  • the present invention provides an assay designed to detect very small amounts of HIV-1 mRNA.
  • the assay format was designed to meet these criteria: (a) because of the desirability of developing a method that can screen a large number of samples, the selected format has to be fast and simple, thus precluding the fractionation of cells or the isolation of nucleic acids, and necessitating the use of solution hybridization; and (b) because nonhybridized probes are amplified by Qß replicase along with hybridized probes, the format must include an extremely efficient means of removing the nonhybridized probes.
  • Hybridization is extremely efficient in solutions of the chaotropic salt, guanidine thiocyanate (Thompson and Gillespie, 1987), and concentrated solutions of guanidine thiocyanate will lyse cells, denature all proteins (including nucleases), liberate nucleic acids from cellular matrices, and unwind DNA molecules, permitting hybridization to occur without interference from cellular debris (Pelligrino, et al. 1987).
  • the "reversible target capture” procedure (Morrisey, et al. 1989) is an efficient means for removing nonhybridization probes. In this improved “sandwich hybridization” technique (Ranki, et al. 1983; Syvanen, et al.
  • probe-target hybrids are bound to the surface of paramagnetic particles. After the particles are washed to remove nonhybridized probes, the hybrids are released from the particles, and then bound to a new set of particles for another washing. Repeating this procedure several times dramatically reduces the concentration of nonhybridized probes (Morrissey, et al. 1989).
  • This invention also concerns the development of extremely sensitive assays for the detection of blood cells infected with pathogenic retroviruses.
  • the assays utilize novel recombinant RNAs that serve as specific hybridization probes and also serve as templates for their own exponential amplification by Qß replicase. Since more than one billion copies of each hybridized recombinant-RNA probe can be synthesized in a short incubation, extreme sensitivities can be achieved.
  • These assays can be used to routinely screen donated blood and to identify asymptomatic individuals who are carriers, to prevent the spread of retroviral diseases.
  • oligonucleotide probes bind to targets and then serve as primers for DNA polymerase. Since as many as a million copies of each target region can be generated, great sensitivity can be achieved. Moreover, probes that are not bound to their targets cannot be elongated, so their presence does not generate a background signal.
  • DNA polymerase is inhibited by many components of the sample.
  • hemoglobin interferes with amplification; consequently, peripheral blood mononuclear cells must be separated from other cells prior to amplification.
  • cellular DNA is isolated prior to analysis.
  • DNA polymerase cannot copy RNA, necessitating an additional reverse transcription step.
  • each cycle of amplification involves incubation at two different temperatures, necessitating the use of a relatively expensive "temperature cycler"; and the 20 or more cycles needed for each assay consume time.
  • the amount of DNA that can be synthesized with a given pair of primers is apparently limited by unidentified factors in the reaction to approximately one million copies of each target.
  • RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis by the polymerase and a heterologous sequence of interest derived from a different RNA molecule inserted at a specific site in the internal region of the recombinant molecule.
  • Kramer, et al. does not teach or suggest that if the inserted sequence is a hybridization probe sequence, that the resulting molecules can be replicated after hybridization to produce multiple copies for detection.
  • RNA-directed RNA polymerase can then be used after hybridization has occurred to produce multiple copies of the replicatable RNA for detection.
  • Chu, et al. do not describe a method in which different recombinant-RNA "probe" sequences can be used simultaneously in the same assay.
  • Qß replicase is highly specific for its own template RNA (Haruna and Spiegelman, 1965c), and will not copy any other RNA in the sample. Amplification can be initiated with as little as one molecule of RNA (Levisohn and Spiegelman, 1968). Incubations are carried out at 37°C and take less than 30 minutes; and the large amount of RNA that is synthesized (typically, 200 nanograms in a 50 microliter reaction) enables its detection by simple colorimetric methods.
  • RNA reporters of the subject invention Quantitation of the number of targets originally present in a sample can occur over a range of target concentrations that exceeds 1,000, 000-fold (Lizardi et al., 1988).
  • the materials required for these assays are inexpensive, and the simplicity of the procedure lends itself to automation. Protocols that permit the simultaneous detection of cells infected with different pathogenic retroviruses in the same sample are effective with the recombinant RNA reporters of the subject invention.
  • the present invention concerns a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising: (a) a recognition sequence for the binding of an RNA-directed RNA polymerase; (b) a sequence required for the initiation of product strand synthesis by the polymerase; and (c) a heterologous RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo- or polynucleotide of interest.
  • the invention also provides a method for determining the presence or concentration of an oligo- or polynucleotide of interest in a sample, comprising the steps of: (a) forming a specific complex between the recombinant-RNA probe molecule described above and the oligo- or polynucleotide of interest, by incubating the sample with the recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest; and (d) detecting the recombinant-RNA probe molecules synthesized in step (c), thereby determining the presence or concentration of the oligo- or polynucleotide
  • the invention herein further provides a method for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample, comprising the steps of: (a) forming specific complexes between a mixture of different types of recombinant-RNA probe molecules described above, each type having a different inserted sequence, and the oligo- or polynucleotides of interest, by incubating the sample with the mixture of recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the recombinant-RNA probe molecules which are hybridized to the oligo- or polynucleotides of interest; (d) separating the mixture of synthesized recombinant-RNAs by hybridizing them to
  • FIG. 1 Structure of plasmid pT7-MDV-poly.
  • the heavy black line represents MDV-1 cDNA.
  • MDV-fal-un (+) RNA contains a 58-nucleotide insert (shown between the arrows) in place of the 3-nucleotide segment, AGU, that occurs in natural MDV-1 (+) RNA (Mills, et al. 1973).
  • the secondary structures that the computer program predicts in the region of the recombinant outside the insert are identical to the secondary structures that were experimentally identified in MDV-1 RNA (Mills, et al.
  • the numbers on the side of the panel indicate the length of each transcript (in nucleotides).
  • Figure 4 Effect of initial RNA concentration on the time-course of recombinant RNA synthesis.
  • a series of 25 ⁇ l Qß replicase reactions were initiated with the following quantities of MDV-fal-un RNA: 140 pg, 1.4 pg, 14 fg, 0.14 fg, and 0 fg, which corresponds to 10 9 , 10 7 10 5 , 10 3 , and 0 molecules of added template.
  • Samples of each reaction were taken every 5 minutes to determine the amount of RNA that had been synthesized. The results demonstrate that the time spent in the exponential phase of synthesis is increased when the initial number of template molecules is decreased (Kramer, et al. 1974).
  • Figure 7 Replicatable HIV-1 hybridization probe.
  • the nucleotide sequence of MDV-hiv (+) RNA was folded into the secondary structures predicted to be most stable by a computer program.
  • the probe sequence (bold letters) is located on the exterior of the molecule, where it is free to hybridize to its target, and where it is less likely to interfere with the sequences and structures required for replication.
  • FIG. 8 Schematic representation of the complex formed when a capture probe links a probe-target hybrid to an oligo(dT) group on the surface of a paramagnetic particle.
  • the replicatable probe is a single-stranded RNA containing a probe sequence (thick line) embedded within the sequence of a replicatable RNA (indicated by the hairpin structures).
  • the replicatable probe is joined by hydrogen bonds (crosshatching) to its complementary target sequence within the target molecule.
  • the target molecule can be an RNA or a DNA.
  • the capture probe is a single-stranded DNA that contains a 5'-probe sequence that is hybridized to a different complementary target sequence within the target molecule.
  • the target sequence for the capture probe is located relatively close to the target sequence for the replicatable probe on the target molecule.
  • the capture probe also contains a 3'-poly(dA) tail that is hybridized to an oligo(dT) group on the surface of the paramagnetic particle.
  • the hydrogen bonds that join the 3'-poly(dA) tail of the capture probe to the oligo(dT) group are relatively weak, permitting the much more stable probe-target hybrid to be released from the particle at higher guanidine thiocyanate concentrations.
  • Figure 9 Kinetic analysis of amplification reactions initiated with replicatable probes isolated from hybridization reactions. Approximately 50 ng of MDV-hiv RNA was synthesized in each amplification reaction. This amount of RNA is sufficient to have been accurately measured by the fluorescence of an intercalating dye, such as ethidium bromide.
  • an intercalating dye such as ethidium bromide.
  • This invention concerns a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising (a) a recognition sequence for the binding of an RNA- directed RNA polymerase; (b) a sequence required for the initiation of product strand synthesis by the polymerase; and (c) a heterologous RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo- or polynucleotide of interest.
  • the recognition sequence for the binding of an RNA-directed RNA polymerase is in an internal region of the recombinant-RNA probe molecule.
  • the insertion site for the heterologous RNA sequence is not near any sequence required for the binding of the RNA polymerase or for the initiation of product strand synthesis.
  • such an insert has a minimal affect on the replicability of the molecule.
  • the insert in the recombinant-RNA probe molecule has a minimal effect upon the secondary and tertiary structure of the molecule.
  • the specific insertion site of the recombinant- RNA probe molecule is at a specific nucleotide.
  • sequence in the recombinant RNA probe molecules of the present invention which is required for the initiation of product strand synthesis is a cytidine-rich 3'-terminal sequence.
  • RNA-directed RNA polymerase useful in the practice of this invention is Qß replicase.
  • RNA variant templates for Qß replicase are a microvariant RNA, a nanovariant RNA, cordycepin-tolerant RNA, or RQ120 RNA or a mutant thereof.
  • a variant RNA template is MDV-1 RNA or a mutant thereof.
  • the MDV-1 RNA is MDV-1 (+) RNA.
  • the MDV-1 RNA is MDV-1 (-) RNA.
  • One feature of the invention herein is to provide a recombinant-RNA probe molecule wherein the heterologous sequence is inserted between nucleotides 63 and 64.
  • Transcripts derived from a recombinant plasmid by incubation with a DNA-directed RNA polymerase are especially useful in the practice of the invention to provide recombinant-RNA probe molecules.
  • the recombinant-RNA probe molecule is a variant RNA template for Qß replicase or a mutant thereof.
  • the variant RNA template is MDV-1 RNA or a mutant thereof, in particular, MDV-1 RNA wherein the heterologous sequence is inserted between nucleotides 63 and 64.
  • the MDV-1 RNA is MDV-1 (+) RNA or MDV-1 (-) RNA.
  • an infectious agent may be a virus, a viroid or virusoid, a prokaryote such as a bacterium, a eukaryote such as a parasitic protozoan, for example a parasitic protozoan that causes malaria.
  • viruses are HIV-1, HIV-2, HTLV-1, and HTLV-2, and an example of a specific nucleic acid sequence within any of these viruses is a highly conserved region in the viral pol gene.
  • the inserted heterologous sequence of the recombinant-RNA probe molecule may be complementary to a specific gene sequence or portion thereof, or to an allele of the specific gene sequence or portion thereof.
  • the invention herein provides a method for determining the presence or concentration of an oligo- or polynucleotide of interest in a sample, comprising the steps of: (a) forming a specific complex between the recombinant-RNA probe molecules, as described above, and the oligo- or polynucleotide of interest, by incubating the sample with the recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest; and (d) detecting the recombinant-RNA probe molecules synthesized in step (c), thereby determining the presence or concentration of the oligo- or polynucle
  • Such a method as described above may be used to generate highly amplified signals in a nucleic acid hybridization assay.
  • the method has the power to generate a signal from a single hybridized molecule, and therefore, could be utilized to devise ultra-high sensitivity DNA (or RNA) detection assays.
  • the method is based on the use of recombinant-RNA templates for Qß replicase and Qß replicase.
  • the oligo- or polynucleotide in the sample may be bound to a solid support.
  • the solid support may be a nitrocellulose or nylon membrane.
  • the oligo- or polynucleotide of interest and the recombinant-RNA probe molecule may be in solution.
  • the unhybridized recombinant RNA molecules may be separated from those that are hybridized to the oligo- or polynucleotides of interest by employing techniques and skills which are well-known in the art.
  • the recombinant RNA molecule hybridized to the oligo-or polynucleotide of interest which in turn is bound to a solid support
  • such separation is readily accomplished by simple washing which does not significantly disrupt the connection to the solid support.
  • a technique known as the sandwich hybridization method may also be used to effect separation of hybridized from unhybridized recombinant-RNA molecules. Chromatographic and electrophoretic techniques may be uses as well.
  • the unhybridized recombinant-RNA probe molecules are removed from the reaction mixture in step (b) by separating hybridized recombinant-RNA probe molecules from the unhybridized probe molecules through the capture of the oligo- or polynucleotide onto a solid support.
  • the hybridized recombinant-RNA probe molecules are separated from the unhybridized probe molecules by (a) capturing the oligo- or polynucleotide of interest onto a solid support comprising paramagnetic particles linked to oligo(dT) groups which are bound to the 3' poly(dA) tails of capture probes, the capture probes also comprising a sequence complementary to a sequence of the oligo- or polynucleotide of interest located close to the sequence of the oligo- or polynucleotide of interest that is specifically complexed to the recombinant-RNA probe molecule by hybridizing the oligo- or polynucleotide of interest to the complementary sequence of the capture probes, (b) placing the resulting reaction mixture in a magnetic field that draws the paramagnetic particles bound to the capture probe bound to the oligo- or polynucleotide of interest specifically complexed to the recombinant-RNA probe molecule to
  • the magnetic beads may be minute iron oxide particles with extensively convoluted surfaces that are siliconized and contain a large number of covalently linked oligo(dT) 14 molecules. These beads are sold by Gene-Trak Systems of Framingham, Massachusetts. Probe-target hybrids are bound to these beads by means of capture probes, which are synthetic oligodeoxyribonucleotides, approximately 40 nucleotides in length, to which oligo (dA) 100-150 tails have been added by incubation with terminal transferase.
  • the oligo(dA) tails enable capture probes to hybridize to the oligo(dT) on the surface of the beads, and the specific sequence at their other end enables them to hybridize to target strands at a location close to where the reporter probe binds.
  • the magnetic beads with the probe-target hybrids bound to their surface, are drawn to the walls of the reaction tube by placing the tube in the strong magnetic field of a magnetic separation device (Gene- Trak Systems).
  • the hybridization solution is withdrawn by aspiration, and replaced by 250 ⁇ L of a fresh 2.5 M GTC solution.
  • the beads are then released from the walls of the tube by lifting the tube out of the magnetic field.
  • the beads are washed by vortexing and pulled back to the walls of the tube.
  • the solution is again removed by aspiration, but this time it will be replaced with a 3.25 M GTC solution.
  • the beads are again released and be incubated for 5 minutes at 37°C.
  • the relatively weak hybrids formed by the oligo(dA) tails of the capture probes and the oligo(dT) 14 on the surface of the beads come apart, releasing the much stronger target-probe hybrids back into solution.
  • the stripped beads are drawn to the sides of the tube, and the released target-probe hybrids are transferred to a new tube containing new beads.
  • the GTC concentration is then adjusted downwards to 2.5 M, and the mixture incubated for 5 minutes at 37oC to alow the target-probe hybrids to be recaptured onto the surface of the new beads.
  • detecting the recombinant-RNA probe molecules which have been synthesized or replicated in step (c) above methods well-known to those in this art may be employed.
  • detection can be by ultraviolet absorbance of replicated RNA, as, for example, by the method of contact photoprinting (Kutateladze, et al. 1979).
  • detecting is carried out by the incorporation of radioactively labelled ribonucleoside 5'- triphosphate precursors into the recombinant-RNA products. In another embodiment, detection is carried out by the incorporation of chemically modified ribonucleoside 5'- triphosphate precursors into the recombinant-RNA products.
  • Biotin or iminobiotin can be incorporated into replicated RNA, which can then be detected by known techniques with an enzyme-avidin or enzyme-streptavidin adduct, which binds to the RNA-bound biotin and catalyzes production of a conveniently detectable chromogen. See Matthews (Matthews, et al. 1985); Leary et al. (Leary, et al. 1983). Incorporation of biotin or iminobiotin into replicated RNA can be accomplished by employing UTP that is biotinylated through a spacer to carbon-5 of the uracil moiety as a substrate for the replicate in the replication reaction. Such UTP's are known compounds.
  • RNAs which include uracils biotinylated through spacer groups joined to the carbon-5 position are templates for Qß replicase catalyzed replication.
  • RNA resulting from the replication process could also be biotinylated employing photobiotin acetate according to the procedure of Forster, et al., (Forster, et al. 1985), and then detected, with an avidin-enzyme adduct-chromogenic compound system, like replicated RNA's synthesized with biotinylated UTP in the replication reaction.
  • the chemically modified ribonucleoside 5'-triphosphate precursors may be biotinylated or the chemically modified ribonucleotide 5'-triphosphate precursors may be fluorescent.
  • detecting is carried out by the binding of RNA-specific chromogenic or fluorogenic dyes to the recombinant-RNA products.
  • RNA resulting from the replication process can be made fluorescent by employing a T4 RNA ligase-catalyzed reaction to append nucleotides modified to be fluorescent to the 3'-end of replicative RNA. See Cosstick, et al., 1984.
  • the fluorescence of the resulting RNA can be employed to detect the RNA by any of the several standard techniques.
  • a reporter substance that binds specifically with nucleic acid
  • Such substances include: chromogenic dyes, such as "stains all" (Dahlberg, et al. 1969); methylene blue (Dingman and Peacock, 1968), and silver stain (Sammons, et al.
  • RNA for example, ethidium bromide (Sharp, et al., 1973; Bailey and Davidson, 1976); and fluorogenic compounds that bind specifically to RNAs that are templates for replication by Qß replicase — for example, a phycobiliprotein (Oi, et al. 1982; Stryer, et al.; U.S. Patent No. 4,520,110) conjugated to the viral subunit of Qß replicase.
  • Detecting may be also carried out by physical methods, such as the absorption of ultraviolet light and the determination of mass by weighing.
  • RNA-directed RNA polymerase In incubating the reaction mixture obtained in step (b), an RNA-directed RNA polymerase is employed.
  • An example of such a polymerase useful in the practice of this invention is Qß replicase.
  • a useful aspect of this invention is provided when the method described above is used to test a sample such as a tissue specimen, for example a blood specimen.
  • a rapid and efficient assay is attained when the conditions for the hybridization step of the method comprise exposing the sample to guanidium thiocyanate. Guanidium thiocyanate prepares DNA from cells in the sample for hybridization in a single step, by simultaneously lysing the cells, denaturing nucleases, and unwinding the DNA from the cellular matrix.
  • This invention also provides recombinant-RNA probe molecules produced by the above-described method and, in particular, recombinant-RNA probe molecules produced by a method where the reaction mixture is incubated with Qß replicase to synthesize additional copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest.
  • Another aspect of the method provided by this invention is attained when the time of incubation in step (c) is sufficiently short so that the number of recombinant-RNA product strands does not exceed the number of polymerase molecules, with the result that the number of recombinant- RNA product molecules is proportional to the logarithm of the number of recombinant-RNA probe molecules originally hybridized.
  • the subject invention provides a method whereby the concentration of the oligo- or polynucleotide of interest detected in step (d) is measured by determining the intensity of a chromogenic or fluorescent signal in a reaction mixture in logarithmic phase at multiple time- points as the reaction proceeds and determining the concentration of the labelled recombinant-RNA products thereby, preparing a standard curve relating the concentration of the oligo- or polynucleotide of interest to the length of time of the reaction using a standard equation, and using the standard curve to determine the concentration of the oligo- or polynucleotide of interest at a known time point in the reaction.
  • This method can be automated.
  • RNA population is a constant for a given set of reaction conditions (Kramer, et al. 1974). If we know how many replicatable probes were initially present in a reaction, and if we know how long that reaction was incubated, then we can predict how many doublings have occurred and how many RNA molecules have been synthesized. Conversely, if we know how long it takes for a particular number of RNA molecules to be synthesized, then we can calculate how many molecules of replicatable probe were present initially. This relationship is summarized by the following equation:
  • N N O 2 ⁇ t/d ⁇
  • N o (-log 2) t + log N
  • log N (-log 2)/d is a constant. If we determine the time it takes for each reaction to synthesize a particular number of RNA molecules, then log N will also be a constant.
  • ethidium bromide is included in the RNA amplification reaction mixture.
  • An ethidium bromide concentration of about 1 ⁇ mol/L gives a good signal, without significantly inhibiting replication (Kramer, et al. 1988). Ethidium bromide becomes
  • a simple instrument periodically monitors the fluorescence of the ethidium bromide in an entire set of amplification reactions.
  • the instrument is programmed to store the kinetic data and to use this data to determine the time for each reaction at which the fluorescence corresponds to the presence of a particular number of RNA molecules (the "endpoint").
  • endpoint the time for each reaction at which the fluorescence corresponds to the presence of a particular number of RNA molecules
  • hybridization reaction each containing a known number of target molecules, permits the establishment of a "standard curve", in which the logarithm of the number of target molecules is inversely proportional to the time at which the endpoint is reached (as described in the second equation).
  • the number of target molecules in each of the unknown samples is then determined by comparing their endpoints with those on the standard curve.
  • This method is readily automatable; it does not require radioactive compounds; the magnitude of the fluorescent signal at the endpoint is the same for all the reactions, and is well above fluorescent background; the assay is accurate; and the logarithmic nature of the standard curve permits the determination of the number of targets in a sample over an extremely wide range of target concentrations.
  • step (c) The method described herein provides another important feature where the time of incubation in step (c) is sufficiently long so that the number of recombinant-RNA product strands exceeds the number of polymerase
  • the subject invention provides a method whereby the concentration of the oligo- or polynucleotide of interest detected in step (d) is determined by
  • RNA synthesized by the fluorescence that occurs when ethidium bromide binds to RNA it is preferable to detect the synthesized RNA by the fluorescence that occurs when ethidium bromide binds to RNA. Since these assays are incubated long enough for the replicase to become saturated with RNA, the amount of RNA synthesized by the end of the incubation period is directly proportional to the logarithm of the number of target sequences that were present in the original sample (Lizardi, et al., 1988). The inclusion of samples
  • RNA molecules increases linearly with time. For example reactions that have been incubated for at least 28 min, have generally passed the saturation point and reached the linear phase of synthesis. A comparison of the amounts of RNA present in each sample at 28 minutes shows that the most RNA is present in those samples that correspond to the hybridization reactions that contained the most targets. Because these reactions were initiated with the greatest number of replicatable probes, they reached the saturation point soonest and had the longest period of time to synthesize RNA in the linear phase.
  • This invention also provides a method for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample, comprising the steps of: (a) forming specific complexes between a mixture of different types of
  • recombinant-RNA probe molecules described above comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence required for the initiation of product strand synthesis by the polymerase, and a
  • heterologous RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo- or polynucleotide of interest, each type having a different inserted sequence, and the oligo- or polynucleotides of interest, by incubating the sample with the mixture of recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the
  • recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotides of interest; (d) separating the mixture of synthesized recombinant-RNAs by hybridizing them to an ordered array of polynucleotides bound to a solid support, where each of the polynucleotides is complementary to one type of synthesized recombinant-RNA; and (e) detecting the recombinant-RNA probe molecules produced in step (d), thereby determining the presence or concentration of each oligo- or polynucleotide of
  • a useful aspect of this invention is provided when the sample is a tissue specimen, such as a blood sample.
  • a particularly efficient method is provided when the conditions for hybridization comprise exposing the sample to guanidine thiocyanate.
  • the solid support provided may be a membrane, for example a nitrocellulose or nylon membrane.
  • a good clinical assay requires speed, specificity and sensitivity.
  • influenza Klebs iella pneumo nia, Neisseria menigitidis ,
  • Staphylococcus aureus, and Streptococcus Pneumonia The patient is typically a young child. Effective treatment requires the identification of the etiolocic agent in a sample of cerebrospinal fluid; and it is essential that antibiotic treatment begin as soon as possible. However, current laboratory techniques require at least 18 hours to identify the agent. Moreover, the sample volume is usually only 50 microliters and often contains less than 50 individual microorganisms, which is well below the limits at which reliable direct detection assays can currently be carried out. The clinical picture is further complicated by a marked increase in the abundance of bacteria that possess antibiotic resistance genes on plasmids.
  • a series of replicatable recombinant-RNA probes molecules can be prepared each of which is specific for a different organism that can cause meningitis.
  • a series of recombinant-RNA probe molecules can be prepared, each of which is specific for different antibiotic resistance genes that could be present in the infectious agent. Using such a series of recombinant-RNA probe molecules a mixture of, for example, 15 different replicatable
  • recombinant-RNA probes are incubated with denatured DNA obtained from a sample of spinal fluid. Only a few types of recombinant-RNA probe molecules species will find targets (for example, one bacterial probe and three resistance gene probes) . After removing the unbound probe molecules, Q ⁇ replicase is used to amplify the remaining probes. After amplification, the mixture of product RNAs is placed in contact with a membrane containing numbered dot-blots, each of which contains denatured DNA
  • T4 polynucleotide kinase and T7 RNA polymerase were purchased from New England Biolabs.
  • Calf intestine alkaline phosphatase T4 DNA ligase, the Klenow fragment of Escherichia coli DNA polymerase I, and bovine pancreatic deoxyribonuclease I were purchased from Boehringer Mannheim.
  • Qß replicase was isolated from bacteriophage Qß-infected E. coli Q13 by the procedure of Eoyang and August (17), with the hydroxyapatite step omitted.
  • ß-cyanoethyl phosphoramidite chemistry (Gait, 1984), in a Microsyn-1450A synthesized (Systec).
  • the oligonucleotides were isolated by preparative gel electrophoresis (Matthes, et al. 1984), eluted from the gel, filtered through nitrocellulose, and purified by chromatography (Lo, et al. 1984) on SEP-PAK C18 cartridges (Waters Associates).
  • Plasmid for Synthesizing MDV-1 (+) RNA by Transcription pT7-MDV contains a promoter for T7 RNA polymerase directed towards a full-length cDNA prepared from MDV-1 RNA (Mills, et al. 1988). This plasmid had been constructed so that transcription from the T7 promoter begins with the first nucleotide of MDV-1 (+) RNA. A Sma I restriction site had been introduced at the other end of the MDV-1 cDNA
  • resulting transcripts lacking the natural 3'-terminal dinucleotide CpA-OH, serve as excellent templates for exponential replication by Qß replicase (Mills, et al.
  • Recombinant RNAs can be generated by inserting any heterologous DNA sequence into one of the unique restriction sites in the polylinker of pT7-MDV-poly, and then utilizing the resulting plasmid as a template for transcription by T7 RNA polymerase.
  • pT7-MDV-fal-un was constructed by inserting a synthetic probe sequence (prepared by annealing dTCGAGACTAACATAGGTCTTAACTTGACTAACA to dTCGATGTTAGTCAAGT TAAGACCTATGTTAGTC) into the Xho I site of the polylinker sequence in pT7-MDV-poly.
  • pT7-MDV-fal-st was constructed by inserting the related synthetic probe sequence
  • nucleotide sequence in the recombinant region of each plasmid was determined by the chain termination procedure (Sanger, et al. 1977), utilizing 7-deaza-deoxyguanosine 5'-triphosphate (Boehringer Mannheim) in place of
  • Sequencing reactions were carried out on total plasmid DNA (Wallace, et al. 1981), utilizing a 20-nucleotide primer (Pharmacia) that was complementary to the T7 promoter sequence (Osterman and Coleman, 1981).
  • Plasmids were isolated from bacteria by the method of Holmes and Quigley (Holmes and Quigley, 1981) and purified by gel filtration chromatography (Bywater, et al. 1983) on Sephacryl S-1000 (Pharmacia). Plasmid DNA was then digested with Sma I or Stu I.
  • RNA bands were visualized by silver staining (Sammons, et al. 1981).
  • RNA in each 2- ⁇ l sample was determined by binding the [ 32 P]RNA to DE81 cellulose discs (Whatman), as described by Maxwell and his coworkers (Maxwell, et al. 1978), and measuring the radioactivity on each disc with a scintillation counter. The size and homogeneity of the RNA in selected samples was determined by polyacrylamide gel electrophoresis. The [ 32 P]RNA bands were detected by autoradiography.
  • pPFR6 Two different plasmids were used as targets.
  • the second plasmid, pUC13 served as a negative control for hybridization of the recombinant RNA probes.
  • Each plasmid was linearized by digestion with BamH I, denatured by incubation in 0.4 NaOH for 60 sec at 42°C, and 0.5- ⁇ g or 1.0- ⁇ g aliquots were bound to a BA83 nitrocellulose membrane (Schleicher and Schuell), according to the dot-blot procedure of Kafatos and his coworkers (Kafatos, et al. 1979).
  • the membrane was cut into sections, each containing a duplicate pair of pPFR6 and pUC13 dot-blots. Each membrane section was prehybridized for 3 hr at 37°C in 5X SSPE (5X SSPE is 900 mM NaCl, 50 mM sodium phosphate (pH 7.4), and 5mM EDTA), 2 mg/ml sodium dodecyl sulfate, 500 ⁇ g/ml heparin (Sigma), and 20% formamide. Each membrane section was then incubated for 4 hrs at 25°C with between 23 ng and 150 ng of the [ 32 P]RNA to be tested and 10 ⁇ g of unlabeled E. coli tRNA carrier (Boehringer
  • RNA samples were tested: MDV-poly,
  • MDV-fal-un MDV-fal-un, MDV-fal-st, and truncated versions of each (transcribed from plasmids cleaved at the Stu I site).
  • the membranes were washed three times (15 min/wash) at 25°C with a solution containing 4X SSPE, 2 mg/ml sodium dodecyl sulfate, and 400 ⁇ g/ml heparin, followed by two 10-min washes at 25°C in IX SSPE, and a 12-min wash at 37 C in 1X SSPE.
  • the hybridized RNA was detected by autoradiography.
  • MDV-fal-un RNA hybridized to dot-blots was eluted by boiling in 200 ⁇ l of water for 60 sec.
  • the integrity of the eluted RNA was analyzed by polyacrylamide gel electrophoresis.
  • the replicability of the eluted RNA was determined by
  • replicase would bind to the nitrocellulose membrane.
  • RNA that would serve both as a specific probe for P. falciparum DNA and as a template for exponential
  • MDV-1 RNA was chosen as the parent molecule because modified MDV-1 RNAs can be synthesized by transcription from recombinant plasmids (Mills, et al. 1988). MDV-1 RNA contains many stable secondary structures (Klotz, et al. 1980; Mills, et al. 1980; Kramer and Mills, 1981), and these secondary structures are required for replication (Mills, et al. 1978; Nishihara, et al. 1983; Priano, et al. 1987). The site which was chosen for inserting probe sequences into MDV-1 RNA was located on the exterior of the molecule (Miele, et al.
  • the first recombinant, MDV-fal-un RNA was likely to possess an unstructured probe region, according to a computer analysis of its sequence (Zuker and Stiegler, 1981).
  • the second recombinant, MDV-fal-st RNA differed from MDV-fal-un RNA in a 5-nucleotide region. As a consequence, it was likely to form a more stable secondary structure in the region containing the probe.
  • Figure 2 shows the nucleotide sequence
  • RNAs were prepared by transcription in vitro: MDV-1, MDV-poly, MDV-fal-un, and MDV-fal-st.
  • nucleotides longer than the MDV-1 RNA from which it was derived it is amplified at essentially the same rate by Qß replicase.
  • Electrophoretic analysis showed that the product of the reaction to which no exogenous template had been added was MDV-1 RNA. This analysis is illustrated in the inset of Figure 4. This was expected, because the method which was used for isolating Qß replicase (Eoyang and August, 1971) does not eliminate all of the contaminating MDV-1 RNA molecules (Kramer, et al. 1974) . Based on the time it took for this reaction to achieve saturation (when the number of RNA molecules equals the number of active replicase molecules), it is calculated that only 40 molecules of MDV-1 RNA were present initially.
  • RNAs were shown to bind specifically to denatured plasmids containing a known quantity of P. falciparum target sequences ( Figure 5). Neither recombinant RNA bound to control plasmids that lacked P. falciparum sequences. Furthermore, MDV-poly RNA, which does not contain a probe sequence, did not bind to either plasmid. Taken together, these results demonstrate that both recombinant RNAs bind specifically to their targets and that, under the hybridization conditions employed, the presence of structure in the probe region has little effect on hybridization.
  • truncated versions of each recombinant probe were prepared by transcription from plasmids that had been linearized by digestion with Stu 1, instead of Sma I. These transcripts were 111-nucleotides long, and contained 63 nucleotides from the 5' end of MDV-1 (+) RNA and 48 nucleotides from the insert region, including the entire probe sequence. Under the hybridization conditions employed, no difference could be observed between the hybridization of the truncated RNAs and the hybridization of the full-length RNAs. This result demonstrates that the constrained topology of the MDV-1 domain (Sammmons, et al. 1981) that surrounds the probe region in the full-length molecule has little effect on the ability of the probe to hybridize to its target. Replication of Recombinant RNAs after Hybridization
  • RNA molecules initially present in each reaction was plotted against the amount of RNA synthesized in 25 minutes (by which time each reaction had completed the exponential phase of synthesis).
  • the amount of recombinant RNA initially added to those reactions can be thought of as representing the amount of probe that would have been bound to targets had this been an actual assay, and the amount of amplified
  • RNA at 25 minutes simulates the signal that would have been detected.
  • the results show that the size of the amplified signal would be in the hundred-nanogram range whether the number of targets was as small as one thousand or as large as one billion.
  • Q ⁇ replicase should be able to detect targets over a range of at least six orders of magnitude.
  • RNAs containing a variety of inserted sequences have recently been constructed. This leads one to believe that, by choosing appropriate inserts, recombinant RNA probes can be prepared that will be able to detect the nucleic acid of any virus, bacterium, or eukaryotic parasite.
  • Amplified recombinant RNAs contain a sequence (the probe) that identifies which target was detected. It should therefore be possible to design diagnostic assays that utilize a mixture of recombinant RNAs, each containing a probe sequence specific for the genome of a different infectious agent. After amplification with Qß replicase, the RNA population would contain replicates of only those probes that had bound to their targets. Subsequent hybridization of these amplified RNAs to a membrane containing an ordered array of DNA dot-blots complementary to each of the probes would permit the simultaneous identification of several different organisms in the same sample.
  • Example 2 Example 2
  • RNA that contained a 30- nucleotide-long probe complementary to a conserved region of the pol gene in human immunodeficiency virus type 1 (HIV-1) mRNA.
  • Test samples were prepared, each containing a different number of HIV-1 transcripts that served as simulated HIV-1 mRNA targets. Hybridizations were carried out in a solution containing the chaotropic salt, guanidine thiocyanate. Probe-target hybrids were isolated by reversible target capture on paramagnetic particles. The probes were then released from their targets and amplified by incubation with the RNA-directed RNA polymerase, Qß replicase.
  • HIV-1 human immunodeficiency virus type 1
  • the replicase copied the probes in an exponential manner: after each round of copying, the number of RNA molecules doubled.
  • the amount of RNA synthesized in each reaction (approximately 50 ng) was sufficient to measure without using radioisotopes.
  • Kinetic analysis of the reactions demonstrated that the number of HIV-1 targets originally present in each sample could be determined by measuring the time it took to synthesize a particular amount of RNA (the longer the synthesis took, the fewer the number of targets originally present). The results suggest that clinical assays involving replicatable hybridization probes will be simple, accurate, sensitive, and automatable.
  • RNA polymerase (EC 2.7.7.6) was purchased from New England Biolabs, Beverly, MA, and calf thymus terminal deoxyribonucleotidyltranferase (EC 2.7.7.31) was obtained from Supertechs, Bethesda, MD.
  • Qß replicase (EC 2.7.7.48) was isolated from bacteriophage Qß-infected Escherichia coli Q13 by the procedure of Eoyang and August (Eoyang and August, 1971), with the hydroxylapatite step omitted.
  • Qß replicase is stable when stored in a glycerol solution at -20 °C: its activity remains unchanged after five years of storage. Single-stranded DNA fragments were prepared, using ß-cyanoethyl phosphoramidite chemistry, on an Applied Biosystems 380A synthesizer, Foster City, CA. Replicatable HIV-I Probes
  • Recombinant MDV-1 RNA containing an inserted HIV-1 probe sequence was synthesized by transcription from a recombinant plasmid.
  • the plasmid was constructed by inserting a synthetic probe sequence (prepared by annealing dGATCACCGTAGCACTGGTGAAATTGCTGCCATTGA to dGATCTO ⁇ TGGCAGCAATTTCACCAGTGCTACGGT) into the Bgl II site of a plasmid that is identical to plasmid pT7-MDV-poly (Lizardi, et al. 1988), except that the polylinker sequence is in the opposite orientation.
  • Single-stranded DNAs containing 3'-poly(dA) tails were synthesized for use in binding probe-target hybrids to oligo(dT) groups on the surface of paramagnetic particles.
  • Four different oligodeoxyribonucleotides (of lengths 24, 40, 40, and 43 nucleotides) were prepared by automated synthesis. Each probe was complementary to a different region of the HIV-1 pol gene near to the target of the replicatable probe.
  • a poly(dA) tail was added to the 3' end of each probe by incubation with terminal deoxyribonucleotidyltransferase (Nelson and Brutlag, 1979).
  • Simulated HIV-1 mRNA targets were purchased from Gene-Trak
  • Each tube contained simulated HIV-1 mRNA targets, MDV-hiv (+) RNA (replicatable probes), and capture probes, dissolved in 70 ⁇ L of 2.5 mol/L guanidine thiocyanate (Fluka Chemical, Hauppage, NY), and placed in a polypropylene "titertube" (Bio-Rad, Richmond, CA). Each tube contained 2 X 10 9 molecules of MDV-hiv (+) RNA, 10 11 molecules of each capture probe, and a different number of target molecules. The number of HIV-1 transcripts in each tube was: 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 10 3 . The tubes were incubated at 37 oC for 18 h.
  • the probe-target hybrids were isolated from the reaction mixture by binding them to oligo(dT) groups on the surface of paramagnetic particles (Morrisey, et al. 1989). These ferric oxide particles ( ⁇ 1 ⁇ m in diameter), purchased from Gene-Trak Systems, possess highly convoluted surfaces coated with silicon hydrides to which numerous oligo(dT) "hairs" have been covalently linked. We added 50 ⁇ L of a suspension of paramagnetic particles to each reaction tube, reducing the guanidine thiocyanate (GuSCN) concentration to 1.45 mol/L.
  • GNSCN guanidine thiocyanate
  • the tubes were incubated at 37oC for 10 min to allow the 3'- poly(dA) tails of the capture probes to hybridize to the oligo(dT) groups on the surface of the particles.
  • the number of oligo(dT) groups available on the surface of the particles far exceeded the number of capture probes present in the reaction. Because the target molecules are hybridized to the 5' end of the capture probes, the probe- target hybrids become linked to the surface of the particles ( Figure 8).
  • the particles are paramagnetic, they do not act as magnets, and they do not cling to each other. However, when placed in a magnetic field, they are drawn to the magnetic source. Accordingly, the paramagnetic particles, with the probe-target hybrids bound to their surface, were drawn to the walls of the titertubes by placing the tubes in the presence of the strong magnetic field provided by a magnetic separation device (which was purchased from Gene-Trak Systems). The supernates, which contained the nonhybridized probes, were then withdrawn from each tube by aspiration, and replaced by 200 ⁇ L of a wash solution containing GuSCN at 1.5 mol/L.
  • a magnetic separation device which was purchased from Gene-Trak Systems
  • the tubes were then withdrawn from the magnetic field and the probe-target hybrids attached to the resuspended particles were washed by vigorous agitation on a multi-tube vortex-type mixer (American Hospital Supply, McGaw Park, IL).
  • the particles were again drawn to the walls of the tubes, the supernates were aspirated, another 200 ⁇ L of 1.5 mol/L GuSCN wash solution was added, and the particles were again agitated and drawn to the walls of the tubes.
  • the supernates were again withdrawn by aspiration, but this time they were replaced with 50 ⁇ L of a release solution containing GuSCN at 3.25 mol/L.
  • the tubes were again removed from the magnetic field, agitated to resuspend the particles, and then incubated at 37 oC for 5 min.
  • the relatively weak hybrids formed between the 3'-poly(dA) tails of the capture probes and the oligo(dT) groups on the surface of the particles came apart, releasing the probe- target hybrids back into solution.
  • the much stronger hybrids formed between the capture probes and the target (and between the replicatable probe and the target) remained intact (Morrissey, et al. 1989).
  • the stripped particles were then drawn to the sides of the tubes, and the supernates (containing the released probe-target hybrids) were transferred to new tubes.
  • the stripped particles were discarded and 50 ⁇ L of a suspension of fresh particles was added to each new tube, reducing the GuSCN concentration to 1.62 mol/L.
  • the tubes were incubated at 37 oC for 10 min to allow the probe-target hybrids to be recaptured by the hybridization of the 3'-poly(dA) tails of the capture probes to the oligo(dT) groups on the surface of the new particles.
  • the reactions were incubated in parallel, and 4- ⁇ L aliquots were withdrawn from each of the seven reactions at one-min intervals between 9 and 29 min of incubation and mixed with 36 ⁇ L of an ice-cold solution containing 120 mmol of NaCl and 20 mmol of EDTA-NaOH (pH 8) per liter.
  • the EDTA in this solution chelated the magnesium in the sample, preventing further replication.
  • the size and homogeneity of the [ 32 P]RNA products in 5- ⁇ L aliquots of each of the 147 samples was determined by electrophoresis through 8% polyacrylamide slab gels in the presence of urea, 7 mol/L (Maniatis and van desande, 1975). Finally, the [ 32 P]RNA products in a 10- ⁇ L aliquot of each of the 147 samples were precipitated by the addition of 190 ⁇ L of an ice-cold solution containing 360 mmol of phosphoric acid, 20 mmol of sodium pyrophosphate, and 2 mmol of EDTA per liter.
  • RNA in each sample was then electrostatically bound to a "Zeta-Probe" quaternary-amine- derivatized nylon membrane(Bio-Rad) in a dot-blot vacuum filtering manifold (Bio-Rad).
  • Each bound sample was washed 10 times with 200 ⁇ L of the ice-cold precipitation solution to remove unincorporated [ 32 P]CTP.
  • the membrane was then air-dried, and the amount of [ 32 P]RNA present in each sample on the membrane was made visible by autoradiography. After the autoradiograph was prepared, the amount of RNA present in each dot-blot was measured in a scintillation counter.
  • the kinetic data can be used to calculate the number of replicatable probes that were present at the beginning of the reaction. If known standards are included among the unknown samples be tested, then these data can be used to determine the number of target molecules originally present in each unknown sample.
  • the results also indicate the limit of detection.
  • the amplification reaction corresponding to the sample containing 10 5 targets achieved saturation at an earlier time than did the amplification reaction corresponding to the sample containing 10 4 targets. However, there was no significant difference in the amplification reactions corresponding to the samples containing 10 4 and 10 3 targets. However, there was no significant difference in the amplification reactions corresponding to the samples containing 10 4 and 10 3 targets. These results indicate that the limit of detection was about 10,000 target molecules. Because electrophoretic analysis of the amplified RNA in each sample indicated that only recombinant RNA was synthesized, the limit of detection was determined by the level of persistence of nonhybridized replicatable probes. We feel that it is important to note that these were only preliminary assays, designed to demonstrate how replicatable probes might be used. Further experiments should lead to alterations in the assay format that will improve the sensitivity.
  • telomere sequences are sorted into separate species by hybridizing to an ordered array of dot-blots, each of which contains DNA complementary to one of the probe sequences.
  • Species-specific hybrization probes for the detection of HTLV-I, HTLV-II, HIV-1, and HIV-2 are derived from a highly conserved region of the viral pol gene, and form a specific hybrid with the corresponding retroviral mRNA. They are each
  • the selected probe sequences are listed in Table 2.
  • the identification of the target region is based on the following sequence determinations: HTLV-I
  • HIV-1 (Ratner et al., 1985; Muesing et al., 1985), and HIV-2
  • the recombinant-RNA probes are prepared by transcription from corresponding recombinant plasmids (Lizardi et al., 1988). Synthetic oligodeoxyribonucleotides containing the probe sequence and appropriate sticky ends cloned into the polylinker region of pT7-MDV-poly are isolated and characterized by restriction mapping and confirmed by nucleotide sequence analysis (Wallace et al., 1981).
  • Transcripts are prepared by incubating Sma I-cleaved plasmids with T7 RNA polymerase, and their sequences confirmed by the chain-termination procedure (Sanger et al., 1977) utilizing reverse transcriptase and a primer that initiates synthesis upstream from the region containing the probe sequence (Lane et al., 1985).
  • the transcripts are incubated with Qß replicase, and the resulting product RNAs analyzed by polyacrylamide gel electrophoresis to confirm their identity and homogeneity.
  • the kinetics of synthesis of Qß replicase reactions initiated with different concentrations of each recombinant-RNA probe is compared to MDV-1 RNA controls.
  • RNA consists of a mixture of the different recombinant RNAs, whose relative abundance reflects the concentration of the different retroviral targets in the sample.
  • This mixture of amplified probes is hybridized to a dipstick containing an ordered array of dot-blots (Kafatos et al., 1979), each of which contains DNA complementary to one of the probe sequences. In this manner, the different recombinant RNAs are sorted out, allowing the amount of each retroviral species to be measured.
  • guanidine thiocyanate in combination with reversible target capture provides a blood sample assay format in which cells do not have to be lysed before the assay is performed, and unhybridized probe is removed before amplification.
  • RNA that is synthesized in these assays is determined by the incorporation of radioactive precursors.
  • Biebricher C. K., Kiekmann, S., and Luce, R. (1982) J. Mol. Biol. 154, 629-648. Structural Analysis of Self-replicating RNA Sythesized by Q-beta Replicase.
  • RNA Replication Required Intermediates and the Dissociation of Template, 67 Product, and Qß Replicase.
  • RNA Coliphages The Role of Secondary Structures during RNA Replication. Priano, C., Mills, D. R., and Kramer, F. R. (1989) Nucleic Acids Res., in preparation. Sequence, Structure, and Evolution of a 77-Nucleotide Template for Qß Replicase. Ranki, M.
  • Nanovariant RNAs Nucleotide Sequence and Interaction with Bacteriophage Qß Replicase.

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Abstract

Une molécule de sonde d'ARN monocaténaire recombiné replicable et hybridable comprend une séquence de reconnaissance permettant de lier une polymérase d'ARN à correspondance ARN; une séquence requise pour lancer la synthèse de brins du produit au moyen de la polymérase; et une séquence d'ARN hétérologue insérée dans un site spécifique dans la région interne de la molécule recombinée, complémentaire d'un oligonucléotide ou d'un polynucléotide à étudier. Des procédés permettent de déterminer la présence d'une concentration d'un oligonucléotide ou d'un polynucléotide à étudier dans un échantillon et en même temps de déterminer la présence ou la concentration de plusieurs oligonucléotides ou polynucléotides différents à étudier dans un échantillon.
PCT/US1991/003634 1990-05-23 1991-05-23 Sondes d'arn recombine hybridable replicable et test d'hybridation WO1991018117A1 (fr)

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WO1994009159A2 (fr) * 1992-10-09 1994-04-28 Amoco Corporation Elements d'insertion et acides nucleiques amplifiables
WO1994010335A2 (fr) * 1992-10-09 1994-05-11 Amoco Corporation Methodes d'analyse
WO1996003528A2 (fr) * 1994-07-27 1996-02-08 Cambridge University Technical Services Limited Oligonucleotides et leur utilisation
US5653939A (en) * 1991-11-19 1997-08-05 Massachusetts Institute Of Technology Optical and electrical methods and apparatus for molecule detection
US5780273A (en) * 1993-04-09 1998-07-14 Amoco Corporation Insertion elements and amplifiable nucleic acids
WO2003070982A1 (fr) * 2002-02-22 2003-08-28 Avaris Ab Complexe comportant au moins deux elements biospecifiques separes par un lieur d'acides nucleiques, par exemple pour l'identification de candidats d'administration de medicaments ; bibliotheques combinatoires de tels complexes
US20200332279A1 (en) * 2006-05-31 2020-10-22 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US12077752B2 (en) 2009-04-03 2024-09-03 Sequenom, Inc. Nucleic acid preparation compositions and methods

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5653939A (en) * 1991-11-19 1997-08-05 Massachusetts Institute Of Technology Optical and electrical methods and apparatus for molecule detection
US5846708A (en) * 1991-11-19 1998-12-08 Massachusetts Institiute Of Technology Optical and electrical methods and apparatus for molecule detection
WO1994010335A3 (fr) * 1992-10-09 1994-08-18 Amoco Corp Methodes d'analyse
WO1994009159A2 (fr) * 1992-10-09 1994-04-28 Amoco Corporation Elements d'insertion et acides nucleiques amplifiables
WO1994009159A3 (fr) * 1992-10-09 1994-06-09 Amoco Corp Elements d'insertion et acides nucleiques amplifiables
WO1994010335A2 (fr) * 1992-10-09 1994-05-11 Amoco Corporation Methodes d'analyse
US5780273A (en) * 1993-04-09 1998-07-14 Amoco Corporation Insertion elements and amplifiable nucleic acids
WO1996003528A2 (fr) * 1994-07-27 1996-02-08 Cambridge University Technical Services Limited Oligonucleotides et leur utilisation
WO1996003528A3 (fr) * 1994-07-27 1996-04-18 Lynxvale Ltd Oligonucleotides et leur utilisation
WO2003070982A1 (fr) * 2002-02-22 2003-08-28 Avaris Ab Complexe comportant au moins deux elements biospecifiques separes par un lieur d'acides nucleiques, par exemple pour l'identification de candidats d'administration de medicaments ; bibliotheques combinatoires de tels complexes
US7824853B2 (en) 2002-02-22 2010-11-02 Avaris Ab Complex comprising at least two biospecific elements separated by a nucleic acid linker e.g. for identification of drug delivery candidates combinatorial library of such complexes
US20200332279A1 (en) * 2006-05-31 2020-10-22 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US11952569B2 (en) * 2006-05-31 2024-04-09 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US12077752B2 (en) 2009-04-03 2024-09-03 Sequenom, Inc. Nucleic acid preparation compositions and methods

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