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WO2005061735A2 - Amplification specifique a un brin - Google Patents

Amplification specifique a un brin Download PDF

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Publication number
WO2005061735A2
WO2005061735A2 PCT/US2004/039666 US2004039666W WO2005061735A2 WO 2005061735 A2 WO2005061735 A2 WO 2005061735A2 US 2004039666 W US2004039666 W US 2004039666W WO 2005061735 A2 WO2005061735 A2 WO 2005061735A2
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strand
nucleic acid
acid molecule
padlock probe
probe
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WO2005061735A3 (fr
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Vinay K. Pathak
David C. Thomas
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US Department of Health and Human Services
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • FIELD The present disclosure relates to analysis of nucleic acid molecules and to methods for amplifying a particular strand of a nucleic acid molecule.
  • BACKGROUND Replication of genetic material for all organisms involves synthesis of two different strands of nucleic acid, the minus-strand and the plus-strand in viruses, and the leading- and lagging-strand in DNA replication.
  • DNA replication the process involves separation of the two strands of the DNA helix, so that the hydrogen-bond donor and acceptor groups on each base become exposed for base pairing. The appropriate incoming single nucleotides are thereby aligned for their enzyme-catalyzed polymerization into a new nucleic acid chain.
  • Replication of viral RNA, such as H1N-RNA is achieved by synthesizing a double-stranded DNA molecule by using reverse transcriptase. Viral RNA is then transcribed from the proviral DNA molecule.
  • PCR polymerase chain reaction
  • U.S. Patent No: 5,744,311 strand displacement amplification
  • transcription- free isothermal amplification U.S. Patent No: 6,033,881
  • repair chain reaction amplification WO 90/01069
  • ligase chain reaction amplification European Patent Appl. 320 308
  • gap filling ligase chain reaction amplification U.S. Patent No: 5,427,930
  • NASBATM RNA transcription-free amplification U.S. Patent No: 6,025,134
  • Padlock probes have been used in DNA diagnostic, and can discriminate gene sequence with single base mutations.
  • padlock probes include two target-complementary regions at the 3' and 5' ends and a generic spacer region. When the 3' and 5' terminal sequences are juxtaposed on a target DNA sequence, the probe ends can be joined by a DNA ligase to from a circular molecule that is catenated with the target strand.
  • Methods of amplification of circularized padlock probes using rolling circle replication and PCR has been described (Thomas et al., Arch. Path. Lab. Med. 123:1170-6, 1999).
  • SUMMARY Provided herein is a novel assay that permits selective site-specific amplification of each strand of a nucleic acid molecule, and in some examples further permits quantitation of the nucleic acid molecule.
  • This assay termed SSA (Strand-Specific Amplification)
  • SSA Single-Specific Amplification
  • padlock probes that specifically hybridize at their 5' and 3' ends to a target nucleic acid molecule, wherein the 5' and 3' ends of the padlock probe are sufficiently proximate to one another to be joined. The 5' and 3' ends of the padlock probe are joined, thereby forming a closed circularized probe.
  • the closed circularized probe is subsequently amplified to provide tandemly repeated padlock probe sequences, for example by using rolling circle amplification, and the products detected, for example by using quantitative real-time PCR.
  • the products are further quantitated to estimate the amount of a particular strand of a nucleic acid molecule in a sample.
  • the SSA technology is used to analyze and monitor infection in cells, such as the cells of a subject having an infection, for example a viral, bacterial, parasitic, or fungal infection.
  • SSA is used to monitor the infection of viruses such as HIN, hepadnaviruses, adenoviruses, and herpesviruses.
  • the infection is analyzed by monitoring the synthesis of a particular nucleic acid strand of the pathogen.
  • the SSA method is used to analyze the impact of a test agent on nucleic acid molecule synthesis, such as reverse transcription of a plus- or minus-strand of a nucleic acid sequence, or synthesis of a leading- or lagging- strand.
  • the SSA method can be used to dete ⁇ nine the effect of anti- pathogenic agents, such as anti-viral, anti-bacterial, anti-fungal, and anti-parasitic agents on the rate of synthesis of a particular nucleic acid strand.
  • the SSA method is used to determine the effect of antiviral drugs on the kinetics of reverse transcription of a virus, such as HIN-1, as well as facilitate development of additional antiviral agents that interfere with specific steps (or replication of the plus- or minus-strand) in reverse transcription.
  • the SSA method can facilitate development of antiviral agents that increase the rate of synthesis of a viral nucleic acid molecule, since such agents would increase the rate of replication errors, which may be detrimental for viral replication and may reduce viral loads in a subject.
  • SSA analysis can also be used to monitor the effect of an anti-neoplastic agent, such as an anti-cancer agent.
  • the ability of the agent to preferentially inhibit or decrease leading- or lagging-strand synthesis of a nucleic acid molecule involved in cancer development or progression can be determined using SSA.
  • the relative amount of leading- or lagging-strand synthesis is compared to a control, such as an amount of leading- or lagging-strand synthesis in a control nucleic acid (such as a housekeeping gene), or an amount of leading- or lagging-strand synthesis observed in the subject prior to starting the anti- neoplastic treatment, or an amount of leading- or lagging-strand synthesis observed in the subject in a prior time point, or combinations thereof.
  • FIG. 1A is a schematic drawing showing an overview of strand-specific amplification (SSA).
  • FIG. IB is a schematic drawing showing a padlock probe hybridized to its target nucleic acid molecule.
  • FIG. 1C is a schematic drawing showing a closed circularized padlock probe hybridized to its target nucleic acid molecule.
  • FIG. 2 is a table showing nucleotide sequences of exemplary HIN-1 padlock probes (SEQ LD ⁇ OS: 1-12). For orientation, a schematic of a typical padlock probe is depicted above the probe sequences.
  • the 5' and 3' arms that hybridize to the target nucleic acid molecule are shown, and the ligation junction within HDV-EGFP for each probe is indicated by a slash.
  • Nucleotide 1 indicates the beginning of the U3 region of the 5' LTR.
  • the 3'-OH and 5'-PO 4 groups at the ligation junction are shown for probe 1 only.
  • the 49-base spacer region that connects the target arms is indicated as "spacer”, and is common to all probes (SEQ ID NO: 13).
  • the underlined nucleotides show the sequences of the dual-labeled probes.
  • probe 1 The location of the FAM (6-carboxy-fluorescein, F) and TAMRA (6-carboxy- tetramethylrhodamine, T) labels on the 5' and 3' ends, respectively, are indicated for probe 1 as an example.
  • the specific locations of the probes are: probe 1, 56 nt 5' of PBS; probe 2, 91 nt 3' of PPT; probe 3, 34 nt 5' of PPT; probe 4, 24 nt 3' of cPPT; probe 5, 36 nt 5' of the cPPT; probe 6, 44 nt 3' of PBS; probe 7,91 nt 3' of PPT; probe 8, 35 nt 3' of PBS; probe 9, 36 nt, 5' of cPPT; probe 10, 26 nt 3' of cPPT; probe 11, 23 nt 3' of CTS; probe 12, 52 nt 5' of PPT.
  • FIG. 3 A is a schematic drawing showing the locations of padlock probes used for SSA analysis of HIN-1 reverse transcription.
  • a schematic outline of HlN-l reverse transcription is outlined. Dashed black lines represent R ⁇ A degraded by the R ⁇ ase H activity of RT and solid lines represent minus- and plus-strand D ⁇ As.
  • R repeat region
  • U5 unique 5' region
  • PBS primer-binding site
  • cPPT central polypurine tract
  • CTS central termination sequence.
  • the approximate locations of padlock probes are indicated with numbers from 1-12 in boxes that are used throughout the figures.
  • FIGS. 3B and 3C are graphs showing exemplary standard curves for padlock probes (B) 3 and (C) 11 used in SSA analysis. Copy numbers of target D ⁇ A (white squares) were calculated based on the standard curve (black squares). Note that even though probe 3 appears to display less efficient amplification than probe 11, because the copy numbers in target DNAs were estimated from the standard curves, the efficiency of amplification does not influence the copy numbers.
  • FIG. 4A are graphs showing SSA analysis of the in vivo rate of HIN-1 reverse transcription using three minus-strand specific padlock probes. A schematic drawing showing the location of the padlock probes is also shown. Each graph represents a different experiment.
  • FIG. 4B are graphs showing SSA analysis of the in vivo rate of minus-
  • FIG. 4C is a graph showing SSA analysis of the in vivo rate of plus-strand initiation using SSA analysis.
  • FIG. 5 A is a graph showing the kinetics of plus-strand D ⁇ A synthesis using plus-strand specific padlock probes (SEQ ID ⁇ OS: 8, 9, 11 and 12).
  • FIG. 5B is a graph showing the kinetics of plus-strand D ⁇ A synthesis after infection with the cPPT " mutant of HDN-EGFP.
  • FIGS. 6A-6B are graphs showing SSA analysis of the effect of RT inhibitors on (A) minus- and (B) plus-strand D ⁇ A synthesis.
  • a schematic drawing showing the location of the padlock probes is also shown. Results are expressed as a percentage of the products accumulated at the 6-hour time point in the absence of test agent. Legends are shown only for probes 3 and 8 but are the same for all probes.
  • FIG. 7 is a graph showing SSA analysis of HIN-1 minus-strand D ⁇ A synthesis in 293 T cells compared to primary T cells. A schematic drawing showing the location of the padlock probes is also shown.
  • SEQ ID NOS: 1-12 show the nucleic acid sequence of exemplary HIN-1 padlock probes.
  • SEQ LD ⁇ OS: 1-6 specifically detect HIN-1 minus-strand D ⁇ A while SEQ LD ⁇ OS: 7-12 specifically detect HIN-1 plus-strand D ⁇ A.
  • SEQ ID NO: 13 shows the nucleic acid sequence of the spacer region of the disclosed exemplary HIN-1 padlock probes.
  • SEQ ID ⁇ OS: 14 and 15 show primer sequences used to amplify HIN-1 padlock probes using rolling circle amplification.
  • SEQ ID ⁇ OS: 16-18 show a primer-probe set specific for the human porphobilinogen deaminase (PBGD) gene.
  • SEQ ID NO: 19 shows a wild-type cPPT nucleic acid sequence.
  • SEQ ID NO: 20 shows a mutated cPPT nucleic acid sequence.
  • amplicons The resulting amplification products are called "amplicons.”
  • To selectively amplify a particular strand of a nucleic acid molecule is to increase the number of copies of one strand of a nucleic acid molecule, such as the plus- or minus- strand, or such as the leading- or lagging-strand.
  • Detect To determine the existence or presence of, for example to determine whether a nucleic acid molecule is present in a sample, or to determine if an amplicon is present following amplification.
  • HIV Human Immunodeficiency Virus
  • ADLS acquired immunodeficiency syndrome
  • HIN subtypes can be identified by particular number, such as HIN-1 and HIN-2. More detailed information about HIN can be found in Coffin et al, Retroviruses (Cold Spring Harbor Laboratory Press, 1997).
  • Hybridization Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al, Molecular Cloning: A Laboratory
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand.
  • Hybridization 5x SSC at 65°C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each Wash twice: 0.5x SSC at 65°C for 20 minutes each High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each Wash twice: lx SSC at 55°C-70°C for 30 minutes each
  • Isolated An "isolated" biological component (such as a nucleic acid sequence or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acid molecules and proteins which have been "isolated” include nucleic acid molecules and proteins purified by standard purification methods.
  • nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.
  • Join To bring together or link.
  • joining two ends of a nucleic acid molecule includes ligating the ends to one another.
  • Label An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy.
  • a label can be attached to a nucleic acid molecule, thereby permitting detection of the nucleic acid molecule.
  • labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fiuorophores, haptens, enzymes, and combinations thereof.
  • Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
  • Lagging-strand Strand of DNA replicated discontinuously in segments called Okazaki fragments.
  • Leading-strand Strand of DNA replicated continuously in the 5' to 3' direction by continuous polymerization at the 3' growing end.
  • Minus strand The reverse complement of a plus sfrand.
  • Nucleic acid molecule or sequence: A deoxyribonucleotide or ribonucleotide polymer in either single- (ss) or double-stranded (ds) form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Double-stranded forms of nucleic acid molecules can have two strands, a first strand and a second strand.
  • first and second strands include, but are not limited to, a plus- and minus-strand, a leading- and lagging- strand, or a 5' to 3' strand and reverse complement of the 5' to 3' strand.
  • Padlock probe A single-stranded DNA molecule that is able to circularize on its target nucleic acid sequence. Padlock probes include three sections, a 5' phosphate terminal end, a spacer region, and a 3' hydroxyl terminal end. The 5' and 3' terminal end sequences can specifically hybridize to a target nucleic acid sequence.
  • a closed circularized padlock probe is a padlock probe with its 5' and 3' ends joined covalently, thus permitting amplification of the padlock probe.
  • Padlock probes are also described in U.S. Patent Nos. 5,912,124; 6,558,928; and 6,225,056.
  • Pathogen A disease-producing agent. Examples include, but are not limited to viruses, bacteria, parasites, and fungi.
  • Plus-strand A virus-specified RNA or DNA that codes for viral proteins.
  • a viral plus strand can serve as a template for the synthesis of a complementary minus-strand RNA by a polymerase.
  • a viral plus strand can serve as a template for minus-strand DNA synthesis by reverse transcription.
  • Primer Nucleic acid molecules that are ten nucleotides or more in length.
  • Longer primers can be at least 15, at least 20, at least 25, at least 30 or at least 50 nucleotides or more in length, such as 10-50 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 15-30 nucleotides.
  • Primers can be annealed to a complementary target nucleic acid strand by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand, and then the primer extended along the target nucleic acid strand by a polymerase.
  • Primer pairs can be used for amplification of a nucleic acid sequence, for example by PCR or other nucleic-acid amplification methods. The specificity of a primer increases with its length.
  • a primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides.
  • primers can be selected that include at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides.
  • a primer includes a label. Quantitating a nucleic acid molecule: Determining or measuring a relative quantity of nucleic acid molecule present, such as the number of amplicons or tandem sequence.
  • nucleic acid molecules such as a target plus- or minus-strand, or target leading- or lagging- strand
  • Real-time quantitative PCR A method for detecting and measuring products generated during each cycle of a PCR, which are proportionate to the amount of template nucleic acid molecule prior to the start of PCR. The information obtained, such as an amplification curve, can be used to quantitate the initial amounts of template nucleic acid molecule.
  • Recombinant A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence.
  • Rolling circle amplification A process (for example an isothermal process) for generating multiple copies of a nucleic acid sequence, such as a padlock probe sequence, wherein the accumulation of products proceeds linearly over time.
  • the resulting product includes tandemly linked copies of the complementary sequence of the template, such as a padlock probe sequence.
  • a closed circularized padlock probe hybridized to its target sequence is incubated with appropriate polymerase and replication primer(s), thereby synthesizing tandemly linked copies of the complementary strand of the padlock probe. Exemplary methods are provided herein, and are also disclosed in U.S.
  • Sample Biological samples containing a nucleic acid molecule, such as genomic DNA, cDNA, RNA, or combinations thereof. Samples can be obtained from the cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, fine needle aspirates, amniocentesis samples and autopsy material.
  • Subject Living multi-cellular vertebrate organisms, including human and veterinary subjects, such as cows, pigs, horses, dogs, cats, birds, reptiles, and fish. Synthesis of a nucleic acid molecule: To replicate a nucleic acid molecule.
  • Target nucleic acid molecule A nucleic acid molecule whose detection, quantitation, qualitative detection, or a combination thereof, is intended.
  • the nucleic acid molecule need not be in a purified form.
  • Various other nucleic acids molecules can also be present with the target nucleic acid molecule.
  • the target nucleic acid molecule can be a specific nucleic acid in a cell (which can include host RNAs and DNAs, as well as other nucleic acid such as viral, bacterial or fungal nucleic acids), the amplification of which is intended.
  • Test agent Any suitable compound or composition can be used as a test agent, such as organic or inorganic chemicals, including aromatics, fatty acids, and carbohydrates; peptides, including antibodies (such as monoclonal, polyclonal, and humanized antibodies), and other specific binding agents; or nucleic acids.
  • test agents include, but are not limited to, any peptide or non-peptide composition in a purified or non-purified form, such as peptides made of D-and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam et al, Nature 354:82-4, 1991), phosphopeptides (such as in the form of random or partially degenerate, directed phosphopeptide libraries; see, for example, Songyang et al, Cell 12:161-1%, 1993).
  • a test agent can also include a complex mixture or "cocktail" of molecules.
  • Therapeutic agent An agent, such as organic or inorganic agents, that can decrease one or more symptoms associated with a disease. For example, an anti- viral therapeutic agent may decrease fever, increase white blood cell count, decrease viral load, or combinations thereof. Such symptoms can be measured by clinical response.
  • SSA Strand-Specific Amplification
  • the 5' and 3' ends of the padlock probe are hybridized to adjacent, or substantially adjacent, target nucleotides, and are ligated to form a covalently closed junction (the square shown in FIG. 1 A and 1C). Only those probes that hybridize to a target sequence can be closed.
  • the closed circular padlock probe is then amplified, for example by using a rolling circle amplification protocol that generates a series of tandemly linked copies of the closed padlock probe.
  • the region representing the region around and including the 5' and 3' ends of the padlock probe can be quantified, for example with a dual-labeled primer-probe set which is complementary to the previous "gap” or "nick” between the 5' and 3' ends of the padlock probe.
  • Padlock probes A padlock probe is a linear, single-stranded DNA molecule that can circularize on its target sequence.
  • An overview of padlock probes can be found in Nilsson et al. (Science, 265:2085-8, 1994; herein incorporated by reference). Circularization is achieved because the 5' and 3' ends of the probe can hybridize and bind to respective target sequences in a target nucleic acid molecule (FIGS. 1 A and IB). In particular examples, the 5' and 3' ends of the probe each recognize immediately adjacent target nucleotides in the target nucleic acid molecule. However, the hybridized ends of the padlock probe can also be separated by a gap which would be filled prior to ligation (as discussed in U.S. Patent No.
  • padlock probes are at least about 50 nucleotides, and in particular examples range from about 50 to about 250 nucleotides in length, such as about 50-100 nucleotides, about 50-75 nucleotides, or about 50-60 nucleotides. However the present disclosure is not limited to padlock probes of particular lengths.
  • Padlock probes can be synthesized using conventional methods, such as using a nucleic acid synthesizer, recombinant molecular biology methods, or combinations thereof. Padlock probes that selectively recognize a first or second strand of a target nucleic acid sequence can be designed using standard molecular biology methods.
  • a sequence to be amplified is identified (such as a plus-strand sequence of a viral nucleic acid molecule) which is ideally not found on the opposite strand (such as the respective minus-strand sequence) and is unique to one site on each strand (with the exception of R regions where two copies can be present).
  • the target sequence is at least 20 nucleotides long (such as at least 30, or at least 40 nucleotides long), and contains a sequence of at least 10 nucleotides at each 5' and 3' end that is complementary to a portion of the target nucleic acid sequence. Ideally, such a region does not have a high GC content.
  • the padlock probe ideally does not have regions that are self-complementary, or a highly repetitive sequence with long runs of single bases, which could inhibit the probe from hybridizing specifically to its target sequence.
  • a padlock probe can be generated that specifically recognizes a particular strand of a nucleic acid molecule, such as a prokaryotic, eukaryotic, or viral nucleic acid sequence.
  • nucleic acid molecules include, but are not limited to, nucleic acid sequences from bacteria (such as E.
  • coli Salmonella, Bacillus or Haemophilus
  • fungi parasites
  • plants yeast
  • mammals such as a mouse, cat, dog, or human
  • viruses such as human immunodeficiency virus (HW), influenza, human papilloma virus (HPV), hepadnaviruses, adenoviruses, and herpesviruses.
  • HW human immunodeficiency virus
  • HPV human papilloma virus
  • HPV human papilloma virus
  • hepadnaviruses hepadnaviruses
  • adenoviruses adenoviruses
  • herpesviruses herpesviruses.
  • Exemplary HF/-1 minus-strand probes are provided in SEQ ID NOS: 1-6
  • exemplary HTV-1 plus-strand probes are provided in SEQ LD NOS: 7-12.
  • the present disclosure is not limited to these particular examples, as those skilled in the art can generate other padlock
  • Padlock probes include three sections: a 5' phosphate terminal end, a spacer region, and a 3' hydroxyl terminal end.
  • the 5' and 3' terminal end sequences can hybridize to the target nucleic acid molecule, and are thus designed to selectively form a specific and stable hybrid with a specific sequence (such as either but not both of a plus- or minus-strand) of a target nucleic acid sequence.
  • terminal end regions can be at least 10 nucleotides in length, such as about 10 to about 40 nucleotides in length, such as about 10-30 nucleotides, about 10-20 nucleotides, about 15-20 nucleotides, such as about 10, about 15, about 20, about 25 nucleotides, about 30 nucleotides, or about 40 nucleotides.
  • the terminal end regions have limited homology with primers used for amplification, to reduce false priming.
  • the sequence of each te ⁇ ninal end region is specific to a particular region of a particular target strand of a nucleic acid molecule. As shown in FIG.
  • the 5' and 3' terminal end sequences 10, 12 of a padlock probe 14 are complementary to a sequence of the target nucleic acid molecule 16, 18 such that when the ends of the padlock probe hybridize or anneal to the target nucleic acid molecule, the 5' and 3' terminal end sequences of the padlock probe are proximate to each other, but separated by a gap or nick 20.
  • the ends being proximate means that they are capable of being joined to one another, for example by ligation.
  • this gap 20 can be about 0-20 nucleotides, such as about 0, about 5, about 10 or about 20 nucleotides.
  • the gap is no more than about 5 nucleotides, no more than about 10 nucleotides, or no more than about 20 nucleotides.
  • the 5' and 3' terminal end sequences will only hybridize to the target nucleic acid molecule if the sequence of interest is present. If the target nucleic acid sequence of interest is not present, the padlock probe would not detectably bind to the target nucleic acid sequence.
  • a spacer region also referred to in the art as the linker region 22 is located between the 5' and 3' terminal end sequences.
  • the spacer region is about 30-100 nucleotides in length, such as about 30- 75 nucleotides, about 30-50 nucleotides, or about 50-100 nucleotides. In particular examples, the spacer region is at least about 30 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, or no more than about 100 nucleotides.
  • the spacer region can include a sequence that permits identification or selective amplification of the padlock probe sequence, for example by including a sequence that is complementary to a replication primer or fluorescent probe.
  • Such a replication primer can be used to amplify a closed padlock probe, for example using a rolling-circle method.
  • the spacer region does not recognize any region of the target sequence.
  • the spacer region is not GC or AT rich, but instead of average sequence composition, with no long runs of bases.
  • sets of padlock probes are provided, which allow for the simultaneous detection of several different target sequences or permit differential detection of different probes that recognize different targets.
  • each padlock probe in a set of padlock probes includes an identical spacer region, but unique 5' and 3' ends for recognizing a target nucleic acid molecule.
  • the identical spacer region peraiits amplification of the closed padlock probe with the same set of primers.
  • the amplicons produced from one padlock probe versus another padlock probe can be distinguished by using labeled probes, each containing a different combination of labels, wherein a particular combination of labels corresponds to a particular padlock probe product.
  • the target nucleic acid molecule In order to hybridize a padlock probe to its target nucleic acid sequence, the target nucleic acid molecule is provided in a single-stranded (ss) fo ⁇ n. If the target nucleic acid molecule is double-stranded (ds), it can be heated, for example to about 94°C, to denature the nucleic acid molecule into ss form. The temperature is then lowered, for example to about 50-55°C to allow the padlock probe 5' and 3' terminal ends to anneal to their target nucleic acid sequence. As described above, the target nucleic acid molecules can come from any source.
  • a target nucleic acid sequence can be a DNA sample, an RNA sample, a mitochondrial DNA sample, a circular DNA sample such as a plasmid or cosmid, a chromosomal DNA sample, a viral DNA sample, or a cDNA sample.
  • different samples are pooled and used as target nucleic acid molecules for the SSA method.
  • genomic and plasmid bacterial DNA can be assayed together in a single procedure. If the targeted nucleic acid molecule of interest is present, the 5* and 3' ends of the padlock probe will hybridize to the nucleic acid molecule, leaving a "gap" or "nick” between the 5' and 3' ends.
  • the ends of the padlock probe are joined in such a way to permit replication of the probe, for example by directly or indirectly ligating the ends to one another.
  • the 5' and 3' ends of the padlock probe are joined by a ligase.
  • the ligase if thermostable, can be included in the initial hybridization reaction mixture.
  • the ligation reaction produces a closed circularized padlock probe (also referred to in the art as an amplification target circle).
  • the ligase used can depend on the target nucleic acid molecule.
  • the ligase ideally forms phosphodiester bonds at nicks in double stranded DNA.
  • exemplary ligases include E. coli DNA ligase, T4 DNA ligase, Taq DNA ligase, and AMPLIGASE.
  • T4 DNA ligase can be used because it can seal gaps in the DNA component of a DNA:RNA hybrid.
  • a reverse transcriptase can be used to transcribe the RNA into a DNA sequence. The ligase used can determine the reagents needed to effect the ligation reaction.
  • the ligase reaction includes ATP or NAD as an energy source, Mg " ⁇ , or combinations thereof.
  • the ligation forms a covalently closed circular padlock probe.
  • the relatively short length of the padlock probes reduces the risk of end-to-end ligation of probes that have not annealed to a target nucleic acid molecule but are free in solution.
  • selection of the ligase and reaction conditions can reduce end-to-end probe ligation. For example, if AMPLIGASE is used, ligation takes place at 60°C. Under these conditions, a maximum of 1 in 1,000,000 probe terminal ends that are not bound to the targeted sequence will be ligated.
  • An example of indirect ligation of the ends is to fill the gap between the 5' and 3' ends of the padlock probe with a gap filler that links the 5' and 3' ends to one another.
  • a gap filler is a nucleotide or small oligonucleotide.
  • Such a method can be used when the gap between the 5' and 3' ends is 1 or more nucleotides in length, such as 1-20 nucleotides in length. For example, if a gap oligonucleotide is used, two ligation reactions are performed to seal the nicks between the oligonucleotides and the ends of the probe to form a closed circularized padlock probe.
  • the gap filler will hybridize onto the target to fill the gap.
  • the ligase then ligates together the adjoining ends of the gap filler and the ends of the probe to form a closed circularized padlock probe.
  • the gap is filled using a polymerase and nucleotide triphosphates before the probe ends are ligated together.
  • FIG. 1C shows a padlock probe following ligation. The 5' and 3' ends of the padlock probe have been joined at 24 to span gap 20 and form a closed circularized padlock probe 26. Because the padlock probe forms a double helix with the target nucleic acid molecule 28, the padlock probe interlocks with the target nucleic acid molecule 28. Following ligation, the closed circularized padlock probe 26 is tethered to the target nucleic acid molecule 28 at the location where the padlock probe hybridized to the target nucleic acid molecule.
  • the closed circularized padlock probes act as templates for the generation of amplicons, such as tandem-sequence nucleic acid molecule.
  • the closed circularized padlock probes are amplified by adding one or more replication primers and extending from the primers using a polymerase, such as DNA polymerase.
  • a polymerase such as DNA polymerase.
  • Exemplary methods for amplification include rolling-circle amplification (RCA) and PCR.
  • DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, DNA polymerase III holoenzyme, phage M2 DNA polymerase, T5 DNA polymerase, Bst DNA polymerase, and PRD1 DNA polymerase.
  • the assay mixture can be optimized for the polymerase selected, and can include deoxynucleoside triphosphates (dNTPs) and Mg "1-1" .
  • dNTPs deoxynucleoside triphosphates
  • Mg "1-1" Mg "1-1" .
  • RCA the replication of circular DNA or RNA produces many copies in tandem of a sequence complementary to the circle. The accumulation of padlock probe complements proceeds linearly over time.
  • DNA polymerase attaches at a primer sequence and produces a strand of tandem- sequence DNA from the closed circularized padlock probe. Each padlock probe that was ligated to form a closed circularized padlock probe produces a tandem-sequence DNA strand.
  • the tandem-sequence DNA generated, an amplicon is a concatamer including repeated sequences complementary to the closed circularized padlock probe, having repeating regions complementary to the spacer region and the target hybridization end regions.
  • the replication primer is an oligonucleotide, such as about 15-30 bases long, which anneals to a complementary region on the amplification target circle.
  • the primer can include at the 5' end a 4-10 nucleotide sequence that is not complementary to the target amplification circle. This non-complementary region of the primer can aid in strand displacement during replication.
  • Including a compatible helicase with the DNA polymerase can also facilitate strand displacement by uncoiling the nucleic acid molecule being amplified.
  • RCA cascade rolling circle amplification
  • two primers and a DNA polymerase having strong sfrand displacement activity such as Bst DNA polymerase, large fragment
  • the primers can recognize any sequence on the padlock probe, such as a region of the spacer.
  • primers are designed to bind to the spacer region of the padlock probe, such that amplification occurs only on ligation of the 5' and 3' ends.
  • sequences complementary to the closed circularized padlock probe can be detected and in some examples also quantified, for example by using real-time quantitative PCR (Q-PCR), semiquantitative PCR, RNase protection assays, and CRCA using energy transfer primers or molecular beacons.
  • Q-PCR real-time quantitative PCR
  • semiquantitative PCR PCR
  • RNase protection assays RNase protection assays
  • CRCA energy transfer primers or molecular beacons.
  • Real-time Q-PCR monitors the entire process of the PCR (real time) rather than merely the end product (such as in RT-PCR and competitive PCR), and thereby permits precise quantification. Further specificity is achieved by using primer pairs and a probe (such as labeled oligonucleotide probes and intercalating dyes), as discussed below.
  • the amplicon sequences do not need to be purified prior to quantitation. Indeed, amplification and detection (and in some examples also quantitation) reactions can be performed in the same reaction chamber, such as the well of a 96 well plate. However, if desired, the amplicon sequences produced from a closed circularized padlock probe can.be purified or isolated, and then quantitated. For example, primers needed to amplify a closed circularized padlock probe, and the primers and probes needed to detect and quantify the amplicon products during PCR, can be included in the same reaction chamber, along with at least two DNA polymerases, one for the amplification of the closed circularized padlock probe, and another for the real-time Q-PCR analysis of the resulting amplicons.
  • the reaction chamber can further include Mg ions and dNTPs.
  • a signal is detected in real time, such as an increase or decrease in fluorescence emission.
  • Several methods can be used to generate a detectable signal for real-time Q- PCR.
  • the detected signal is plotted as a function of cycle number, and provides a good indication of the amount of PCR product that was generated during each cycle of PCR.
  • Exemplary signals include fluorescent and radioactive signals.
  • fluorescence signals that are proportional to the amount of PCR product can be generated by fluorescent dyes that are specific for dsDNA or by sequence- specific labeled oligonucleotide probes.
  • Exemplary fluorophores that are specific for dsDNA include, but are not limited to, ethidium bromide, YO-PRO-1, and SYBR green I. All of these molecules fluoresce when associated with dsDNA exposed to the appropriate wavelength of light. For example, SYBR green I can be excited with blue light with a wavelength of about 480 nm, and its emission monitored at about 520 run. This emitted signal can be detected, and the number of amplicons produced quantitated, for example by analysis of the resulting amplification curve. When using this exemplary method, a modified oligonucleotide or hybridization probe is not needed. For sequence-specific detection, labeled oligonucleotide probes can be used.
  • the label is a fluorophore.
  • the fluorescence signal intensity can be related to the amount of PCR product by a product-dependent decrease of the quench of a reporter fluorophore, or by an increase of the fluorescence resonance energy transfer (FRET) from a donor to an acceptor fluorophore.
  • FRET fluorescence resonance energy transfer
  • FRET is the radiationless transfer of excitation energy by dipole-dipole interaction between fluorophores with overlapping emission and excitation spectra. Because the FRET and the quench efficiency are strongly dependent on the distance between the fluorophores, the PCR-product-dependent change in the distance between the fluorophore can be used to generate the sequence-specific signals.
  • sequence-specific detection formats can be used to practice the disclosed method.
  • quenchers include, but are not limited to TAMRA (6- carboxy-tetramethylrhodamine), DABCYL (4-(4-dimethylaminophenylazo)benzoic acid), and the Black Hole Quencher (BHQ, Biosearch Technologies, Novato, CA).
  • BHQ a non-fluorescent quencher (NFQ) can quench most commonly used fluorophores, and does not fluoresce itself.
  • NFQ non-fluorescent quencher
  • Donor probes are 3'-terminally labeled with a reporter fluorophore (such as 6-carboxy-fluorescein, FAM), and the acceptor probes are 5' terminally labeled with an acceptor fluorophore (such as a cyanine dye (for example Cy3 or Cy5), TET (6- carboxy-4,7,2',7'-tefrachloro-fluorescein), TAMRA; and ROX (6-carboxy-
  • a reporter fluorophore such as 6-carboxy-fluorescein, FAM
  • acceptor fluorophore such as a cyanine dye (for example Cy3 or Cy5)
  • TET 6- carboxy-4,7,2',7'-tefrachloro-fluorescein
  • TAMRA 6-carboxy-fluorescein
  • ROX 6-carboxy-fluorescein
  • N,N,N',N'-teframethylrhodamine N,N,N',N'-teframethylrhodamine.
  • the donor fluorophore is excited, so that no acceptor fluorescence is detected from unbound probes.
  • the probes hybridize adjacently on the target nucleic acid molecule (single stranded) and the excitation energy transferred from the donor to the acceptor fluorophore. This FRET is detected, and quantitation of the amplicons can be made by analysis of the resulting amplification curve.
  • 5' nuclease fluorogenic target-specific oligonucleotide probes are utilized.
  • a TaqMan probe (Applied Biosystems, Foster City, CA), which includes a reporter fluorophore at the 5' end, and quencher internally or at the 3' end.
  • An exemplary 5' reporter fluorophore is FAM and an exemplary 3' quencher fluorophore is TAMARA.
  • Intact probes do not produce a fluorescent signal, because they are quenched.
  • the TaqMan probe which is complementary to the amplicon sequence, is bound to the single-stranded PCR product like the primers.
  • Taq DNA polymerase Upon reaching the probe, Taq DNA polymerase cuts the probe, releasing the quencher from the reporter fluorophore, which now fluoresces after excitation with the appropriate wavelength of light.
  • the TaqMan probe itself hybridizes to its target-nucleic acid molecule, and the 5' to 3' exonuclease activity of Taq DNA polymerase releases the quencher as described above. The signal generated by the reporter fluorophore is detected, and quantitation of the amplicons can be made by analysis of the resulting amplification curve.
  • a target-specific oligonucleotide TaqMan probe is about 20-40 nucleotides, has a GC content of about 40-60%, has no repeated sequence motifs, and in some examples has no runs of a single nucleotide.
  • a particular TaqMan probe includes a fluorophore on its 5' end, and a minor groove- binding (MGB) moiety on the 3' end.
  • MGB minor groove- binding
  • Another example of fluorogenic target-specific oligonucleotide probes is molecular beacons.
  • Molecular beacons include a reporter fluorophore on one end, and a quencher (such as DABCYL) on the other end.
  • the probe In its free state, the probe forms a hairpin structure whereby the reporter is close to the quencher. However, during the annealing phase of the PCR, the loop can hybridize to the PCR product, thereby opening the stem and relieving the quenching. The fluorescence emitted by the reporter molecule is detected during each annealing cycle, and quantitation of the amplicons can be made by analysis of the resulting amplification curve.
  • sunrise primers commercially available as Ampliflour hairpin primers.
  • Sunrise primers include a 5 '-terminal haiipin, labeled with a reporter fluorophore and a quencher. The hairpin keeps the reporter and quencher together. As the forward primer, the sunrise primer is extended. This extended product serves as a template for the reverse primer in the next step. Eventually, the polymerase opens the hairpin, and a ds PCR product is formed, in which the reporter and quencher are separated. The fluorescence emitted by the reporter is detected, and quantitation of the amplicons can be made by analysis of the resulting amplification curve. Yet another example of fluorogenic target-specific oligonucleotide probes is scorpion primers.
  • Scorpion primers include self-complementary sequences that fonn a 5 '-terminal stem-loop structure, which the loop sequence is complementary to the amplicon sequence. The 3' end serves as the primer. The stem region is labeled with a reporter fluorophore and a quencher. The primer is extended, thereby forming a template for the reverse primer. The stem then opens and the loop binds to the product, separating the reporter and quencher. In contrast to sunrise primers, the reverse extension is blocked by a hexethylene glycol group, to ensure that the reporter of the scorpion primer remains quenched in unspecific products like primer dimers.
  • Light-up probes can also be used as fluorogenic target-specific oligonucleotide probes.
  • Light-up probes are peptide nucleic acid molecules that use thiazole orange as the fluorophore. A quencher is not required. Upon hybridization of the light-up probe with the target nucleic acid molecule, duplex or triplex molecules are formed with increased fluorescence intensity of the fluorophore.
  • RNA cleavage oligo design software is available from Applied Biosystems, and Primer3 is a free program from the Massachusetts Institute of Technology (MIT).
  • Determining the amount of template by PCR can be performed as a relative quantitation, or as an absolute quantitation (also referred to as standard-curve quantitation).
  • Relative quantitation describes changes in the amount of a target nucleic acid molecule compared to that of a control nucleic acid molecule. Quantitation can be done relative to the control(s) by subtracting the cycle threshold of the control from the cycle threshold of the target nucleic acid molecule.
  • Absolute quantitation provides a number of nucleic acid molecule targets present in the sample in relation to a specific unit.
  • a standard curve (5 or 10-fold serial dilution) of "knowns" is used to quantify the "unknown" target nucleic acid molecule of interest.
  • the unknown amount of target nucleic acid molecule can be quantified by deriving the value from a standard curve generated with known samples from any source.
  • a known sample is a plasmid ' for the target nucleic acid molecule, and the standard curve is generated based on a serial dilution of a starting amount, which is quantified, for example by using a spectrophotometer, fluorometer, or Pico Green.
  • a known sample is a synthetic oligonucleotide for the entire amplicon.
  • a known sample is a cell line with a known copy number or expression level of the nucleic acid molecule of interest.
  • the amplification curves obtained during real-time Q-PCR are evaluated.
  • the amplification step is monitored through the fluorescence of dsDNA-specific dyes (like SYBR green I) or of sequence-specific probes, such as those described above.
  • the resulting amplification curves generally have three phases: an initial lag phase, an exponential phase, and a plateau phase.
  • the template copy number can be estimated from the number of cycles needed for the signal to reach an arbitrary threshold. Ideally, the threshold intersects the amplification curve in the exponential phase, in which the signal increase correlates with product accumulation.
  • the disclosed SSA method can be used to identify agents that increase or decrease the kinetics of nucleic acid molecule strand synthesis, such as synthesis of a plus-strand, minus-strand, leading-strand, or lagging-strand.
  • an agent that decreases plus-or minus strand synthesis of a viral nucleic acid molecule is a potential anti- viral agent
  • an agent that increases plus-or minus strand synthesis of a viral nucleic acid molecule is a potential anti-viral agent, because such agents may increase the rate of replication errors, thereby mutating and inhibiting the virus.
  • an agent that decreases leading- or lagging-strand synthesis of a bacteria- or fungal-specific nucleic acid molecule is a potential anti-bacterial or anti-fungal agent, respectively.
  • an agent that decreases leading- or lagging-strand synthesis of a cancer-specific nucleic acid molecule is a potential anti-cancer agent.
  • the screening method includes contacting a sample including a target nucleic acid molecule, with one or more test agents.
  • the sample can include a cell, ⁇ such as a cell expressing the target nucleic acid molecule.
  • the cell is isolated.
  • the sample is a cell- free system that includes the target nucleic acid molecule and agents needed for its synthesis.
  • nucleic acid molecules are isolated from a cell prior to analysis. Following exposure to the test agent, the target nucleic acid molecule is amplified using the SSA method described above, wherein a decrease in synthesis of the target nucleic acid molecule in the presence of the test agent is an indication that the test agent decreases synthesis of the target nucleic acid molecule, and wherein an increase in synthesis of the target nucleic acid molecule in the presence of the test agent is an indication that the test agent increases synthesis of the target nucleic acid molecule.
  • a test agent that decreases synthesis of the target nucleic acid molecule is a candidate for use as an anti-pathogenic agent or anti- cancer therapeutic.
  • a test agent that increases synthesis of the target nucleic acid molecule is a candidate for use as a mutagenic agent or anti-viral therapeutic.
  • An increase or decrease in synthesis can be determined by a comparison to a reference standard, such as an amount of synthesis of a housekeeping nucleic acid molecule, or to a control (for example an amount of synthesis in the absence of the test agent).
  • Any suitable compound or composition can be used as a test agent, such as organic or inorganic chemicals, including aromatics, fatty acids, and carbohydrates; peptides, including monoclonal antibodies, polyclonal antibodies, and other specific binding agents, or nucleic acid molecules.
  • Therapeutic agents identified with the disclosed approaches can be used as lead compounds to identify other agents having even greater therapeutic activity.
  • the disclosed SSA method can also be used to monitor an infection in a subject, for example a subject having a pathogenic infection, such as a bacterial, viral, parasitic, or fungal infection (or combinations thereof). Similar methods can be used to monitor the effectiveness of a therapeutic agent administered to a subject, such as an anti-viral, anti-bacterial, or anti-fungal composition.
  • a pathogenic infection such as a bacterial, viral, parasitic, or fungal infection (or combinations thereof).
  • Similar methods can be used to monitor the effectiveness of a therapeutic agent administered to a subject, such as an anti-viral, anti-bacterial, or anti-fungal composition.
  • the kinetics of synthesis of a particular strand of a nucleic acid molecule or presence of a particular strand of a nucleic acid molecule is determined for a target nucleic acid molecule, such as a bacterial, viral, parasitic, or fungal nucleic acid molecule (or a combination thereof).
  • a target nucleic acid molecule such as a bacterial, viral, parasitic, or fungal nucleic acid molecule (or a combination thereof).
  • the kinetics of synthesis of a particular strand of a nucleic acid molecule or presence of a particular strand of a nucleic acid molecule can be determined for a target nucleic acid molecule following a therapy, in order to determine whether the therapy is effective, to determine whether the subject is recovering from the infection, or both.
  • the monitoring method includes amplifying a target nucleic acid molecule from the subject using the disclosed SSA method.
  • Nucleic acid molecules fi-om the subject can be obtained from any source that contains nucleic acid molecules, such as blood or saliva.
  • a decrease in the kinetics or presence of a target nucleic acid molecule indicates that the therapy is effective, that the subject is recovering from the infection, or both.
  • an increase or no change in the kinetics or presence of a target nucleic acid molecule indicates that the therapy is not effective, that the subject is not recovering from the infection, or both.
  • a change in synthesis can be determined by a comparison to a reference standard, such as an amount of synthesis of a housekeeping nucleic acid molecule, or to a control.
  • a reference standard such as an amount of synthesis of a housekeeping nucleic acid molecule
  • padlock probes allow measurements of the kinetics of minus-strand D ⁇ A transfer (probes 1 and 2), minus-strand D ⁇ A synthesis (probes 3, 4, 5 and 6), plus-strand D ⁇ A synthesis initiation at the polypurine tract (PPT; probes 2 and 7) and central polypurine tract (cPPT; probes 5 and 11), plus-strand D ⁇ A transfer (probes 6 and 8) and plus-strand D ⁇ A synthesis (probes 8, 9, 11 and 12) and detection and quantification of the central flap (probe 10).
  • Methods for generating a padlock probe to a desired sequence is known (for example see ⁇ ilsson et al, Science. 265:2085-8, 1994; Landegren and ⁇ ilsson, Ann. Med.
  • padlock probes useful for the methods disclosed herein can be prepared for any nucleic acid sequence of interest.
  • Padlock probes containing a 5 ' phosphate group were chemically synthesized by standard phosphoramidite chemistry and purified by polyacrylamide gel electrophoresis by Integrated D ⁇ A Technologies, Inc. (Coralville, IA).
  • the HIV-l padlock probes (SEQ ID ⁇ OS: 1-12) are 83-mers including a common spacer region of 49 bases: 5 ' - TTGCGACTCGTCATGTCTGAACTCTAGTGATCTTAGTGTCAGGATAGCT- 3' (SEQ LD NO: 13), with target arms directed against various minus- and plus- strand sites in pHDV-EGFP as indicated in FIGS. 2 and 3.
  • the ligation junction of each probe on the targeted strand is indicated.
  • EXAMPLE 2 Hybridization and Ligation of Padlock Probe This example describes methods used to hybrizide a padlock probe to its target nucleic acid molecule and subsequently ligate the 5' and 3' ends of the padlock probe, to form a circularized padlock probe. Briefly, after infecting cells with HIN-1, total cellular D ⁇ A (including nascent HIN-1 D ⁇ A) is isolated, denatured, and hybridized to a strand-specific padlock probe generated in Example 1. The probe termini are ligated to form a circularized probe that is amplified using cascade rolling-circle amplification (RCA). The products of this reaction can be quantified by real-time PCR using dual-labeled TaqMan probes (see Example 3).
  • RCA cascade rolling-circle amplification
  • virus particles were prepared as follows. For most studies, virus was prepared from a 293T-based cell line, which contains an undetermined number of integrated proviruses derived from pHDV-EGFP. Because the viral D ⁇ A is stably integrated into the producer cells, contamination of samples with transfected D ⁇ A was avoided. Another potential source of D ⁇ A contamination was reinfection of the viral producer cells, leading to reverse transcription and formation of viral D ⁇ A products after transfection with VS V-G- expressing plasmid. To suppress this possible source of D ⁇ A contamination, the producer cells were maintained in the presence of AZT to suppress reinfection of the producer cells. To generate virus, HIV-GFP2 cells were plated at 2 x IO 6 cells per 100-mm- diameter dish.
  • the cells were pretreated with fresh medium containing 1 ⁇ M AZT for 10 hours before transfection with 4 ⁇ g of NSN-G- expressing plasmid (Yee et al, Proc. Natl Acad. Sci. USA 91 :9564-8, 1994) using the CalPhos transfection kit (Clontech), washed once with 6 ml of medium and further treated with fresh medium containing 1 ⁇ M AZT. After 7 hours, transfection solution containing 1 ⁇ M AZT was removed by washing cells once with AZT-free medium and fresh medium was then added to the cells. The virus was collected 17 hours later and concentrated 10-fold by ultracentrifugation at 20,000 x g for 1 hour.
  • the virus was treated with D ⁇ asel (30 units/ml, 10 n M final concentration of MgCl ) for 1 hour at room temperature, divided into 1-ml aliquots and frozen at -80°C.
  • Virus was also produced following transient transfection of 293T cells with pHDV-EGFP or pHJN-GFP-cPPT and VSV-G-expressing plasmid, using the MBS Mammalian Transfection Kit (Sfratagene).
  • the treatment of virus produced from the transfected cells with D ⁇ asel (105 U/ml, 10 n M MgCl 2 , 3 hours at room temperature) also resulted in very low background levels, indicating little contamination with transfected D ⁇ A.
  • a padlock probe was hybridized to the 5' and 3' ends of its denatured target D ⁇ A and circularized by freatment with a thermostable D ⁇ A ligase (New England Biolabs, Beverly, MA).
  • the 10- ⁇ l reaction mix contained 1 x Taq DNA ligase buffer (New England Biolabs), IO 9 molecules of padlock probe, 12 units of Taq DNA ligase, and 4 ⁇ l of sample containing total cellular DNA extracted from infected cells (see above).
  • EXAMPLE 3 Rolling Circle Amplification and Quantification with Real-Time PCR Following hybridization and ligating the padlock probe, the closed circularized padlock probes were amplified using rolling circle amplification (RCA), and the products quantified using real-time PCR.
  • RCA rolling circle amplification
  • Ligation reactions obtained in Example 2 were diluted to 100 ⁇ l with water and 5 ⁇ l aliquots were added to a reaction mixture (25 ⁇ l, final volume) containing 20 n M Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 200 ⁇ M dNTPs, 500 nM each RCA-23 (5'- ACTAGAGTTCAGACATGACGAGT-3 '; SEQ ID NO: 14) and REV-21 (5'- GATCTTAGTGTCAGGATAGCT-37 SEQ ID NO: 15) primers, 100 nM dual- labeled probe (see FIG.
  • the primers SEQ ID NOS: 14 and 15 were chemically synthesized by Integrated DNA Technologies, Inc. with a 5'-OH and used without further purification except desalting. An initial incubation was performed at 63 °C for 8 minutes to perform the two-primer rolling circle amplification reaction (thereby amplifying the closed circularized padlock probes), followed by a 2-minute incubation at 94°C, which effectively denatured Bst DNA polymerase, while simultaneously activating
  • Platinum Taq DNA polymerase used in real-time PCR used in real-time quantitative PCR.
  • the dual-labeled probes (synthesized by Integrated DNA Technologies, Inc.) used for real-time quantitative PCR are represented by the underlined sequences of their cognate padlock probes listed in FIG. 2, and in each case were end-labeled with 5'-FAM (6- carboxyfluorescein) and 3 '-TAMRA (6-carboxy-tetramethyl-rhodamine).
  • 5'-FAM 6- carboxyfluorescein
  • 3 '-TAMRA 6-carboxy-tetramethyl-rhodamine
  • Copy numbers of target DNA were calculated using the ABI PRISM 7700 Sequence Detection System software based on standard curves generated with known amounts of pHDV-EGFP (for example see FIGS. 3B and 3C). The correlation coefficients were >0.99, indicating that the output signals are linear with respect to input DNA.
  • EXAMPLE 4 Determination of the Rate of HIV-Minus-Strand DNA Synthesis This example describes methods used to determine the rate of synthesis of an HIV- 1 -minus-strand. Similar methods can be used to determine the rate of synthesis of any strand of any target nucleic acid molecule, using the appropriate padlock probes and primers. In addition, the rate of plus- and minus-strand synthesis (or leading- and lagging-strand synthesis) can be compared using the methods described in this and in Example 7. A time-course of infection with HIV-l vector pHDV-EGFP (Unutmaz et al, J. Exp. Med. 189:1735-1746, 1999) was performed as follows.
  • the retroviral vector pHDV-EGFP expresses HIV-l Gag-Pol but does not express Vif, Vpr, Vpu, Env or Nef.
  • VSV-G vesicular stomatitis virus envelope protein G
  • VSV-G vesicular stomatitis virus envelope protein G
  • 2 x IO 6 or 6 x IO 5 293T cells (human kidney cells, American Type Culture Collection, Manassas, VA) were plated per 100-mm or 60-mm-diameter dish, respectively, the day before infection.
  • a 1-ml tube of virus was thawed and diluted 20-fold in fresh media.
  • a diluted virus solution (2 ml or 0.6 ml) was used to infect each 100-mm or 60-mm-diameter dish, respectfully. After infection for 30 minutes, the virus was removed, cells were washed twice with 4 mis of phosphate buffered saline to remove any residual virus, and fresh media was added to the plate.
  • Total DNA (including nascent HIV-l DNA) was isolated using the QiaAmp DNA Blood Kit (Qiagen) and resuspended in 400 ⁇ L of H 2 O.
  • SSA analysis was done four times for each primer-probe set using 4 ⁇ L of DNA.
  • the accumulation of minus-strand DNA products was determined with padlock probes 3, 5, and 6 (SEQ LD NOS: 3, 5 and 6, respectively) at each time point (see map above graph in FIG. 4A). Analysis of reverse transcription products after 6, 12, and 24 hours indicated that the amounts of products peaked at 6 hours.
  • the copy numbers of the reverse transcription products that accumulated 6 hours after infection was set to 100% and the accumulation of the products at earlier time points was expressed as a percentage of the copy number of products that accumulated at the 6-hour time point.
  • samples were measured by real-time PCR, using a primer-probe set specific for the human porphobilinogen deaminase (PBGD) gene (Genbank access number M95623).
  • the forward primer was 5 '-AGGGATTCACTCAGGCTCTTTCT-3 '
  • Table 1 Time course for synthesis of HIN-1 plus-strand products detected by probe 9 a
  • b Copy numbers were calculated based on standard curves generated with known amounts of pHDV-EGFP in the presence of 25 ng of uninfected 293T cell D ⁇ A.
  • C PBGD copy numbers were determined as described herein and normalized to the 0-h value, set as 1.00 (actual copy number was 313,385).
  • d HTV-l copy numbers were normalized by multiplying the original copy numbers (column 2) by the normalized values for PBGD (column 3).
  • e Final HIV-l copy numbers were calculated by subtracting the 0-h value (313) from the normalized copy numbers (column 4). Values were calculated as the percentage of the final copy numbers divided by the 6-h copy number (2,065,930) and placed in column 2 of Table 2.
  • SSA analysis was performed with probes 3, 5 and 6; two representative results are shown in FIG. 4A.
  • the distance between the curves representing the kinetics of accumulation of the products provided an estimate of the average time required for reverse transcriptase to copy the template between two probes.
  • the average distance between the linear portions of the curves was determined by measuring the distance when the amounts of products accumulated were 5, 10, 20 and 30% (shown as arrows).
  • the average time required for minus-strand DNA synthesis to be extended fi-om probe 3 to probe 6 (7,031 nucleotides (nt)) was approximately 105 and 109 minutes for time courses 1 and 2.
  • the average rate of minus-strand DNA synthesis was not uniform throughout the course of replication because the rate between probes 5 and 6 (101 + 30 nt/minute), wliich represented the 5 ' half of the genome, was approximately twofold faster than the rate between probes 3 and 5 (52 ⁇ 23 nt/minute), wliich represented the 3' half of the genome (P ⁇ 0.002; t test).
  • EXAMPLE 5 Kinetics of HIV-l minus- and plus-strand DNA Transfer This example describes methods used to determine the amount of time needed to transfer HIV-l minus- and plus-strand DNA using the disclosed SSA method.
  • the kinetics of minus-strand DNA transfer (FIG. 4B, left graph) were determined using the same methods described for minus-strand DNA synthesis (Example 4) except that the accumulation of products detected by probes 1 and 2 (see map above the left graph of FIG. 4B) were determined over a 3 hour time course.
  • the kinetics of product accumulation detected by probes 1 and 2 and probes 6 and 8 were compared to estimate the average time for HIN minus- and plus-strand D ⁇ A transfer, respectively, using the methods described above.
  • plus-strand DNA transfer was determined by analyzing the accumulation of products detected by probes 6 and 8 over a 6 hour time course, which are located 3' of the PBS and are specific for the minus and plus strand, respectively (FIG. 4B, right panel). On average, plus-strand DNA transfer took about 28 minutes. Since about 100 nt of DNA synthesis occurred in about 1 minute during progression of reverse transcription from probe 6 to probe 8, a longer delay of about 27 minutes (28 minutes - 1 minute) was associated with plus-strand DNA transfer.
  • EXAMPLE 6 Kinetics of HIV-l plus-strand DNA synthesis Initiation This example describes methods used to determine the kinetics of plus-strand initiation at the PPT and cPPT of HIV-l. Initiation of plus-strand DNA synthesis at the PPT (FIG. 4C, left graph) was determined by the same methods described above for minus-strand DNA synthesis except that the accumulation of products was detected with probes 2 and 7 (see map above FIG. 4C) were compared over a 3 hour time course. These probes are located 3 ' of the PPT and are specific for the minus- and plus-strand, respectively. It took about 12 minutes for progression of reverse transcription fi-om probe
  • FIG. 4C right graph
  • EXAMPLE 7 Determination of the Rate of HIV-Plus Strand DNA Synthesis This example describes methods used to determine the kinetics of HIN-1 plus-strand synthesis. Similar methods can be used to dete ⁇ nine the rate of synthesis of any strand of any target nucleic acid molecule, using the appropriate padlock probes and primers. To determine the kinetics of plus-strand D ⁇ A synthesis in vivo, the kinetics of accumulation of products detected by plus-strand-specific probes S, 9, 11 and 12 (FIG. 5A) after infection with wild-type HDN-EGFP were compared using the methods described above.
  • probes 11 and 12 accumulated with faster kinetics than products detected by probes 8 and 9, indicating that plus- strand D ⁇ A synthesis initiation at the cPPT and subsequent plus-strand D ⁇ A synthesis through probe 11 and 12 sites occurred about 30 minutes before minus- strand D ⁇ A synthesis reached the PBS to allow plus-strand D ⁇ A transfer and synthesis of products detected by probes 8 and 9 (FIG. 5 A).
  • Probes 8 and 9 are 5' of the cPPT and are separated by 4,060 nt whereas probes 11 and 12 are 3' of the CTS and are separated by 2,759 nt.
  • probes 8 and 9 accumulated with very similar kinetics, as did those of probes 11 and 12, indicating a very fast rate of plus-strand D ⁇ A synthesis or the presence of multiple sites for D ⁇ A synthesis initiation.
  • a cPPT * mutant was generated by introducing four purine-to-pyrimidine substitutions within the cPPT sequence as follows.
  • a restriction fragment (Sbfi-SaH) containing the cPPT was subcloned into the pHDV- EGFP plasmid to generate pHIV-GFP-cPPT " and sequenced to confirm the presence of the desired mutations and absence of undesired mutations.
  • the mutations in the cPPT did not significantly influence its ability to complete a single cycle of replication as determined by analysis of GFP expression in infected cells.
  • the products detected with probe 12 accumulated with faster kinetics than the products detected by probe 11 (FIG. 5B), indicating that plus-strand DNA synthesis was initiated at one or more sites between probes 11 and 12.
  • Plus-strand DNA synthesis initiation at the cPPT was abrogated by the mutations in the cPPT, because probe 11 products accumulated with kinetics similar to those for probe 8 and 9 rather than with the faster kinetics observed when the cPPT was wild type (FIG. 5 A).
  • the copy numbers of reverse transcription products detected with probe 1 at 6 hours were set to a value of 1 (average value: 2-6 x IO 6 copies) and the copy numbers of products detected with various minus- and plus-strand probes (see FIG.
  • Probe 10 was designed to detect the central flap (FIG. 3), and two copies of probe 10 product were expected to be generated during reverse transcription; one copy would result from initiation at the cPPT and a second copy would result from plus-strand strong-stop DNA transfer followed by extension of plus-strand DNA synthesis, leading to displacement of the DNA initiating at the cPPT.
  • the rate of synthesis of a particular sfrand of a nucleic acid molecule can be selectively determined.
  • the rate of nucleic acid molecule synthesis can be compared between particular nucleic acid molecules using the methods described herein.
  • the method also permits quantification of each nucleic acid strand during reverse transcription and used it to measure the abundance of reverse transcription products generated at distinct steps over the time course of a viral infection.
  • EXAMPLE 8 Effect of Reverse Transcriptase Inhibitors on HIV DNA Synthesis
  • RT reverse transcriptase
  • RT reverse transcriptase
  • Similar methods can be used to determine the rate of synthesis of any strand of any target nucleic acid molecule in the presence of one or more test agents (such as a therapeutic agent) or compositions including one or more test agents, using the appropriate padlock probes and primers.
  • RT inhibitors 2',3'-dideoxyinosine (ddl), 3'-deoxy-2',3'-didehydrothymidine (d4T) and AZT
  • ⁇ RTI nonnucleoside RT inhibitor
  • Efavirenz Efavirenz
  • RT inhibitors concentrations were used: EFV, 9 nM; ddl, 50 ⁇ M; d4T, 4 ⁇ M; AZT, 1 ⁇ M.
  • infection efficiencies were reduced 95-99% relative to a minus- inhibitor control infection, as determined by flow cytometry 36 hours after infection.
  • the RT inhibitors had a minimal effect on the early minus-strand DNA products detected by probe 3 (FIG. 6A, left panel); in contrast, the late minus-strand DNA products detected by probe 6 accumulated to variable levels (FIG. 6A, right panel).
  • the probe 6 products In the presence of AZT and d4T, the probe 6 products accumulated to approximately 5% of the control levels; in comparison, the probe 6 products accumulated to approximately 15 and 55% of the control in the presence of EFV and ddl, respectively.
  • minus-strand DNA synthesis was substantially inhibited by AZT and d4T, moderately inhibited by EFV, and minimally inhibited by ddl.
  • the accumulation of plus-strand DNA products detected by probes 8 and 9 (FIG. 6B, left panels) was decreased to a similar extent as compared to the probe 6 products (FIG. 6A, right panel), whereas the accumulation of products detected with probes 11 and 12 (FIG.
  • ddl and EFV only decreased minus-strand DNA synthesis by 45% and 85%, respectively, at concentrations that inhibited viral replication by >95%, indicating that these RT inhibitors also impaired plus-strand DNA synthesis, hihibition of plus-strand DNA synthesis could not be observed because it is initiated at multiple sites and reduction in plus-strand products is not cumulative, as is the case for minus-strand DNA synthesis.
  • EXAMPLE 9 Determining the Kinetics of HIV-l Replication in Primary Cells This example describes methods used to determine the kinetics of HIV-l replication of a minus-strand in a primary cell. One skilled in the art will appreciate that similar methods can be used to determine the kinetics of replication of any nucleic acid sfrand from any primary cell, such as a cell obtained from a human or veterinary subject.
  • Peripheral blood mononuclear cells (PBMCs) and macrophages were isolated from healthy donors through Histopaque gradients (Sigma), activated by 2 .
  • ⁇ g/ml phytohemagglutimn for 3 days, and maintained in RPMI medium containing 10% fetal bovine serum and 200 units/ml recombinant interleukin-2 for 3-4 days.
  • Macrophages were grown from elutriated monocytes on non-tissue culture-treated plates in RPMI medium plus 10% human AB serum for 7 days.
  • CD4 + cells were purified from the PBMCs using antibodies that specifically recognize these cells (Dynabeads kit, Dynal Corp.) according to the manufacturer's instructions. After isolation, the cells were resuspended in RPMI media with IL-2 at a concentration of about 1 million cells per ml.
  • PBMC cells were infected with HIN-1 vector pHDV-EGFP as described in Example 4.
  • the methods and padlock probes (probes 3 and 6) described in the examples above were used, except that the incubation was increased to 1 hour.
  • primary cells grow in suspension, in contrast to the 293T cells used in the above examples which attach to plates. Therefore, washing of the cells to replace media or remove virus used centrifugation of the cells instead of rinsing cells attached to a plate. The kinetics was measured over a period of 10 hours. However, longer or shorter periods can be used.
  • An example of the time course of infection is provided in FIG. 7.
  • primary cells containing nucleic acids can be obtained from a subject, such as a human subject, using routine methods known in the art.
  • primary cells can be obtained from a sample from a subject, such as a blood sample or saliva sample, hi one example, human PBMCs are collected by apheresis and subsequently purified. The PBMCs can be used as a source of other cells, such as CD4+ cells.
  • PBMCs can also be isolated by counterflow centrifugal elutriation or using a Ficoll-Paque Plus isolation solution (Amersham Pharmacia Biotech, Uppsala, Sweden).
  • PBMCs can be isolated from a 1 ml sample of venous EDTA-blood by density gradient centrifugation (LymphoPrep; AXIS-SHIELD Poc AS, Oslo, Norway), washed in phosphate-buffered saline, and resuspended in 5 ml of culture medium.
  • the cell concentration can be determined by cell counting (such as a Coulter ZF cell counter, Analis, Namur, Belgium).
  • PBMCs are diluted in medium to the desired concentration, such as 10° cells/ml and spun to obtain pellets which can be stored at -80°C until use.
  • EXAMPLE 10 Determining the Kinetics of Nucleic Acid Replication in Mutant Pathogens This example describes methods that can be used to determine the effects of one or more mutations in a pathogen, such as a virus, bacterium, fungus, or parasite. Although particular examples are described for HIV, one skilled in the art will recognize that padlock probes can be generated for any pathogen of interest and the methods disclosed herein utilized.
  • a pathogen such as a virus, bacterium, fungus, or parasite.
  • a pathogenic protein such as a viral protein (for example RT, NC, Vif, Vpr, Nef, and so on) or c ⁇ -acting elements (such as PBS, PPT, cPPT, CTS, and so on), on the kinetics of various steps nucleic acid replication.
  • a pathogenic protein for example RT, NC, Vif, Vpr, Nef, and so on
  • c ⁇ -acting elements such as PBS, PPT, cPPT, CTS, and so on
  • the rate of minus-strand or plus-strand synthesis in mutant HIV-1 can be determined using the methods and probes described in Examples 4 and 7, respectively, but instead of using HIN-1 vector pHDV-EGFP, an HIV-l vector containing the desired mutation would be used.
  • EXAMPLE 11 Determining the Rate of Synthesis of a Pathogenic Nucleic Acid
  • a pathogenic nucleic acid such as a bacterial, parasitic, or fungal nucleic acid.
  • the desired pathogen such as a bacteria, parasite, or fungus can be grown in culture using standard methods known in the art. Samples of the pathogen can be obtained during the desired time course, such as every 10 minutes, every 30 minutes, every 60 minutes, every 120 minutes, every 4 hours, every 12 hours, every 24 hours, or combinations thereof.
  • Nucleic acids, such as DNA are isolated from the pathogen using methods known in the art.
  • the isolated nucleic acids are then used in the disclosed SSA analysis (for example as described in Examples 1-9), along with the appropriate padlock probes and primers.
  • methods of generating padlock probes and primers are known in the art.
  • the amount of nucleic acid strand synthesis of a particular strand of a nucleic acid can be compared to a control, such as an amount of synthesis observed in a pathogenic housekeeping gene (such as genes involved in basic metabolic functions, such as glycolysis, the Krebs cycle, the pentose phosphate pathway, the biosynthesis of aromatic amino acids, phenylpropanoids, and ethylene), an amount of synthesis of a particular strand of a nucleic acid observed prior to a treatment (for example an anti- fungal or antibacterial treatment), or an amount of synthesis of a particular strand of a nucleic acid observed at a previous time, or combinations thereof.
  • a control such as an amount of synthesis observed in a pathogenic housekeeping gene (such as genes involved in basic metabolic functions,
  • Pathogenic housekeeping genes are known in the art, and ideally have expression levels that remain relatively constant in different experimental conditions.
  • EXAMPLE 12 Determining the Rate of Synthesis of a Mammalian Nucleic Acid
  • a mammalian nucleic acid such as a mammalian nucleic acid involved in carcinogenesis, such as ⁇ 53 or BRCA1.
  • the disclose methods can be used to monitor the effect of an anti-neoplastic agent.
  • a sample containing nucleic acids is obtained from the mammalian subject, such as a human or veterinary subject.
  • DNA is isolated from the sample prior to conducing the assay.
  • cells in the sample are merely lysed prior to the assay.
  • samples are obtained over a period of time, such as hourly, daily, weekly, bi-weekly, monthly, bi-monthly, yearly, bi-yearly, or combinations thereof.
  • the lysed cells or isolated nucleic acids are then used in the disclosed SSA analysis (for example as described in Examples 1-9), along with the appropriate padlock probes and primers.
  • methods of generating padlock probes and primers are known in the art.
  • the amount of leading- or lagging-strand synthesis observed in the subject can be compared to a control, such as an amount of leading- or lagging-strand synthesis observed in a housekeeping gene (such as porphobilinogen deaminase (PBGD); mitochondrial ATP synthase 6 (mATPsy ⁇ ); and glyceraldehyde-3- phosphate dehydrogenase (GAPDH)), an amount of leading- or lagging-strand synthesis observed in the subject prior the subject receiving a treatment (for example an anti-neoplastic treatment), or an amount of leading- or lagging-strand synthesis observed in the subject at a previous time, or combinations thereof.
  • PBGD porphobilinogen deaminase
  • mATPsy ⁇ mitochondrial ATP synthase 6
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • EXAMPLE 13 Assay for Measuring the Effect of Restriction Factors on Replication of HIV-l This example describes methods used to measure the kinetics of reverse transcription of HIV-l in cells expressing the host restriction factor TRIM5 alpha (Stremlau et al, Nature 427:848-53, 2004).
  • Reverse transcription products measured by probes 3 and 6 were reduced to 5% of human or empty- vector control levels in the cells containing the rhesus TRTM5alpha factor. These results confirm that the restriction factor from rhesus monkeys blocks either initiation of or an early step in the reverse transcription process during infection by HIV-l, while the factor from human cells is ineffective at blocking replication. The effects of such factors on the kinetics of reverse transcription can also be measured by SSA.
  • EXAMPLE 14 Diagnosing or Monitoring an Infection
  • a subject having a pathogenic infection such as a bacterial, viral, parasitic, or fungal infection (or combinations thereof).
  • Similar methods can be used to monitor the effectiveness of a therapeutic agent administered to a subject, such as an anti-viral, anti-bacterial, or anti-fungal composition (or combinations thereof).
  • the kinetics of synthesis of a leading- or lagging-strand of a nucleic acid molecule or presence of a particular strand of a nucleic acid molecule is determined for a target nucleic acid molecule, such as a bacterial, viral, parasitic, or fungal nucleic acid molecule (or a combination thereof).
  • a target nucleic acid molecule such as a bacterial, viral, parasitic, or fungal nucleic acid molecule (or a combination thereof).
  • a subject is suspected of having an infection, and the method is used to diagnosis the subject as having a particular infection.
  • a subject is receiving or received treatment for an infection, and the SSA method is used to determine whether the therapy is effective, to determine whether the subject is recovering from the infection, or both.
  • the diagnostic and monitoring methods include amplifying a target nucleic acid molecule from the subject using the disclosed SSA method.
  • Nucleic acid molecules from the subject can be obtained from any source that contains nucleic acid molecules, such as blood or saliva.
  • the presence of nucleic acid replication by the pathogen of interest indicates that the subject is infected with the pathogen of interest.
  • a decrease in the kinetics or presence of a target nucleic acid molecule indicates that the therapy is effective, that the subject is recovering from the infection, or both.
  • an increase or no change in the kinetics or presence of a target nucleic acid molecule indicates that the therapy is not effective, that the subject is not recovering from the infection, or both.
  • a change in synthesis such as an increase or decrease (such as an increase or decrease of at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, or even at least 95% as compared to a standard or a control), can be determined by a comparison to a reference standard, such as an amount of synthesis of a housekeeping nucleic acid molecule, or to a control, such as an amount of leading- or lagging-strand synthesis observed in the subject prior the subject receiving a treatment (for example an anti-pathogenic treatment), or an amount of leading- or lagging-strand synthesis observed in the subject at a previous time, or combinations thereof.
  • a reference standard such as an amount of synthesis of a housekeeping nucleic acid molecule
  • a control such as an amount of leading- or lagging-strand synthesis observed in the subject prior the subject receiving a treatment (for example an anti-pathogenic treatment), or an amount of leading- or lagging-strand synthesis observed in the subject at
  • EXAMPLE 15 Screening Assay for Agents that Alter Nucleic Acid Synthesis
  • This example describes methods that can be used to screen test agents for their ability to modify synthesis of a nucleic acid molecule, such as increasing or decreasing such synthesis. Such methods can be used to identify anti-pathogenic agents, such as anti-viral, anti-fungal, anti-bacterial, and anti-parasitic agents.
  • the disclosed SSA method can be used to monitor specific steps in transcription and reverse transcription, the method can be used to identify agents that interfere with or enhance specific steps in transcription and reverse transcription.
  • a novel method for amplifying a particular strand of a target nucleic acid molecule is provided.
  • screening assays can be used to identify and analyze agents that decrease or increase synthesis of a particular strand of a target nucleic acid molecule, such as a plus-strand, minus- strand, leading-strand, or lagging-strand.
  • screening assays can be used to identify agents that decrease synthesis of a particular strand of a target nucleic acid molecule, such as a nucleic acid sequence associated with infection (such as a viral nucleic acid molecule) or a nucleic acid sequence associated with cancer.
  • a nucleic acid sequence associated with infection such as a viral nucleic acid molecule
  • the present disclosure is not limited to the particular examples disclosed herein.
  • Agents identified via the disclosed assay can be useful, for example, in selectively or non-selectively decreasing or even inhibiting synthesis of a particular sfrand of a target nucleic acid molecule by more than an amount of synthesis in the absence of the agent, such as a decrease of at least about 10%, at least about 20%, at least about 50%, or even at least about 90%.
  • This decrease in synthesis can serve to ameliorate symptoms associated with a disorder.
  • a decrease in synthesis of a viral plus- or minus-strand can serve to ameliorate symptoms associated with viral infection, such as fever.
  • a decrease in synthesis of a leading- or lagging-strand can serve to ameliorate symptoms associated with cancer, such as reduction in the size of a tumor.
  • Agents identified via the disclosed assay can also be useful, for example, in increasing synthesis of a particular strand of a target nucleic acid molecule by more than an amount of synthesis in the absence of the agent, such as a increase of at least about 10%, at least about 20%, at least about 50%, or even at least about 90%.
  • This increase in synthesis can serve to ameliorate symptoms associated with a disorder.
  • an increase in synthesis of an HIV-l plus- or minus-strand can serve to increase the mutation rate of HIV- 1, ultimately resulting in reducing viral loads, and ameliorate symptoms associated with viral infection, such as fever.
  • the basic principle of the assay system used to identify agents that modulate synthesis of a target nucleic acid molecule involves preparing a reaction mixture containing the target nucleic acid molecule.
  • a reaction mixture containing the target nucleic acid molecule.
  • there is more than one target nucleic acid sequence for example at least two, at least three, at least four, or at least 10 different target nucleic acid sequences.
  • the mixture is then exposed to the test agent.
  • a cell-free assay is used.
  • the reaction mixture is an in vitro system that includes the target nucleic acid molecule as well as other proteins and agents needed for synthesis.
  • the test agent is then added to the reaction mixture, and its effect on synthesis dete ⁇ nined using the disclosed SSA method.
  • Intact cells can also be used to screen test agents for their ability to increase or decrease synthesis.
  • a cell expressing the target nucleic acid molecule(s) for example a recombinant cell expressing a recombinant target nucleic acid molecule, can be used to screen for an agent that increases or decreases synthesis using the SSA methods disclosed herein.
  • the target nucleic acid sequence is cloned into a vector, which is used to transfect the cell. Controls are incubated without the test agent or with a placebo. Exemplary controls include agents known not to alter synthesis of the target nucleic acid molecule. The kinetics of synthesis of the target strand is then dete ⁇ nined using the SSA methods disclosed herein.
  • test agents found to increase or decrease synthesis of the target sfrand of the nucleic acid molecule can be formulated in therapeutic products (or even prophylactic products) in pharmaceutically acceptable formulations, and used for specific freatment or prevention of an infection.

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Abstract

Cette invention concerne un nouveau procédé permettant d'amplifier spécifiquement un brin particulier d'une molécule d'acide nucléique désirée, appelé amplification spécifique à un brin (SSA). Cette invention concerne également des procédés permettant de cribler des agents d'essais, de surveiller une infection et de surveiller l'efficacité d'un agent thérapeutique à l'aide du procédé spécifique à un brin.
PCT/US2004/039666 2003-12-04 2004-11-24 Amplification specifique a un brin Ceased WO2005061735A2 (fr)

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US20250051842A1 (en) * 2022-05-11 2025-02-13 10X Genomics, Inc. Compositions and methods for gene expression library analysis
US12467081B2 (en) 2020-11-23 2025-11-11 Pleno, Inc. Encoded endonuclease assays
US12497651B2 (en) 2023-01-05 2025-12-16 Pleno, Inc. Encoded dual-probe endonuclease assays

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JP4108118B2 (ja) * 1993-03-26 2008-06-25 ジェン−プローブ・インコーポレイテッド ヒト・免疫不全ウイルス１型の検出
CA2439477C (fr) * 2001-02-27 2014-04-15 Virco Bvba Amplification de sonde circulaire a l'aide d'amorces de transfert d'energie

Cited By (4)

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Publication number Priority date Publication date Assignee Title
US12467081B2 (en) 2020-11-23 2025-11-11 Pleno, Inc. Encoded endonuclease assays
US20250051842A1 (en) * 2022-05-11 2025-02-13 10X Genomics, Inc. Compositions and methods for gene expression library analysis
US12325877B2 (en) * 2022-05-11 2025-06-10 10X Genomics, Inc. Compositions and methods for gene expression library analysis
US12497651B2 (en) 2023-01-05 2025-12-16 Pleno, Inc. Encoded dual-probe endonuclease assays

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