WO2010011901A1

WO2010011901A1 - Detection of rna-dna duplex molecules

Info

Publication number: WO2010011901A1
Application number: PCT/US2009/051657
Authority: WO
Inventors: Joe Hernandez; Venkatakrishna Shyamala; Colette Cote; Nabarun Chakraborty
Original assignee: Innovative Biosensors, Inc.
Priority date: 2008-07-24
Filing date: 2009-07-24
Publication date: 2010-01-28

Abstract

Provided are methods and compositions for detecting a RNA-DNA duplex in a sample.

Description

Detection of RNA-DNA Duplex Molecules

BACKGROUND

Nucleic acid detection assays arc useful is in many fields. Such assays are valuable in diagnosing human and animal maladies and are also valuable in research and commercial activities.

SUMMARY

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration showing example aspects of the described methods for detecting a RNA-DNA duplex in a sample.

Figure 2 is a graph showing results of relative light units (RLU) over time of an example duplex CANARY® protocol.

DETAILED DESCRIPTION Methods for detecting a RNA-DNA duplex in a sample are described. A sample refers to any composition or solution comprising a RNA-DNA duplex, or in need of testing to determine whether it comprises a RNA-DNA duplex. A RNA- DNA duplex can be formed in a sample comprising a DNA molecule or molecules by contacting the DNA with a test RNA molecule or molecules that selectively hybridizes at stringent conditions to the DNA molecule. Similarly, a RNA-DNA duplex can be formed in a sample comprising a RNA molecule or molecules by contacting the RNA with a test DNA molecule or molecules that selectively hybridizes at stringent conditions to the RNA molecule. Optionally, a plurality of test RNA or DNA molecules can be hybridized to one or more target DNA or RNA molecule to form a DNA-RNA duplex with multiple DNA or RNA molecules and/or multiple DNA-RNA duplexes wherein the DNA-RNA duplexes include different DNA or RNA molecules. The hybridizing portion of the hybridizing nucleic acids can be at least 10 (e.g., 20, 25, 30, 50, 100, 1000, or 2000) nucleotides in length. Optionally, the hybridizing portion of the hybridizing nucleic acid is 10, 30 or 2000 nucleotides in length. The hybridizing portion of the a test RNA or DNA molecule can be at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding a target RNA or target DNA molecule. Hybridization of a test nucleic acid to a target nucleic acid can be performed under stringent conditions. Nucleic acid duplex stability can be expressed as the melting temperature or Tm, which is the temperature at which a test nucleic acid dissociates from a target nucleic acid. This melting temperature can be used to define stringency conditions. If a target sequence is substantially identical to a test sequence, then the lowest temperature at which only homologous hybridization with a particular concentration of salt (e.g., SSC or SSPE) can be determined. Assuming that a 1% mismatch results in a I⁰C decrease in the Tm, the temperature of the final wash in the final hybridization reaction is reduced accordingly (for example, if test sequences having > 95% identity with the target are sought, the final wash temperature is decreased by 5⁰C). The parameters of salt concentration and temperature can be varied. Additional guidance regarding such sample conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubcl ct al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Optionally, to prepare RNA-DNA duplex, varying concentrations of RNA with ssDNA in transcription buffer from Ambion® mMessage Machine Kit [0.1X final] (Applied Biosystems, Austin, TX), and 0.65U RNase Inhibitor (NEB #AM2682) (Applied Biosystems, Austin, TX) can be mixed. The reagent mixture can be incubated at 95⁰C 2 minutes to denature the nucleic acid, and then at 65⁰C 30 minutes followed by 5O⁰C 30 minutes for annealing nucleic acid. The reaction mix can be diluted in assay buffer for use in the CANARY® (Innovative Biosensors, Rockville, MD) assay. Also, for use as controls, mixes can be prepared with RNA alone, ssDNA alone and dsDNA. Thus, a target DNA or RNA molecule present in a sample can be selectively hybridized with a test RNA or DNA molecule. Optionally, the test RNA or DNA molecule is complementary to a portion of the target RNA or DNA molecule and hybridizes along the full length of the target RNA or DNA molecule. Optionally, the test RNA or DNA molecule is complementary to a portion of the target RNA or DNA molecule and hybridizes along a portion of the target RNA or DNA molecule.

The sample comprising a RNA-DNA duplex can be contacted with a sensor cell expressing on its sensory cell surface a RNA-DNA binding antibody or antibody fragment. The antibody or antibody fragment binds with the RNA-DNA duplex in the sample and the binding of the sensor cell with the RNA-DNA duplex is detectable. The detected binding indicates the presence of a RNA-DNA duplex in the sample. Sensor cells, and methods and devices for using the same, are described in U.S. Patent 6,087,114, 6,248,542, 5,541,309, 5,360,728, 5,139,973 and 5,126,276, which are incorporated herein in their entirety. Figure 1 is a schematic illustration showing example aspects 100 of the described methods for detecting a RNA-DNA duplex in a sample. A target DNA or RNA 102 is provided. If the target to be detected is DNA, a test RNA molecule is contacted with the DNA target to form a RNA-DNA duplex sample. If the target to be detected is RNA, a test DNA molecule is contacted with the RNA target to from a RNA-DNA duplex sample. The test RNA or DNA molecule can be complementary to the target DNA or RNA molecule along both the full length (104 and 110) of the test and target molecule. In other examples, the test RNA or DNA molecule can be complementary to portions of the target DNA or RNA molecule (106 and 108). In this latter case, the test RNA or DNA molecule can be shorter than the target DNA or RNA molecule. In either case, a RNA-DNA duplex 108 or 1 10 can be contacted with a sensor cell 116. The sensor cell can comprise a RNA-DNA duplex binding antibody or fragment 118 on its surface. When a duplex (108 and/or 110) is bound by the antibody or fragment 118, as shown at 114 and 112, the sensor cell can produce light which can be detected to indicate presence of the target RNA or DNA in the sample. The sensor cell and the RNA-DNA duplex can be contacted in a liquid matrix. For example, liquid matrix can include, but is not limited to, homogeneous liquid including organic (alcohols, benzene, acids etc.) and inorganic (water etc.) liquid, and heterogeneous liquid constituting one or more organic or inorganic solutes or liquids, which is or are dissolved or mixed or suspended into another organic or inorganic liquid. Optionally, a solid matrix can be incorporated to facilitate higher assay sensitivity. Examples of solid matrices include, but are not limited to, plates, slides, dishes, beads, particles, cups, strands and chips.

The given liquid matrix can be used to transfer a duplex and biosensor cell and to provide a platform for signal generation. A solid surface can be introduced to strategically concentrate duplex target in order to obtain higher sensitivity. For instance, duplexes can be captured first immunologically or mechanically. The captured cells can then presented to the sensor cells for detection.

Sensor cells include biomarker cells for use with the BioFlash® system based on the CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) technology (Innovative Biosensors, Inc., Rockville, MD). BioFlash® can be used to rapidly detect the presence of a RNA-DNA duplex by detecting the binding of the RNA-DNA duplex with a sensor cell comprising a RNA-DNA duplex binding antibody or antibody fragment. The BioFlash® system can comprise an engineered biosensor expressing membrane bound RNA-DNA duplex specific antibodies and a calcium sensitive bioluminescent molecule. Binding of the antibody by a RNA-DNA duplex leads to elevation of intracellular calcium and light emission. The amplified light output can be detected by, for example, using a luminometer. When a RNA-DNA duplex to be detected binds to the antibody on the surface of a sensor cell, calcium ions move into the cytosol as described in Wilson ct al., J Exp Med 166:601-606 (1987). The increased cytosolic calcium concentration causes an emitter molecule to emit a photon, which can be read by the optical detector.

A suitable emitter molecule is any molecule that emits a photon in response to elevated cytosolic calcium concentrations, including bioluminescent and fluorescent molecules. One emitter molecule, the bioluminescent aequorin protein, is described in Button et al., Cell Calcium 14:663-671 (1993); Shimomura et al, Cell Calcium 14:373-378 (1993); and Shimomura, Nature 227: 1356-1357 ( 1970). Aequorin generates photons by oxidizing coelenterazine, a small chemical molecule. Coelenterazine diffuses through cellular membranes, so coelenterazine or an analog thereof can be added to the culture medium surrounding the cells. Alternatively, genes encoding enzymes that make coelenterazine can be introduced into the cells. Also, bio luminescent green fluorescent protein (GFP) can be used (see Chalfie, Photochem Photobiol 62:651-656 (1995)). Optionally, the cell cytosol contains both GFP and aequorin. In response to elevated calcium in the cytosol, aequorin donates energy to GFP in an emissionless energy transfer process. GFP then emits the photon. Alternatively, the emitter molecule can be a calcium-sensitive fluorescent molecule (e.g., indo-1) which is illuminated by a wavelength of light suitable to induce fluorescence.

Aequorin, or any other emitter molecule, can be introduced into the cell by methods well know in the art. If the emitter molecule is a protein (as is the case with aequorin), the cell can contain an expression vector encoding the protein (i.e., a nucleic acid or virus which will produce the emitter molecule when introduced into a cell). An expression vector can exist extrachromosomally or integrated into the cell genome. A sensor cell with surface-bound antibodies can be cither prokaryotic or eukaryotic. Upon binding of a KNA-DNA duplex to the antibodies, the cell mobilizes calcium ions into the cytosol. An example of a sensor cell is a B cell (i.e., a B cell from a cold or warm-blooded vertebrate having a bony jaw) which can be genetically engineered to express one or more surface-bound monoclonal antibodies. It also can be produced by, for example, immunizing an animal with the antigen to be detected and harvesting the B cell from the immunized animal. The harvested B cells can be further immortalized and screened for production of a surface monoclonal antibody specific for the antigen to be detected. The B cells are optionally genetically engineered to express a calcium sensitive bioluminescent cytosolic molecule. Another useful cell type that can be used is a fibroblast that optionally can be adhered to a substrate or device. However, fibroblasts do not contain the signal transduction machinery to transfer a signal from the cytoplasmic portion of a surface antibody via a signal cascade to calcium stores in the cell. To overcome this problem, a chimeric surface antibody can be expressed in the fibroblast. This chimeric antibody contains a cytoplasmic amino acid sequence derived from a polypeptide (e.g., a fibroblast growth factor receptor) that can transduce a signal from the inner surface of the plasma membrane of the fibroblast to intracellular calcium stores. Thus, when an antigen binds to the extracellular portion of the chimeric antibody to cause antibody aggregation on the surface, calcium mobilization is induced. A similar strategy using chimeric antibodies can be employed for any other non B-cell type. The fibroblast can also be genetically engineered to express a calcium sensitive bioluminescent cytosolic molecule.

Growth of the cell can be controlled by any means well known in the art, including providing anti -mitotic drugs (e.g., α-amanitin) or growth factors (e.g., fetal bovine serum) in the medium. Cells can also be genetically engineered to grow at a determined rate. As discussed above, any cell can be used where binding of a RNA- DNA duplex to the antibody on the surface of the cell leads to an increase in calcium concentration in the cytosol. In fact, the cell can be a non-living, manufactured unit as long as it satisfies the above factors.

An antibody that specifically binds to a RNA-DNA duplex to be detected is a molecule that binds to the RNA-DNA duplex, but docs not substantially bind other antigens or epitopes in the sample. For example, the antibody does not substantially bind an RNA or DNA that is not duplexed. Such antibodies can be chimeric (i.e., contain non-antibody amino acid sequences or contain portions from a different species) or single chain (i.e., the complementarity determining region of the antibody is formed by one continuous polypeptide sequence).

Polyclonal cells expressing antibodies can be prepared by immunizing a suitable animal with the antigen to be detected. The cells producing antibody molecules directed against the antigen can be isolated from the animal (e.g., from the blood) and further purified by well-known techniques, such as panning against an antigen-coated petri dish. Surface antibody-producing cells can be obtained from an animal and used to prepare a monoclonal population of cells producing surface antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature 256:495-497 (1975); Kozbor et al., Immunol Today 4:72 (1983); or Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., pp. 77-96 (1985). The technology for producing cells expressing monoclonal antibodies is well known (sec, e.g., Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N. Y.), with modifications to select for surface antibodies rather than secreted antibodies. Any protocol used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a cell producing a surface monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977; Kenneth, In Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner, Yale J Biol Med 54:387-402 (1981).

As an alternative to preparing monoclonal cells, a nucleic acid encoding a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the antigen to thereby isolate immunoglobulin library members that bind the antigen. Kits for generating and screening phage display libraries arc commercially available. Additionally, examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271 ; PCT Publication WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs ct al., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); Griffiths et al., EMBO J 12:725-734 (1993).

After the desired member of the library is identified, the specific sequence can be cloned into any suitable nucleic acid expresser and transfected into a cell such as a fibroblast. The expresser can also encode amino acids operably linked to the antibody sequence as appropriate for the cell which is to express the antibody. As discussed above, the cytoplasmic transmembrane sequence of a fibroblast growth factor receptor can be linked to a single-chain antibody specific for the antigen to be detected, so that the cell mobilizes calcium when contacted with the antigen. Although separate recombinant heavy chains and light chains can be expressed in the fibroblasts to form the chimeric antibody, single chain antibodies also are suitable (see, e.g., Bird et al., Trends Biotechnol 9:132-137, 1991; and Huston et al., Int Rev Immunol 10:195-217, 1993).

As used herein, the term, antibody fragment means a binding fragment of an antibody. Binding fragments include single chain fragments, Fv fragments and Fab fragments. The term Fab is refers generically to double chain binding fragments of intact antibodies having at least substantially complete light and heavy chain variable domains sufficient for antigen-specific bindings, and parts of the light and heavy chain constant regions sufficient to maintain association of the light and heavy chains (e.g., including Fab' and F(ab')₂ fragments). Fab fragments can be formed by complexing a full-length or substantially full-length light chain with a heavy chain comprising the variable domain and at least the CHl domain of the constant region. While various antibody fragments can be obtained by digesting an antibody, one of skill in the art will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology.

Thus, the term antibody fragment, as used herein, includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies {e.g., single chain Fv). Single chain antibodies or fragments are also useful in the described methods. Methods for producing single chain antibodies are described, for example, in U.S. Pat. No.

4,946,778. Techniques for the construction of Fab expression libraries are described by Huse et al, Science (1989) 246:1275-1281; these techniques facilitate rapid identification of monoclonal Fab fragments and can be used to detect monoclonal Fab fragments with the desired specificity for RNA-DNA duplex molecules. Suitable antibodies and antibody fragments include those that are obtained using methods such as phage display. The antibodies or antibody fragments can be polyclonal or monoclonal. Monoclonal antibodies are prepared from hybridoma cells secreting the desired antibody and screening methods are known in the art and examples are described below. Monoclonal antibodies can be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture, including the hybridoma technique originally developed by Kohlcr and Milstein {Nature (1975) 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et a!., Immunology Today (1983) 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Monoclonal antibodies also can be produced in germ-free animals as was described in U.S. Pat. No. 5,091,512. The antibodies and antibody fragments can be humanized or fully human {i.e., produced by transgenic animals that make human antibodies).

The antibodies and antibody fragments described herein can be screened for binding to a RNA-DNA duplex. The terms bind, binds, and binding, when referring to an antibody or other binding moiety, are intended to indicate a binding reaction or affinity that is determinative of the presence of a target antigen (i.e., a RNA-DNA duplex), optionally, in the presence of a heterogeneous sample, population of proteins and/or other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target antigen {i.e., a RNA-DNA duplex) and do not bind in a significant amount to other components present in a test sample.

A variety of immunoassay formats may be used to select antibodies or fragments that are specifically immunoreactive with a RNA-DNA duplex. For example, solid-phase ELTSA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen. Typically a specific or selective reaction is at a statistically significant level above the background signal. For example, at least 1.5 to twice the background signal or noise or more than 10 times the background signal or noise. Specific binding between an antibody or other binding agent and an antigen generally means a binding affinity of at least 10⁶ M^"1. Further examples of specific binding affinity include, but are not limited to at least 10⁷ M^"1, at least 10^s M^"1, at least 10⁹ NT¹, and at least 10¹⁰ M^'1. Specific binding between an antibody or other binding agent and an antigen can also be described in terms of their dissociation constant KD. The antibodies and antibody fragments described herein can bind with a KD of at least 1 μM, at least 500 μM, at least 300 μM, at least 100 μM, at least 50 μM, at least 30 μM, at least 10 μM, or at least 3 μM.

Methods for the detection of a RNA-DNA duplex in a sample include contacting the sample with an antibody or antibody fragment that binds to the RNA- DNA duplex. Antibodies that bind a RNA-DNA duplex and methods of making the same are known in the art. For example, United States Patent 4,833,084 describes a monoclonal antibody, secreted by hybridoma ATCC HB 8730, specific for a RNA- DNA duplex. Further, Fliss et al., Production and Characterization of Anti-DNA- RNA Monoclonal Antibodies and Their Application in Listeria Detection, Appl and Envt Micro (1993) 59(8): 2698-2705, describes production of RNA-DNA duplex antibodies. The antibodies or antibody fragments that specifically bind to a RNA-DNA duplex can be provided in a kit. The kit can comprise a container for containing the antibody or antibody fragment. Such a kit can also contain components necessary to perform the assays described above or other assays. For example, the kit can comprise sensor cells such as those described above. These kits can further include apparatus for collecting, storing, and/or disposing of a sample. Kits as described herein can include instructions, for example, for performing an assay or preparing a sample. Such kits can contain one or more apparatus, matrix (liquid or solid) and chemical reagent needed to perform an assay or contain only certain elements of an assay. Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular modification of a method of detecting a RNA-DNA duplex or is disclosed and discussed and a number of modifications that can be made to the method of detecting a RNA-DNA duplex are discussed, each and every combination and permutation of the RNA-DNA duplex and the detection method are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention except as and to the extent that they are included in the accompanying claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Example 1

A hybridoma expressing antibody that specifically binds to a RNA-DNA duplex was obtained. The antibody was expressed on the surface of the B-CeIl to form a CANARY® sensor cell. Target RNA (or DNA) and complementary DNA (or RNA) were hybridized together. A 2Kb length RNA-DNA duplex and a short 30 base pair length RNA-DNA oligo duplex were prepared. The hybridized RNA-DNA duplexes were presented to the sensor cell to perform a CANARY® assay. The CANARY® assay was carried out in a liquid matrix, which is the conventional matrix for a standard CANARY® assay. Antibodies expressed on the B-cell sensor cell surface bound the RNA-DNA duplexes and triggered a scries of intracellular chemical and biochemical changes resulting in detectable photon emission. The photon emission was detected using standard optical equipment.

In vitro RNA Prep for PBP2a (~2kb message, polyA tailed) using

Ambion® (Austin, TX) mMessage Machine T7 Ultra kit #AM1345 lμl (lμg) template (pDisplay PBP2a+6X His linearized with Xhol), lOμl T7 2X NTP/ARCA (Anti-reverse cap analogue), 2μl 1 OX T7 Reaction Buffer, 2ul T7 Enzyme mix and 5μl dH2O was mixed and incubated 2hrs at 37°C. DNA was digested by adding lμl Turbo DNase 1, mixing well, and incubating for 15 minutes at 37⁰C. For the addition of poly A tail, 36μl dH2O, 20μl 5X E. coli Poly (A) polymerase (E-PAP) Buffer, lOμl 25mM MnC12, lOμl ATP Solution was added. 4μl E-PAP was added and mixed gently. The mixture was incubated 45 minutes at 37⁰C and transferred to ice. lOμl ammonium acetate was added and mixed. Extraction was performed with lOOμl phenol/chloroform/isoamyl alcohol by vortexing and spinning 5 minutes at 14K. Aqueous portion was transferred to a new tube and 1 volume isopropanol was added and mixed well. Chill -2O⁰C ON. The mixture was spun for 15 minutes at 14K. Then the mixture was washed with 50μl 70% EtOH, spun for 5 minutes at 14K and decanted. The mixture was then spun at 1 minute at 14K and decanted. The sediment was resuspended in 20μl dH2O. lμl was checked (add lOμl formaldehyde + dyes and incubate 10' at 75⁰C to prep for gel) on 1.5% agarose/lX TBE gel. Quantization by reading 260 OD in spectrophotometer was performed.

Lambda Exonuclease III ssDNA Prep

Using Roche's (Mannheim, DE) Expand ® Long Range dNTPack #04829034001 Asymmetric PCR was performed by limiting the concentration of the kinased primer. The strand primed by the kinased primer was preferentially degraded by lambda exonuclease 111 to obtain enriched ssDNA. Kinased primer was prepared by incubating forward primer petpbp2a f xhol at lOOpmol with lμl Polynucleotide Kinase (PNK), lμl 1OX PNK Buffer, lμl 1OmM dATP, dH2O to lOμl for 30 minutes at 37⁰C. Enzyme was heat killed for 20 minutes at 65⁰C. Primer was diluted to lpmol/μl for use.

1 μl Template (pDisplay PBP2a+6X His), 1.2μl Forward kinased primer petpbp2a f xhol atlpmol/μl, 2μl Reverse primer petpbp2a r kpnl at 30pmol/μl, lOμl 5X PCR Buffer, 2.5μl dNTPs, 1.5μl 3% DMSO, 0.7μl Enzyme, and 31. lμl dH2O was mixed. Amplification was performed at Ix (92⁰C 2 minutes), 1Ox (92⁰C 10 seconds, 5O⁰C 15 seconds, 68⁰C 30 seconds), 2Ox (92⁰C 10 seconds, 5O⁰C 15 seconds, 68⁰C 30 seconds + 5 seconds per cycle), Ix (68⁰C 7 minutes) and 8⁰C hold.

PCR product was column purified using Qiaquick® PCR Purification Kit (Qiagen #28104) (Hilden, DE) using manufacturer's spin column instructions and elute in 35μl dH2O. A check of 3.5μl on 1.5% agarose/TAE gel was performed.

Lambda Exonuclease III Digestion (NEB #M0262S) 40μl PCR product, lOμl 1OX Exo Reaction Buffer, 2μl lambda exonuclease III, 48μl dH2O were mixed and split into 8 PCR tubes (~12.5μl per tube). The tubes were incubated 30 minutes at 37⁰C and 10 minutes at 75⁰C to heat kill enzyme. The samples were column purified using Qiaquick® PCR Purification Kit using manufacturer's spin column instructions and elute in 50μl dH2O (Qiagen, Hilden, DE). 5μl was checked on 1.5% agarose/lX TAE gel and concentration was determined by reading OD 260/280 nm in a spectrophotometer.

Hybridization Conditions

10¹⁰ RNA, 10⁸ ssDNA, transcription buffer from Ambion® mMessage Machine Kit [0.1X final] (Austin, TX), 0.65U RNase Inhibitor (NEB #AM2682), and dH2O were mixed. Also mixes were made for RNA alone, ssDNA alone and dsDNA alone. Samples were incubated 95⁰C 2 minutes, 65⁰C 30 minutes followed by 5O⁰C 30 minutes. Samples were diluted in assay buffer for use in the CANARY® (Innovative Biosensors, Rockville, MD) assay.

Controls

Assay Buffer alone (250μl AB, no target) Cells plus AB (250μl AB, no target)

RNA:DNA--2μl in 250μl AB (f.c. lOclO RNA hybridized to 10c8 ssDNA or dilutions thereof)

RNA only-2μl in 250μl AB (f.c. lOel O RNA following above hybridization conditions) ssDNA only--2μl in 250μl AB (f.c. 10e8 DNA following above hybridization conditions) PIasmid (dsDNA) only-2μl in 250μl AB (f.c. 10e8 plasmid DNA following above hybridization conditions)

Biotinylated Oligo-Streptavidin beads complex was prepared as follows: Mix the biotinylated RNA oligos with DNA or the biotinylated DNA oligos with RNA in 0.1 or 1 X transcription buffer and water. Incubate for 5 minutes at 95⁰C followed by 30 minutes at 50⁰C. Add lOμg beads per sample and incubate 30 minutes at room temperature.

Oligo sequences:

HC Biotin RNA: 5 V5Biosg/rCrCrG rArCrG rCrArG rCrArC rUrUrG rArGrA rCrCrA rCrArC rCrArU rGrCrA rArCrU rArCrA rUrArC-3 '

HC CompDNA: 5'-GTA TGT AGT TGC ATG GTG TGG TCT CAA GTG CTG-3'

HCDNAOLIGO: 5'-/5Biosg/CCG ACG CAG CAC TTG AGA CCA CAC CAT GCA ACT ACA TAC-(fam) HCRNA: 5'-rGrUrA rUrGrU rArGrU rUrGrC rArUrG rGrUrG rUrGrG rUrCrU rCrArA rGrUrG rCrUrG-3'

CANARY Assay

• Master mixes were made and aliquot 250μl per tube. 1 OK cells were added to cap and spin 8 seconds before placing in the Berthold luminometer (Bundoora, AU). Relative Light Units (RLU) were measured for 60 seconds and plotted. Mean

RLU from 40s to 50s used for further analysis.

• When master mixes were not made, 2μl hybrid mix were added to 250μl AB. 1OK cells were immediately added to cap, spin 8 seconds and immediately place tube in the luminometer. Relative Light Units (RLU) were measured for 60 seconds and plotted. Mean RLU from 40s to 50s used for further analysis.

Two small scale B cell preparations (SS19 and SS21) of three clones (C21- 11 A, B and C) were made to evaluate the response to full length RNA/DNA target and select nucleic acid controls. Protocol: Samples were prepared by loading with coelenterazine according to standard CANARY® (Innovative Biosensors, Rockville, MD) small scale protocol.

Targets were full-length RNA alone or exo/ssDNA alone or full-length RNA/DNA hybridized in 0.1X transcription buffer then diluted in 250μl assay buffer. The concentration of all conditions was RNA/DNA: 10el0/10e8 copies/assay. Samples were spun for 8 seconds. Integral RLU values are based on 40-50 seconds. A Signal to Noise (S :N) ratio of >3 was considered positive.

Conclusion: Significant signal was observed upon providing 2 kb length RNA-DNA duplex (AB+RD). The signal with DNA alone (AJB+DNA) or RNA alone (AB+RNA) was not significant.

Repeated the small scale assay (SS22) of clone C21-11A with a target of biotinylated oligos + streptavidin beads. [ccl065:89-97]

Protocol: Samples were prepared following the above mentioned protocols. Targets were constituted using various combination of (+/- biotin)RNA oligo, (+/- biotin)DNA oligo + beads or in combination + beads in 250μl assay buffer. Also FL- RNA+DNA was used as control. The concentration of all conditions was RNA/DNA: 10el0/10e8 copies/assay. Samples were normalized to 1OK cells and were spun for 8 minutes prior to assaying.

Conclusion: Results indicate good signal with biotinylated DNA/RNA oligo+beads labeled as bDR (biotinylated DNA+ RNA) or bRD (biotinylated RNA+ DNA). The previously described 2 kb long RNA-DNA duplex was used as positive control (AB+RD) and yielded robust signal. No signal was seen with mock beads (AB+beads) and with RNA oligo alone or DNA oligo alone controls.

Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim docs not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

Any patents or publications mentioned in the specification are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting a RNA-DNA duplex in a sample, comprising:

a) contacting a sensor cell expressing on the sensory cell surface a RNA-DNA antibody or antibody fragment with a sample comprising a RNA-DNA duplex, wherein the antibody or antibody fragment binds with the RNA-DNA duplex; and

b) detecting binding of the sensor cell with the RNA-DNA duplex, binding indicating the presence of a RNA-DNA duplex in the sample.

2. The method of claim 1, further comprising forming the RNA-DNA duplex.

3. The method of claim 2, wherein the RNA-DNA duplex is formed by hybridizing a test RNA molecule to a target DNA molecule that selectively hybridizes at stringent conditions to the RNA molecule.

4. The method of claim 1 , wherein the RNA-DNA duplex is formed by hybridizing a test DNA molecule to a target RNA molecule that selectively hybridizes at stringent conditions to the DNA molecule.

5. The method of claim 2, wherein the RNA-DNA duplex is formed by hybridizing a test RNA or DNA molecule to a target DNA or RNA molecule, wherein at least 10 base pairs of the test molecule is complementary to the target molecule.

6. The method of claim 5, wherein the RNA-DNA duplex is formed by hybridizing a test RNA or DNA molecule to a target DNA or RNA molecule, wherein at least 30 base pairs of the test molecule is complementary to the target molecule.

7. The method of claim 5, wherein the RNA-DNA duplex is formed by hybridizing a test RNA or DNA molecule to a target DNA or RNA molecule, wherein at least 2 kilo base pairs of the test molecule is complementary to the target molecule.

8. The method of claim 1, wherein the RNA-DNA duplex contacts the sensor cell in a liquid matrix.

8. A sensor cell expressing on its surface a RNA-DNA antibody or antibody fragment.

9. The sensor cell of claim 8, wherein the cell is configured to emit a detectable signal when the RNA-DNA antibody or antibody fragment binds with a RNA-DNA duplex molecule.