WO2011026135A1 - Compositions and methods for rapid detection and analysis of nucleic acids - Google Patents

Abstract

Disclosed are methods, compositions and apparatus for detection and/or determination of concentration of target molecules. Further disclosed are methods and compositions for determining presence of or quantifying target nucleic acid agents.

Description

81825-392660

COMPOSITIONS AND METHODS FOR RAPID DETECTION AND ANALYSIS

OF NUCLEIC ACIDS

RELATED APPLICATIONS

[001] The present application claims the benefit under 35 U.S. C. § 119(e) of U.S.

Provisional Patent Application Serial No. 61/238,531, filed August 31, 2009, which is incorporated herein by reference in its entirety for all purposes.

FIELD

[002] Embodiments of the present invention relate to methods, compositions and/or apparatus for the detection of presence and/or determination of concentration of nucleic acids or other target molecules in a sample. Certain embodiments report assessing concentration of nucleic acids or other target molecules in biological sample such as a fluid sample from a subject. Methods, compositions and/or apparati disclosed herein can be effective for detection and/or concentration measurement over a wide range of concentrations of target nucleic acids.

BACKGROUND

[003] Detection and characterization of nucleic acids is an essential segment of medicinal, clinical, and biological research. Currently, one of the most common methods for nucleic acid detection relies on complementary interaction of a homogenous single-stranded probe population and target nucleic acid(s) within a complex mixture of heterogeneous nucleic acid sequences. Hybridization methods were initially developed in the 1970's, and are now used for a variety of purposes including research, characterization of a variety of diseases, such as breast cancer and lymphoma, forensic analysis, infectious agent detection, mutagenic and evolutionary analysis.

[004] Several methods are commonly used in the research and diagnostics including: Southern blotting, Northern blotting, dot blots, micro-arrays, in situ hybridization, Polymerase Chain Reaction (PCR), real-time PCR and melting-curve analysis. The stringency/specificity of these techniques relies upon common characteristics that can modify the thermodynamically favored structure of paired nucleic acids. These factors include the nucleic acid sequence, variation of probe strand length, base content, temperature, and buffer composition. The use of modified nucleic acids such as peptide 81825-392660 nucleic acids (PNA) and locked-nucleic acids (LNA) among others can be used to increase specificity of interaction between target sequences and specialized probes.

SUMMARY OF THE INVENTION

[005] Certain embodiments of the present invention generally relate to the detection and/or determination of concentration of nucleic acids or other target molecules using complex formation with a target nucleic acid and a primary binding agent conjugated to molecular detection elements and secondary binding agents conjugated to capture molecules. In some embodiments, a primary binding agent may be a complementary oligomeric sequences of DNA, RNA, peptide nucleic acid (PNA), or chemical

modifications of these oligomeric sequences that may directly bind to a target nucleic acid molecule. In other embodiments, capture molecules may be modified to permit sequestration of target sequences to a polymer in solution, thereby reducing the electrophoretic mobility of the target nucleic acid sequences.

[006] Some embodiments herein concern application of an electrophoretic field to a vertical electrophoresis chamber allowing separation of complexed binding agents, target molecules, and polymer from the remainder of the one or more samples. In accordance with these embodiments, application of an electrical filed can permit removal of nonspecific sample constituents and sequestration of captured target nucleic acid sequences. These captured complexes may be immobilized by a polymer allowing for visualization and quantification of target nucleic acids. The captured target nucleic acid sequences may be removed for further analysis or procedures. In certain embodiments, a captured target nucleic acid may be analyzed by PCR or other amplification technique known in the art. Alternatively, a captured target nucleic acid may be used to generate complimentary binding molecules for diagnostic or therapeutic purposes.

[007] In certain embodiments, these methods may be combined with methods and apparatus disclosed in the Enhanced Velocity Electro Immunoassay (EVEIA) for the detection and quantification of nucleic acids in biological fluids (previously described in US Patent Application No. 12/023,450, filed January 31, 2008 and U.S Patent Application No. 12/700,677 ^', filed February 04, 2010, incorporated herein by reference in their entirety for all purposes). Some of these embodiments concern separating target nucleic acid complexes 81825-392660 disclosed herein using EVEIA systems in order to isolate and analyze the target nucleic acid.

[008] Other embodiments disclosed herein concern compositions containing one or more complex formed with a target nucleic acid. In accordance with these embodiments, complexes can include, but are not limited to, a target nucleic acid associated with a binding and capture element, as well as certain uncharged polymers.

[009] Certain embodiments disclosed herein include binding agents. Binding agents can include, but are not limited to, nucleic acids, aptamers, nucleic acid derivatives or combinations thereof. In other embodiments, the binding agents are DNA or RNA oligomers. In accordance with these embodiments, the binding agents can be chemically modified DNA or RNA oligomers. Binding agents may be peptide nucleic acid (PNA) oligomers. In other embodiments, capture molecules and detection molecules include, but are not limited to, one or more of DNA or RNA oligomers, chemically modified DNA or RNA oligomers, peptide nucleic acid (PNA) oligomers or combinations thereof. A target molecule contemplated herein may be a chromosomal DNA fragment, or a messenger RNA (mRNA), or amplified segments of the DNA or mRNA. In yet other embodiments, a target molecule may be a siRNA or miRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate certain embodiments disclosed herein. Embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0011] Fig. 1 represents a schematic of an exemplary method of certain embodiments disclosed herein where a detectable agent can be conjugated to a first oligonucleotide to form a signal generating oligonucleotide.

[0012] Fig. 2 represents a schematic of an exemplary method of certain embodiments disclosed herein where a biotin derivative can be conjugated to a second oligonucleotide to form a capture oligonucleotide.

[0013] Fig. 3 represents a schematic of an exemplary method of certain embodiments disclosed herein where a signal generating oligonucleotide, a capture oligonucleotide, and a 81825-392660 nucleic acid target are combined to form a ternary complex along with the excess oligonucleotides.

[0014] Fig. 4 represents a mixture when the complex of Fig. 3 (302) is mixed with neutravidin, which can bind with biotin-containing components to form a neutravidin- ternary complex, a neutravidin-capture oligonucleotide complex, as well as excess signal generating oligonucleotide and excess neutravidin.

[0015] Fig. 5 represents an exemplary schematic where voltage applied to the reaction chamber can generate a mixture illustrated in Fig. 4 and move by electrophoresis into a region containing exemplary uncharged polymers conjugated to for example, biotin.

Components of the exemplary mixture from Fig. 4 that contain neutravidin can bind to the polymers conjugated to biotin.

[0016] Fig. 6 represents a schematic similar to Fig. 3, except using the FRET technique.

The acceptor oligonucleotide can include an oligonucleotide complementary to the nucleic acid target, conjugated to an acceptor dye. The capture oligonucleotide can include an oligonucleotide complementary to the nucleic acid target, conjugated both to a donor dye, and to a biotin molecule. An acceptor oligonucleotide, a capture oligonucleotide, and a nucleic acid target are combined to form a ternary complex along with the excess oligonucleotides.

[0017] Fig. 7 represents a figure with similarities to Fig. 3, except using a hairpin molecule quenching technique. A hairpin oligonucleotide can include a hairpin oligonucleotide having one arm complementary to a nucleic acid target, conjugated to a traceable dye on one arm of the hairpin, and conjugated to a quencher on the other arm of the hairpin. The capture oligonucleotide can include an oligonucleotide complementary to the nucleic acid target, conjugated to a biotin molecule. The hairpin oligonucleotide, the capture oligonucleotide, and the nucleic acid target can be combined to form the ternary complex along with the excess oligonucleotides. The formation of this complex destabilizes the hairpin structure, separating the traceable dye from the quencher, thereby activating the traceable dye.

[0018] Figs. 8A-8C represents schematics of certain exemplary embodiments disclosed herein concerning identifying and quantifying target nucleic acids.

Description of Illustrative Embodiments

Definitions 81825-392660

[0019] Terms that are not otherwise defined herein are used in accordance with their plain and ordinary meaning.

[0020] As used herein, "a" or "an" may mean one or more than one of an item.

[0021] As used herein, "about" can mean plus or minus 10%, for example, about 10 minutes can mean from 9 to 11 minutes.

DETAILED DESCRIPTION

[0022] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments of the invention. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the details outlined herein, but rather that concentrations, times and other details may be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0023] Embodiments herein provide for methods and compositions for detection of presence and/or determination of concentration of nucleic acids, nucleic acids linked to other molecules or other target molecules in one or more samples. Methods disclosed herein may be used for diagnostics, identification (e.g. organism, contaminant, biowarfare agent, forensic analysis etc), disease prognosis or other purposes that involve identification and quantification of novel nucleic acid sequences.

[0024] In certain embodiments, a particle (which may be a molecule) moving through a fluid experiences a frictional force proportional to its velocity, its size (as described by its hydrodynamic radius), and the viscosity of the fluid. The formal expression of this is known as the Stokes equation.

[0025] Embodiments herein provide for methods and compositions for identifying and quantifying target nucleic acid molecule(s). In certain embodiments, the EVEIA assay (previously described and identified in US Patent Application No. 12/023,450, filed January 31, 2008, incorporated herein by reference in its entirety) may be applied to capturing nucleic acid complexes in a solution or non-solid phase (e.g. solution phase) during separation of these complexes. In certain embodiments, separation may be through electrophoresis of samples. In accordance with these embodiments, certain types of 81825-392660 electrophoresis can occur through a vertical density gradient contained within a transparent electrophoretic channel.

[0026] In some embodiments, nucleic acid samples contemplated of use herein can include, but are not limited to, any sample from a subject such as a human or animal, samples obtained from a swab, samples from soil, surfaces or water, samples from food or other consumables, or samples from a culture such as a bacterial, viral or cell culture.

[0027] Some embodiments described herein concern sample chambers of use for sample separation. In accordance with these embodiments, a sample chamber can house a conductive density gradient containing, for example, three layers. These layers are from top to bottom, a sample layer, and a capture layer where target nucleic acid molecule complexes accumulate within the column, and a stacking layer. The three layers are held in place by the density differences within the sample column. In one embodiment, the sample layer is comprised of 2% sucrose, the capture layer 5% sucrose and the stacking layer 7% sucrose. Those skilled in the art will recognize that other density modifying agents can be used to generate the distinct layers of the density gradient within the column. Similarly, those skilled in the art will recognize that a linear gradient rather than a step gradient could be used for this method. The capture layer can be further modified by the addition of a large uncharged polymer that may be modified to detect and capture cognate target molecules. In certain embodiments, the capture layers contain essentially uncharged polymer modified with biotin. Other embodiments concern capture layers having linear polyacrylamide (LPA) modified with biotin. In yet other embodiments, uncharged polymers may include one or more of LPA, dextran, other polysaccharides, linear polyacrylamide, polyethylene glycol or the like. In certain embodiments, essentially uncharged polymers have a very low net charge to mass ratio (e.g. less than one net charge per 10,000 Daltons), where net charge is positive charges minus negative charges, within the pH ranges used for the assay (e.g. pH 6 to pH 11). Those skilled the in the art will recognize that additional polymers with no net charge could be used within the capture layer.

[0028] In certain embodiments, for the capture of target nucleic acids, two or more binding agents can be added to a biological sample. Some biological samples of interest may include serum that may contain the target nucleic acid of interest. In other embodiments, 81825-392660 binding of target molecules to one or more binding agents can include one binding agent conjugated to a capture molecule and another conjugated to a detection molecule forming a complex. In one embodiment, a capture molecule can be PNA. In accordance with these embodiments, the PNA can be a specific sequence complementary to the target nucleic acid sequence and conjugated to a biotin molecule. In this example, the detection molecule is a PNA of specific sequence complementary to the target sequence conjugated to a fluorescent molecule. Those skilled in the art will recognize that other signals can be used for detection of a target nucleic acid including, but not limited to radio label, enzyme, colorimetric, or other technique that can be directly or indirectly quantified.

[0029] Some embodiments herein concern after nucleic acid hybridization complexes are formed where a molar excess of an additional agent may be added to facilitate binding of the complexes to modified uncharged polymers. For example, neutravidin can be added to the sample and the sample applied to a column. In accordance with these embodiments, neutravidin can function to couple target nucleic acid-binding agent complex to biotinylated uncharged polymer in the capture layer through a biotin-neutravidin interaction. Then application of an electrophoretic field to the sample chamber can drive migration of the sample through the chamber. Here, a mass increase of the target nucleic acid-binding agent-polymer complex formed as the hybridization complexes of interest reach the capture layer can significantly decrease charge-to-mass ratio resulting in slowed migration or to a cessation of migration. In certain embodiments, continued electrophoresis of these complexes can wash the non-specific sample to the stacking layer, and the captured complex can be visualized using methods known in the art. In one embodiment, capture nucleic acid hybridization complexes can be visualized by laser excitation of the fluorescent dye conjugated to the detection oligonucleotide. In addition, fluorescence of the fluorescent dye can be quantified. Quantification of fluorescence may be performed by any means known in the art. In certain embodiments, fluorescence may be quantified by image analysis using a CCD camera and in certain embodiments, software programs known in the art, such as EVEIA software ( described in US Patent Application No. 12/023,450, filed January 31, 2008, incorporated herein by reference in its entirety for all purposes).

[0030] As disclosed herein, standard Watson-Crick base pairing interactions can create double stranded nucleic acid complexes consisting of target sequence bound to capture and 81825-392660 detection oligonucleotides allowing the capture and detection of the desired target sequence. In certain embodiments, heating or other sample treatment may be required to optimize hybridization of nucleic acid binding agents to the nucleic acid target. In accordance with these embodiments, for double stranded nucleic acid targets a heating step may be used to separate annealed strands used for capture and detection from the target nucleic acid. In addition, salt, organic solvent, or other buffer constituents known in the art may be supplied to the sample for use in adjusting stringency of hybridization to the target nucleic acid. Capture and detection oligonucleotides can be modified by various chemistries to afford greater chemical stability and provide different charge characteristics from standard nucleic acids (RNA and DNA) to favor use in this system or other EVEIA system. For example, peptide nucleic acids (PNAs) can be used to create nucleic acid complexes with less charge facilitating easier capture within the capture layer.

Additionally, many modified nucleic acids (including, but not limited to, PNAs, LNAs, 2'- OMe, etc.) can be used to create stronger duplex formations which can enhance binding of capture and detection oligonucleotides to target sequences over competing endogenous complementary sequences (e.g. for double stranded targets). In certain embodiments, this may be more important when capture and detection oligonucleotides are designed for shorter targets, such as siRNA or miRNA, that require high stringency coupled with a shorter length (8-13nt). Moreover, modified nucleic acids can have greater chemical stability to serum nucleases compared to standard nucleic acids. In certain embodiments, target nucleic acid sequences can be of lengths of about 16 to about 10,000 nt. Larger lengths may be broken down by enzymatic or mechanical methods to lengths within this range, if needed. For example, chromosomal DNA may be sheared using sonication to lengths ranging from 50 to lOOOnt. Also, methods for using restriction enzymes to cut DNA sequences to known sizes are known to those skilled in the art.

[0031] In other embodiments, for detection of longer nucleic acid targets (e.g. greater than 24 nts) multiple detection oligos targeting various regions of the nucleic acid of interest could be used as a method to amplify signal output. In accordance with these embodiments, this could allow detection of very small amounts of viral or bacterial derived nucleic acids (e.g. about 3000 molecules or more). In other embodiments, options for detection could be to use double stranded DNA binding dyes such as SYBR Green I or other binding dyes as probes for detection of bound captured complementary targets. In yet other 81825-392660 embodiments, another option for detection could use fluorescence resonance energy transfer (FRET, see Example section) techniques to reduce background issues, for example by increasing the difference between the excitation wavelength and the emission wavelength, and by making the fluorescence emission may be dependent on the formation of a full complex. FRET techniques depend on the presence of two dyes, a donor dye and an acceptor dye, in close proximity. Some examples of donor and acceptor dyes include, but are not limited to, Alexa Fluor® 633 and Alexa Fluor® 647; Cy3 and Cy5, respectively, or other indicator/quencher molecules known in the art. Other donor and acceptor dyes are known in the art. Excitation of the donor dye can lead to energy transmission to the acceptor dye followed by emission, without transfer of a photon between the two dyes. Emission by the acceptor dye drops with the sixth power of the distance between the two dyes. As disclosed herein, one of the FRET dyes could be conjugated to a capture oligonucleotide, and another could be conjugated to a detection oligonucleotide. The formation of the capture-oligonucleotide/target- oligonucleotide/detection-oligonucleotide complex brings the FRET dyes into close proximity for efficient emission. In other embodiments, another option for detection could use nucleic acid hairpins linked to fluorescent probes and quenching agents (as previously disclosed) as probes for detection of bound captured complementary targets. In its unbound state, formation of a hairpin brings the fluorescent probe and quenching agent into close proximity, thereby greatly reducing fluorescence. When bound to the target oligonucleotide of interest, the hairpin unfolds, thereby removing the quencher from close proximity to the fluorescent probe, increasing fluorescence for detection of the complex. In certain embodiments, compositions of complexes (e.g hairpin loop complexes; uncharged polymer complexes described herein) are contemplated of use for these and other systems of detection and quantification of target nucleic acids. In certain embodiments, double- stranded regions of a hairpin may be 2 nt to any size that may be contained within the length of the target nucleic acid sequence, e.g. for a target sequence of 50nt with a loop of 10 nt (double stranded), the double stranded sequence may be 20 base pairs long. In certain embodiments, the double stranded region may include mismatches, bulges, etc. Structures of hairpin loops are well known by those knowledgeable in the area. A loop region of a hairpin may be of any size 4 nt or longer.

Affinity Probe Capillary Electrophoresis (APCE) 81825-392660

[0032] In certain embodiments of the present invention, capillary electrophoresis can be used. Capillary electrophoresis involves applying a voltage, by means of positively and negatively charged electrodes, across a long capillary filled with an ionic buffer causing charged molecules to migrate toward one electrode or the other. Positively-charged ions migrate toward the negative electrode (the cathode), dragging the bulk solution with them. This electro-osmotic flow can cause all ions to migrate toward the cathode (although at different rates) and can be used as a single detector that can analyze all ionic species regardless of charge as they flow past. Electro -osmotic flow can be eliminated or even reversed by changing the electrical characteristics of the capillary wall.

[0033] In some embodiments, for APCE, a target molecule can be incubated with a binding agent that has been conjugated to a detection molecule, usually a fluorescent probe. The mixture is then injected into the capillary, and a voltage is applied, setting up electro- osmotic flow. The free target, the free binding agent, and the target/binding agent complex can migrate at different rates (augmented by the electro-osmotic flow), and can therefore be detected as separate peaks by a fluorescent detector (the free target should not yield a signal).

Enhanced Velocity Electro Immunoassay (EVEIA) for Target Molecule Detection and Quantitation

[0034] Certain embodiments of the present application provide a rapid, high sensitivity method for detecting and determining the concentrations of target molecules in samples involving vertical stacked electrophoresis. Because the electrophoretic part of the EVEIA technique takes place in containers of much larger volume and cross-sectional area than microcapillaries, the claimed methods allow the detection of target molecules that are present in low concentration in a sample. In certain embodiments, electrophoresis may occur in a tube, channel or other container with a minimum hydrodynamic radius (twice the cross-sectional area divided by the circumference) of 0.5 mm or higher.

[0035] In some embodiments, methods and compositions disclosed herein may be used to target nucleic acid based diseases or disease progression in a subject. In accordance with these embodiments, target nucleic acids may be identified or quantified in a sample in order to assess health of a subject. In other embodiments, compositions and methods disclosed 81825-392660 herein may be directed to target DNA-based infectious diseases for example, for diagnostic or disease progression purposes. These methods may be used in place of or in addition to method including, but not limited to PCR and isothermal amplification methods already on the market. In certain examples, like bacteremia, blood culture (about a 16 hr process) generally precedes amplification (due to the sometimes very low CFUs in blood). Methods disclosed herein may be used to detect bacteremia-associated nucleic acids in order to rapidly diagnose a subject. In some embodiments, amplification can be performed not only to generate the specific signal, but also to generate a readily detectable quantity of that signal. Sensitivity of direct hybridization methods (no amplification) is lacking with many platforms (for example, one company struggled with this in their attempts to produce an MRSA assay using "thin-film" technology). The LOD achieved with a synthetic target may be used to determine a sample processing route used to detect target nucleic acids (e.g. DNA) in a blood culture extract (Blood cultures = ca. 30% whole blood, 70% culture media, 1E8-5E9 CFU/ml). Some of these methods may include amplification. In accordance with these embodiments, unlike with amplification-based methods, nucleic acid samples may be fragmented into small segments (for example, for less charge and mass in the capture zone and more-effective strand separation) for example by digestion, sonication and pressure changes are possibilities among other methods known in the art.

[0036] In certain embodiments, compositions and methods disclosed herein may be used to identify bioagents, biowarfare agents, biocontaminants in foods or water, or for other identification purposes.

[0037] In other embodiments, kits are contemplated of use for detecting one or more nucleic acid. In accordance with these embodiments, a kit can include any composition disclosed herein as well as container means for housing such components. Other components contemplated of use in a kit can include control samples (e.g. negative and/or positive control samples) for comparing and assessing concentrations of nucleic acid molecules in a sample. In certain embodiments, detectible agents and quenching agents may be included as part of the kit for detecting a target nucleic acid and optionally, quantifying the concentration of the target nucleic acid.

EXAMPLES 81825-392660

[0038] The following examples are included to illustrate various embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods, compositions and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes may be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

[0039] In one exemplary method, schematics are used to represent some embodiments of the present invention (see for example Figs. 1-7).

[0040] Fig. 1 represents a schematic of an exemplary method of certain embodiments disclosed herein where a detectable agent, Fluorescein (101) is conjugated to a first oligonucleotide (102) to form the Signal Generating Oligonucleotide (103). The individual components represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0041] Fig. 2 represents a schematic of an exemplary method of certain embodiments disclosed herein where a biotin derivative (201) is conjugated to a second oligonucleotide (202) to form a Capture Oligonucleotide (203). The individual components are meant to represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0042] Fig. 3 represents a schematic of an exemplary method of certain embodiments disclosed herein where a Signal Generating Oligonucleotide (103), the Capture

Oligonucleotide (203), and the Nucleic Acid Target (301) are combined to form the Ternary Complex (302) along with the excess Oligonucleotides (103) and (203). Drawings are not to scale, and individual figures represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0043] Fig. 4 represents a mixture when the complex of Fig. 3 (302) is mixed with neutravidin (401), which binds with biotin-containing components to form a neutravidin- ternary complex (402), a neutravidin-capture oligonucleotide complex (403), as well as excess Signal Generating Oligonucleotide (103) and excess neutravidin (401). Drawings are not to scale, and the individual figures are meant to represent the presence of particular components in a reaction, not the stoichiometric amounts of each component. 81825-392660

[0044] Fig. 5 represents in one example where a voltage applied to the reaction chamber can generate mixture from Fig. 4 and move by electrophoresis into a region containing exemplary uncharged polymers that include, but are not limited to, dextran or other polysaccharide, linear polyacrylamide, polyethylene glycol conjugated to for example, biotin. Other agents may be used in place of or in addition to biotin, for example, covalent chemical interacting agents with reverse complement to a target nucleic acid sequence. Components of the exemplary mixture from Fig. 4 that contain neutravidin can bind to the polymers conjugated to biotin. In some embodiments, uncharged polymers can range in size from hundreds of thousands to millions of Daltons. Complexes formed with these uncharged polymers can have reduced electrophoretic mobilities compared to any components in the resulting mixture from Fig. 4 and those not conjugated to uncharged polymers. Drawings are not to scale, and the individual figures represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0045] Fig. 6 represents a schematic similar to represented in Fig. 3, except using the FRET technique. The Acceptor Oligonucleotide (601) can include an oligonucleotide complementary to the Nucleic Acid Target, conjugated to an acceptor dye (601a). The Capture Oligonucleotide (602) is comprised of an oligonucleotide complementary to the Nucleic Acid Target, conjugated both to a donor dye (602a), and to a biotin molecule. The Acceptor Oligonucleotide (601), the Capture Oligonucleotide (602), and the Nucleic Acid Target (603) are combined to form the Ternary Complex (604) along with the excess oligonucleotides (601) and (602). The formation of this complex brings the donor dye (602a) into close proximity to the acceptor dye (601a), activating fluorescent signaling. Drawings are not to scale, and individual figures represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0046] Fig. 7 represents a figure similar to Fig. 3 analogous to Fig. 3, except using a hairpin quenching technique. The Hairpin Oligonucleotide (701) can include a hairpin oligonucleotide having one arm complementary to a Nucleic Acid Target, conjugated to a fluorescent dye (701 (a)) on one arm of the hairpin, and conjugated to a fluorescent quencher (701(b)) on the other arm of the hairpin. The formation of the hairpin brings a fluorescent dye (701 (a)) into close proximity to a quencher (701(b)), thus inhibiting or reducing signal of the fluorescence. A hairpin oligonucleotide may be designed with 81825-392660 mismatched bases to destabilize it compared with binding to the Nucleic Acid Target (703).

The Capture Oligonucleotide (702) can include an oligonucleotide complementary to the

Nucleic Acid Target, conjugated to a biotin molecule. The Hairpin Oligonucleotide (701), the Capture Oligonucleotide (702), and the Nucleic Acid Target (703) can be combined to form the Ternary Complex (704) along with the excess Oligonucleotides (701) and (702).

The formation of this complex destabilizes the hairpin structure, separating the fluorescent dye (701 (a)) from the quencher (701(b)), thereby activating it. Drawings are not to scale, and the individual figures represent the presence of particular components in a reaction, not the stoichiometric amounts of each component.

[0047] Fig. 8A-8C represent schematics of a binding agent associated with a target DNA partitioned on an Eveia column (previously disclosed) using LPA-PNA (8A) or biotinylated PNA (8B). The general methodology represented in Fig. 8B is equivalent to an antibody sandwich assay, with the capture and detect oligos substituting for the antibodies.

An off-column denaturation/renaturation and maintenance of temperature at approx 3-5 degrees below Tm is needed. A schematic of biotinylated PNA capture probe use is represented in Fig. 8C.

[0048] This probe may be obtained with an N- or C-terminal lysine (in place of biotin); this would increase functional group flexibility (via conjugation) but, due to our current purification limitations with a 4.5-5 kDa molecule, opted for pre-conjugation to biotin. The

O-linker groups impart increased water solubility.

Examples for Oligo detection:

(10 nM oligo used for Tm calculations)

Tm in 50 mM NaCl = 46.2°C

Tm in 20 mM NaCl = 38.0⁰C

Tm in 10 mM NaCl = 31.1°C

Delta G for strongest heterodimer (50 mM NaCl, 0.25 uM oligo) = -3.14 kcal/mole(low; safe; couple bp's) The addition of A647 can need HPLC purification and a minimum

Synthesis scale of 100 nmole.

Example 2

[0049] The following describes one exemplary experiment implementing one embodiment of the present invention. This experiment employs an embodiment using automated

EVEIA instrument as described in PCT/US08/75894 (U.S. Patent Application No. 81825-392660

12/677,270, incorporated by reference in its entirety). In this example, an instrument having a single column is loaded in an automated fashion by a robotic, single-head pipetter. Although sample preparation is done off-line, loading of all three layers, loading of the sample, setting of the electrophoresis voltage level, laser illumination and image capture and analysis are all computer controlled.

OLIGONUCLEOTIDES

[0050] This example uses a target nucleic acid sequence including a section of the mecA gene that encodes penicillin-binding protein 2a (PBP2a; PBP2'), conferring resistance to b- lactam antibiotics (including methicillin). This nucleic acid sequence could be used as a diagnostic for MRSA (multidrug-resistant Staphylococcus aureus). This 58-mer single- strand DNA was synthesized and purified by Integrated DNA Technologies (IDT), and was not purified further before use:

[0051] 5 -GAA GTG GTA AAT GGT AAT ATC GAC TTA AAA CAA GCA ATA GAA TCA TCA GAT AAC ATT-3' (SEQ. ID NO: 1); MecA ssDNA target sequence (57mer).

[0052] The underlined and bolded region at the 5' end is complementary to the PNA capture probe, while the underlined and bolded region at the 3' is complementary to the detection probe.

[0053] As a negative control, the above target sequence was randomized: 5'-GAC CAA TGA CAC AGT GGA TCC TCT GAG GAG CTT ACT AGT TTC ATA CAT GTG GAC ATC-3' (SEQ. ID NO:2)

[0054] The negative control sequence was prepared in an identical manner as the target sequence. The detection probe is a 24-mer single-strand DNA sequence with the fluorescent dye Alexa 647 attached at its 5 ' end, synthesized and purified by IDT, and was not purified further before use: 5'-Alexa647-GTT ATC TGA TGA TTC TAT TGC TTG- 3' (SEQ. ID NO:3)

[0055] The capture probe is a 15-mer PNA sequence with a biotin attached at the N- terminus end via an O-linker. The O-linker contributes to solubility in aqueous reagents, and is only available for addition at the N-terminal end. The capture probe was synthesized and purified by Biosynthesis, Inc: N-terminus-Biotin-00-AAT ACC ATT TAC CAC -C- terminus (SEQ. ID NO:4), O-linker, which contributes to solubility, available for N- terminal addition only. Estimated costs for a PNA is around $500 and oligonucleotides is 81825-392660 around $300-500 but no conjugations or purifications are needed for these elements.

Therefore, these processes can be cost effective and rapid compared to other methods known in the art.

[0056] Reverse compliment of target sequence: (detection probe underlined; PNA capture probe bolded) 5'-AAT GTT ATC TGA TCC TAT TGC TTG TTT TAA GTC GAT ATT ACC ATT TAC CAC TTC-3'(SEQ. ID NO:5).

SAMPLE PREPARATION

[0057] 1. Combine target nucleic acid sequence (variable concentration), Alexa-647- conjugated detection probe (1 nM final), and biotinylated PNA capture probe (1.5 nM) in 20 mM NaCl, 20 mM Tris, pH 8.0, with a final sucrose concentration of 2.75%.

[0058] 2. Incubate at 35°C for 5-10 min. Add neutravidin (25-50 nM final) just prior to loading onto column.

COLUMN PREPARATION

[0059] This exemplary column consists of three aqueous layers, with the relative positions maintained by density. All layers are comprised of 25 mM Tris pH 8.0, 0.008% NP-40 (a detergent), 0.004% sodium azide (an antimicrobial), and varying levels of sucrose, which provide the density differences. The lower layer has a sucrose concentration of 7%. 300 microliters are typically loaded onto the proper position on the automated instrument, of which 255 microliters are loaded into the column. The middle layer has an initial sucrose concentration of 5%, but is diluted by 10% with a solution of linear polyacrylamide conjugated to biotin. 50 microliters are loaded onto the proper position on the automated instrument, of which 30 microliters are loaded into the column on top of the lower layer in a very careful manner so that the layers do not mix.

[0060] The upper layer has a sucrose concentration of 2%. 300 microliters are typically loaded onto the proper position on the automated instrument. 200 microliters are loaded into the column on top of the middle layer in a very careful manner so that the layers do not mix. 40 microliters of the sample mixture are then loaded into the upper layer, approximately 5 mm below the surface, so that the sample mixture (with a sucrose concentration of 4%) floats down on top of the middle layer. 30 microliters of upper layer are then loaded on top of the existing upper layer so that the column is filled, allowing electrical contact between the upper and lower reservoirs for electrophoresis.

ELECTROPHORESIS AND DATA SAMPLING 81825-392660

[0061] Electrophoresis was monitored online in order to keep the power dissipation in the column at approximately 525 milliwatts by varying the voltage, in order to maintain the temperature at 28C-30C. The voltage varied from 330 to 400 volts, while the current varied from 1.6 to 1.3 milliamps. The column was irradiated with a 635 nM laser (power at laser outlet aperture: 50 milliwatts), every 15 seconds for 1 second intervals. The image of the irradiated column was captured by a CCD camera, with filters to detect light in the range of 661 nm to 690 nm, and block it otherwise, thereby detecting the presence and location of Alexa 647 conjugated to the detection probe.

[0062] All of the COMPOSITIONS and METHODS disclosed and claimed herein may be made and executed without undue experimentation in light of the present disclosure. While the COMPOSITIONS and METHODS have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variation may be applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the METHODS described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

81825-392660 WHAT IS CLAIMED IS:

1. A method for detecting a nucleic acid target molecule comprising:

obtaining a sample that may contain the nucleic acid target molecule;

subjecting the sample to an apparatus having an applied electrical field in presence of a first binding agent conjugated to a detection molecule and a second binding agent conjugated to a capture molecule, wherein the nucleic acid target molecule, if present in the sample, binds to the first and second binding agents to form a complex;

separating the complex from uncomplexed molecules using the electrical field and detecting the presence of the complex formed by the nucleic acid target molecule and first and second binding agents.

2. The method of claim 1, wherein the apparatus having an applied electrical field comprises a vertical stacked electrophoresis apparatus.

3. The method of claim 2, further comprising:

performing the electrophoresis in the presence of one or more uncharged polymers, the one or more uncharged polymers are capable of binding to the capture molecule to further associate with the complex.

4. The method of claim 1, wherein the binding agents are nucleic acids, aptamers, nucleic acid derivatives or combinations thereof.

5. The method of claim 4, wherein the binding agents are DNA or RNA oligomers.

6. The method of claim 4, wherein the binding agents are chemically modified DNA or RNA oligomers.

7. The method of claim 4, wherein the binding agents are peptide nucleic acid (PNA) oligomers. 81825-392660

8. The method of claim 1, wherein the capture molecule and detection molecule comprise one or more of DNA or RNA oligomers, chemically modified DNA or RNA oligomers, peptide nucleic acid (PNA) oligomers or a combination thereof.

9. The method of claim 1, wherein the target molecule is a chromosomal DNA fragment, or a messenger RNA (mRNA), or amplified segments of DNA or mRNA

10. The method of claim 1, wherein the target molecule is a siRNA or miRNA.

11. The method of claim 1, wherein the electrophoresis is performed in a tube, channel or container with a minimum hydrodynamic radius of 0.5 mm.

12. The method of claim 11, wherein the sample is loaded at the top of the tube, channel or container and the electrophoresis is performed in a gradient of increasing density or viscosity from top to bottom of the tube, the gradient comprising at least two phases of different density or viscosity.

13. The method of claim 12, wherein the gradient comprises a sample layer, a capture layer and a stacking layer.

14. The method of claim 13, wherein the capture molecule is biotin.

15. The method of claim 14, wherein the electrophoresis is performed in the presence of avidin, streptavidin, neutravidin or a combination thereof.

16. The method of claim 14, wherein the electrophoresis is performed in the presence of a neutral or uncharged polymer conjugated to biotin.

17. The method of claim 1, wherein the complex comprises a target molecule, a first binding agent, a second binding agent, a multivalent biotin binding agent selected from avidin, streptavidin or neutravidin and one or more polymers conjugated to biotin. 81825-392660

18. The method of claim 17, wherein formation of the complex results in an increase of the mass to charge ratio and a decrease in electrophoretic mobility of the target molecule and complex.

19. The method of claim 18, wherein the complex is concentrated at the stacking layer.

20. The method of claim 1, further comprising, measuring amounts of a detection molecule in the complex to determine concentration of the target molecule in the sample.

21. The method of claim 1, wherein the detection molecule is an oligonucleotide conjugated to one or more of a fluorescent, luminescent, chemiluminescent, radioactive molecule; or an enzyme that produces a fluorescent, luminescent, chemiluminescent or colored product.

22. The method of claim 1, wherein the first binding agent is an oligonucleotide hairpin.

23. The method of claim 22, wherein the oligonucleotide hairpin has a segment complementary to the target oligonucleotide, with two arms of the hairpin conjugated to a fluorescent probe and a fluorescent quencher.

24. The method of claim 1, wherein the first binding agent is conjugated to a donor or acceptor dye of a FRET pair, and the second binding agent is conjugated to the cognate dye in the FRET pair to the dye conjugated to the first binding agent.

25. A composition comprising: a target nucleic acid molecule, a first binding agent conjugated to a detection molecule, a second binding agent conjugated to a capture molecule, wherein the nucleic acid target molecule binds to the first and second binding agents to form a complex; and one or more uncharged polymers capable of binding to the capture molecule of the complex.

26. The composition of claim 25, wherein the capture agent comprises biotin. 81825-392660

27. The composition of claim 25, wherein the first binding agent is an oligonucleotide hairpin.

28. The composition of claim 27, wherein the oligonucleotide hairpin has a segment complementary to the target nucleic acid, with two arms of the hairpin conjugated to a fluorescent probe and a fluorescent quencher.

29. A kit for assessing a sample for presence of a target nucleic acid comprising: a first binding agent conjugated to a detection molecule and a second binding agent conjugated to a capture molecule, wherein the first binding agent and the second binding agent are capable of binding to a target nucleic acid if the target nucleic acid is present in the sample; and one or more uncharged polymers capable of binding to the capture molecule.

30. The kit of claim 29, further comprising one or more of positive or negative control nucleic acid molecules.