Nucleic acid amplification using molecular photo-switches

Abstract

The present disclosure provides methods that enable sequence-specific nucleic acid amplification in-situ using photo-switchable molecular constructs. The sample comprising target nucleic acids is exposed to pulsed photon fluxes with different wavelengths to actuate the photo-switchable molecular constructs that trigger and / or halt specific biochemical reactions resulting in amplifying the specific nucleic acid sequences in a linear or exponential fashion.

Description

[0001] WSGR Docket No: 63452-705.601

[0002] NUCLEIC ACID AMPLIFICATION USING MOLECULAR PHOTO-SWITCHES CROSS-REFERENCE

[0003]

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 721,129 filed on November 15, 2024, which is entirely incorporated herein by reference.

[0004] FIELD OF USE

[0005]

[0002] Provided herein are methods and compositions for nucleic acid amplification, nucleic acid amplification testing, DNA detection, RNA detection, molecular diagnostics, genetic research, DNA sequencing and cloning.

[0006] BACKGROUND

[0007]

[0003] To detect nucleic acids in a sample, some methods utilize molecular amplification processes such as polymerase chain reaction (PCR) to increase the copy number of the target nucleic acids. Such tests are conventionally categorized as nucleic acid amplification tests (NAATs). NAATs methods have a variety of different performance criteria which include, but are not limited to, analytical sensitivity, specificity, limit of detection (LoD), quantification range, detection dynamic range (DDR), and turnaround time (TAT).

[0008]

[0004] Today, there are a variety of NAAT methods for nucleic acid detection which use specific enzymes, reagents, and temperature profiles to amplify and detect specific sequences. However, there is a need for improved methods and molecular structures, that once included in specific NAAT methods, can improve their performance criteria.

[0009] SUMMARY

[0010]

[0005] Provided herein are systems and methods for nucleic acid amplification. Also provided herein are systems and methods for sequence-specific nucleic acid amplification. Also provided herein are systems and methods for analyte detection.

[0011]

[0006] According to one aspect of the present disclosure, disclosed herein is a system for sequence-specific nucleic acid amplification, comprising: (i) a reaction chamber; (ii) an aqueous sample enclosed in the reaction chamber, comprising: (a) a nucleic acid template; (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; the one or more photo-switchable moieties being configured to (1) switch to a first conformation when radiated by a first excitation light beam with a first wavelength; and (2) switch to a second conformation when radiated by a second excitation light beam with a second wavelength; and (c) one or more enzymes configured to interact with the nucleic acid temple and / or the plurality of oligonucleotide constructs; and (iii) an excitation light source configured to emit at least the first WSGR Docket No: 63452-705.601

[0012] excitation light beam with the first wavelength and the second excitation light beam with the second wavelength.

[0013]

[0007] According to one aspect of the present disclosure, disclosed herein is a method for sequence-specific nucleic acid amplification, comprising: (i) radiating an aqueous sample enclosed in a reaction chamber with a first excitation light beam having a first wavelength, wherein the aqueous sample comprises: (a) a nucleic acid template; (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; and (c) one or more enzymes configured to interact with the nucleic acid temple and / or the plurality of oligonucleotide constructs; (ii) changing, via the radiating in (i), one or more photo-switchable moieties of a first oligonucleotide construct of the one or more oligonucleotide constructs into a first conformation; and changing one or more photo-switchable moieties of a second oligonucleotide construct of the one or more oligonucleotide constructs into the first conformation; and (iii) subsequent to (ii), ligating, via the one or more enzymes, the first oligonucleotide construct with the second oligonucleotide construct; thereby providing a ligated construct, wherein the ligated construct remains bound to the nucleic acid template.

[0014]

[0008] According to one aspect of the present disclosure, disclosed herein is a method of detecting a presence or absence of at least one analyte in a sample, comprising: (i) radiating an aqueous sample with a first excitation light beam having a first wavelength, wherein the aqueous sample comprises: (a) a sample suspected to comprise at least one analyte; (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; (c) a ligase enzyme configured to interact with the at least one analyte and the plurality of oligonucleotide constructs; (ii) radiating the aqueous sample with a second excitation light beam having a second wavelength; (iii) radiating the aqueous sample with a third excitation light beam having a third wavelength; and (iv) detecting the presence or absence of a fluorescent resonance energy transfer (FRET) signal, wherein the FRET signal is configured to be generated when a first oligonucleotide construct and a second oligonucleotide construct of the plurality of oligonucleotide constructs are ligated in the presence of the at least one analyte.

[0015]

[0009] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. WSGR Docket No: 63452-705.601

[0016] Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0017] INCORPORATION BY REFERENCE

[0018]

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [OH] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

[0021]

[0012] FIG. 1 shows a scheme depicting duplex formation using photo-isomerization of azobenzene as a mechanism to control photo-switching of DNA hybridization.

[0022]

[0013] FIG. 2 shows a scheme depicting the building blocks of a photo-triggered ligation chain reaction: (1) template; (2) four complementary probes; and (3) a DNA ligase enzymatic system.

[0023]

[0014] FIG. 3 shows a scheme depicting photon-triggered ligation of oligonucleotides with sequences X and F, respectively, using a sequence complementary sequence.

[0024]

[0015] FIG. 4 shows a scheme depicting photon-triggered ligation of oligonucleotides with sequences X and F, respectively using a sequence complementary sequence.

[0025]

[0016] FIG. 5 shows a scheme depicting photon-triggered ligation of oligonucleotides with sequences X and F, respectively using X + F amplicon.

[0026]

[0017] FIG. 6 shows a scheme depicting photon-triggered ligation of oligonucleotides with sequences X and F, respectively using X + F amplicon.

[0027]

[0018] FIG. 7A shows a scheme depicting using a fluorescent resonance energy transfer (FRET) moiety to detect amplification by placing the donor and acceptor on the same allele. FIG. 7B shows a scheme depicting using a FRET moiety to detect amplification by placing the donor and acceptor on different allele.

[0028]

[0019] FIG. 8 shows a computer system that is programmed or otherwise configured to implement methods provided herein. WSGR Docket No: 63452-705.601

[0029] DETAILED DESCRIPTION

[0030]

[0020] The underlying principle of this invention is to enable nucleic acid amplification by applying specific wavelengths of light to control the properties of nucleic acid molecules, specifically their base pairing (hybridization) thermodynamics and kinetics.

[0031]

[0021] To take advantage of light as a control mechanism for biochemical reactions in general, and specifically for nucleic acid amplification, the methods may rely on molecular constructs that can alter their states or conformations by absorbing wave-length specific photons. Such constructs, generally referred here as “photo-switches” or molecules with photo-switchable moieties, are a unique class of molecules that can change their structural geometry and / or chemical properties upon interacting with electromagnetic waves, i.e., absorption of photons.

[0032]

[0022] There are many known molecular constructs in the art that can be used as the building block for photo-switches. Examples are azobenzene [reference 1], spiropyran [reference 2], merocyanine [reference 3], diarylethene [reference 4], spirooxazine [reference 5], fulgide [reference 6], hydrazone [reference 7], nobormadiene [reference 8], thioindigo [reference 9], acrylamide-azobenzene-quatemary ammonia [reference 10], donor-acceptor Stenhouse adducts [references 11-12], stilbene [reference 13], etc.

[0033]

[0023] The use of light as a stimulus in a method to control molecular reaction has unique advantages: (1) unlike other molecular stimuli (e.g., introducing another molecule, heating, or applying electrochemical potential, etc.), light does not contaminate and / or alter the microenvironment of the reaction system; (2) spatiotemporal control of the reaction can easily be accommodated in a controlled fashion; and (3) photon wavelengths can be precisely tuned to interact with very specific molecular structures.

[0034]

[0024] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0035]

[0025] The present invention may be understood more readily by reference to the following detailed description of the aspects and embodiments of the invention, as well as the Examples described herein.

[0036] Definitions

[0037]

[0026] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and so forth unless otherwise indicated. WSGR Docket No: 63452-705.601

[0038]

[0027] As used herein, the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary from ± 1% to ± 15% of the stated number or numerical range, such as, for example, within ± 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the designated amount and preferably within ± 10%.

[0039]

[0028] When ranges are used herein for physical properties, such as temperatures or peak values, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included.

[0040]

[0029] The term “real-time detection system” as used herein generally refers to a system that performs real-time analysis on an aqueous biological or biochemical sample without stopping or interfering with the interaction of molecules within the system, and the sample includes one or more analytes.

[0041]

[0030] The term “quencher” as used herein generally refers to a compound (e.g., a small molecule dye) that quenches a signal emitted from a fluorescent compound, e.g., a fluorescent intercalator or a fluorescence reporter. In some embodiments, the quencher absorbs excitation energy from the fluorescent compound (e.g., fluorescent intercalator or fluorescent reporter) and dissipates the energy that is absorbed from the fluorescent compound as heat (not a new fluorescence signal).

[0042]

[0031] The term “analyte” as used herein, generally refers to a molecular species to be detected. Non-limiting examples include small molecules, such as organic compounds drugs, hormones, lipids, steroids, or metabolites; polynucleotides such as deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, and peptide nucleic acid (PNA) molecules; polypeptides such as proteins, peptides, antibodies, antigens, enzymes, and receptors; as well as tissues, organelles, and other receptor molecules.

[0043]

[0032] The term “reporter” or “reporter molecule” as used herein, generally refers to a molecular structure that can be attached to a molecule (e.g., an analyte) or be associated with a molecule or construct (e.g., a duplex of a double stranded nucleic acid) covalently or non-covalently, to permit detection of the molecule, distinguishable or traceable by providing a characteristic which may not be intrinsic to the analyte molecule. Examples of reports or reporter molecules or labels may include luminescent molecules (e.g., fluorophores), reductionoxidation (redox) species, or enzymes. In some cases, labels may comprise fluorophores with long lifetimes, such as, for example, lanthanide chelates and transition metal chelates, which may be luminescent or phosphorescent. WSGR Docket No: 63452-705.601

[0044]

[0033] The term “nucleotide,” as used herein, generally refers to a molecule that can serve as the monomer, or subunit, of a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleotide may be a deoxynucleotide triphosphate (dNTP) or an analog thereof (e.g., a molecule having a plurality of phosphates in a phosphate chain, such as 2, 3, 4, 5, 6, 7, 8, 10, or more phosphates). A nucleotide may generally include adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide may include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T, or U, or complementary to a purine (e.g., A or G, or variant thereof) or a pyrimidine (e.g., C, T, or U, or variant thereof). A subunit can enable individual nucleic acid bases of group of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TC, AC, CA, or uracil counterparts thereof) to be resolved. A nucleotide may be labeled or unlabeled. A labeled nucleotide may yield a detectable signal, such as an optical, electrostatic, or electrochemical signal.

[0045]

[0034] The terms “polynucleotide,” “oligonucleotide,” “nucleotide,” “nucleic acid,” and “nucleic acid molecule” generally refer to a polymeric form of nucleotides (polynucleotides) of various lengths, either ribonucleotide (RNA) or deoxyribonucleotides (DNA). Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA including genomic DNA and messenger RNA. The length of the sequence can be any length that can be amplified into nucleic acid amplification products, or amplicons, for example, up to about 20, 40, 100, 200, 300, 400, 500, 600, 00, 800, 21000, 1200, 1500, 2000, 5000, 12000, or more than 10000 nucleotides in length.

[0046]

[0035] As used herein, the term “amplicon” generally refers to a molecular species that is generated from the amplification of a nucleotide sequence, such as through PCR or ligase chain reaction. An amplicon may be a polynucleotide such as RNA or DNA or mixtures thereof, in which the sequence of nucleotides in the amplicon may correlate with the sequence of the nucleotide sequence from which it was generated (i.e. either corresponding to or complimentary to the sequence). The amplicon can be either single stranded or double stranded. In some cases, the amplicon may be generated by using one or more primers that is incorporated into the amplicon. In some cases, the amplicon may be generated in a polymerase chain reaction or PCR amplification, wherein two primers may be used to produce either a pair of complementary single stranded amplicons or a double-stranded amplicon. In some case, the amplicon may be generated by ligation of two oligonucleotides bound to a template nucleic acid catalyzed by a ligase enzyme.

[0047]

[0036] As used herein, the term “fluorescence resonance energy transfer” or FRET generally refers to non-radioactive energy transfer between chemical and / or protein fluorophores. WSGR Docket No: 63452-705.601

[0048] Fluorescent resonance energy transfer (FRET) is a process in which one fluorophore (the acceptor) can be promoted to an excited electronic state through quantum mechanical coupling with and receipt of energy from an electronically excited second fluorophore (the donor). This transfer of energy results in a decrease in visible fluorescence emission by the donor and an increase in fluorescent energy emission by the acceptor.

[0049]

[0037] For FRET to occur efficiently, the absorption and emission spectra between the donor and acceptor have to overlap. Dye pairs are characterized by their spectral overlap properties. Emission spectrum of donor must overlap acceptor absorption spectrum. Extent of overlap determines the efficiency of energy transfer. Extent of overlap also determines the optimal distance for which the assay is sensitive. Where the overlap of spectra is large, the transfer is efficient, so it is sensitive to longer distances. The selection of donor / accept or depends upon the distances considered. Significant energy transfer can only occur when the donor and acceptor are sufficiently closely positioned since the efficiency of energy transfer is highly dependent upon the distance between donor and acceptor fluorophores, for example, within 10 angstroms (A). The fluorophores can be chemical fluorophores and / or protein fluorophores. In some embodiments n, the first component of the FRET pair may be a lanthanide such as a chelate of europium (III) (Eu(III)) or terbium(III) (Tb(III)) which serves as a fluorescent donor and the second component of the FRET pair may be an activated ester of cyanine 5 (Cy5) or tetramethylrhodamine (TMR) which serve as energy acceptors of Eu(III) and Tb(III). Other first and second component pairs that may find use for FRET include, for example, fluorescein and rhodamine; FITC and rhodamine; and fluorescein and trinitrophenyl, and other components as are known in the art. The FRET pair can be selected based on their wavelength of their absorption or emission bands.

[0050]

[0038] As used herein, the term “fluorophore” refers to a chemical compound, which when excited by exposure to a particular wavelength of light, emits light at a different wavelength. As used herein, the term “fluorophore” refers to any molecule known in the art that can be used for FRET, including dyes, fluorescent proteins, and quantum dots.

[0051]

[0039] As used herein, the term “excitable distance” refers to the distance at which FRET can occur between a donor molecule and an acceptor molecule. In some embodiments, the excitable distance can be when the donor molecule and the acceptor molecule are within from about 3 A to about 10 A, from about 10 A to about 20 A, from about 20 A to about 30 A, or from about 30 A to about 40 A of each other. In some embodiments, the excitable distance can be within 10 A. In some embodiments, for fluorescent proteins, the excitable distance can be from about 10 A to about 100 A, or about 50 A to about 90 A. WSGR Docket No: 63452-705.601

[0052]

[0040] As used herein, a “FRET pair” refers to two fluorophores attached with different reporter molecules (donor and acceptor molecules). These two fluorophores can be within an excitable distance, and since fluorophores have overlapping donor emission and acceptor absorption spectrums, and suitable dipole orientation, they can emit FRET signals.

[0053]

[0041] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0054]

[0042] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0055]

[0043] In this disclosure, specific optical real-time detection methods and real-time detection systems using such methods are described. These methods / systems may rely on time-resolved fluorescence systems and the corresponding biomolecular constructs involving time-resolved optical reporters. In some embodiments, the real-time methods may use affinity-based biosensor formats that include one or more capturing / detection probes that are immobilized on a solid surface, hereinafter referred to as a solid-phase capturing probe. In some embodiments, the systems disclosed herein is a real-time detection system.

[0056] Sequence-specific nucleic acid amplification system

[0057]

[0044] In some embodiments, the present disclosure provides a sequence-specific nucleic acid amplification system. The system may comprise an aqueous sample. In some embodiments, the aqueous sample may comprise a nucleic acid target (e.g., with a known sequence) that needs to be amplified (i.e., chemically copied / synthesized). In some embodiments, the aqueous sample may comprise a plurality of nucleic acid oligonucleotide constructs (also called “oligonucleotide constructs” or “a plurality of oligonucleotide constructs”) with known sequences and concentrations with photo-switchable properties. In some embodiments, the aqueous sample may comprise an enzymatic reaction system (also called “one or more enzymes”) capable of interacting with the target and / or oligonucleotide constructs. The system may comprise a vessel (also called “reaction chamber”) that contains or encloses the aqueous sample. The system may comprise a temperature control system (also called “temperature control”) that controls the temperature of the vessel as well as the sample. The system may comprise an excitation light source for illuminating the sample with a pulsed and / or time varying photon fluxes with two or WSGR Docket No: 63452-705.601

[0058] more specific output spectra (e.g., with different wavelength). The system may comprise a control system (also called “control” or “controller”) to operate and coordinate the performance of the excitation light source and / or the temperature control system.

[0059] Reaction chamber

[0060]

[0045] The term “reaction chamber,” or “vessel,” as used herein, generally refers to a physical system that confines an aqueous sample and optionally other components. In some embodiments, a reaction chamber encloses an aqueous sample. In some embodiments, the reaction chamber may comprise a nucleic acid template. In some embodiments, the reaction chamber may comprise a plurality of oligonucleotide constructs. In some embodiments, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, each of the one or more photo-switchable moieties is configured to switch to two different conformations when radiated by two different excitation light beams with two different wavelengths. In some embodiments, each of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. The reaction chamber may allow the photons of light of the two excitation light beams to reach the oligonucleotide constructs or their products residing inside the chamber. In some embodiments, the reaction chamber may comprise a temperature control to set and dynamically change the temperature within the chamber, such as, the temperature of the aqueous solution.

[0061]

[0046] In some embodiments, the reaction chamber can have a volume ranging from about 0.1 nanoliter (nL) to about 10 milliliters (mL). In some cases, the reaction chamber may have a volume ranging from about 1 microliter (pL) to about 100 pL. In some embodiments, the reaction chamber may have a volume that is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 nL. In some embodiments, the reaction chamber may have a volume that is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pL. In some embodiments, the reaction chamber may have a volume that is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL.

[0062]

[0047] The reaction chamber can have a temperature ranging from about 4 °C to about 110 °C.

[0063] The temperature of the reaction chamber can be controlled with accuracies as about ±0.01 °C, ±0.02 °C, ±0.03 °C, ±0.04 °C, ±0.05 °C, ±0.06 °C, ±0.07 °C, ±0.08°C, ±0.09°C, ±0.1°C, ±0.2°C, ±0.3°C, ±0.4°C, ±0.5°C, ±0.6°C , ±0.7°C , ±0.8°C , ±0.9°C or±l°C. In some embodiments, the temperature of the reaction chamber may range from about 25 °C to about 95 °C, and the accuracy of controlling the temperature can be controlled to within ±0.1°C. WSGR Docket No: 63452-705.601

[0064] Excitation light source

[0065]

[0048] The term “light,” as used herein with respect to the reaction chamber, generally refers to the photon flux confined within specific wavelengths and applied to the reaction chamber for a duration of time. The wavelengths of light can be from about 200 nanometers (nm) to about 2000 nm. In some embodiments, the wavelength of light is from about 200 nm to about 400 nm, from about 300 nm to about 500 nm, or from about 400 nm to about 600 nm. In some embodiments, the wavelength of light is from about 300 nm to about 400 nm. In some embodiments, the wavelength of light is more than 400 nm. In some embodiments, the total optical power of the light can be from about 0.01 mW / cm2to about 10,000 mW / cm2, from about 0.1 mW / cm2to about 1000 mW / cm2, from about 1 mW / cm2to about 1000 mW / cm2, from about 0.2 mW / cm2to about 500 mW / cm2, from about 0.5 mW / cm2to about 200 mW / cm2, or from about 1 mW / cm2to about 100 mW / cm2. The duration of light exposure time can be from about 0.1 second (sec) to about 10,000 sec, from 0.25 sec to about 5,000 sec, from about 0.5 sec to about 1,000 sec, from about 0.75 sec to about 500 sec, from about 1 sec to about 100 sec.

[0066]

[0049] The term “excitation light source,” as used herein, generally refers to the combination of devices that in concert generates photons of light within defined wavelengths and controls its power to be applied to the nucleic acid constructs. The light source system may include a photon source that can be a light-emitting diode (LED), laser source, incandescent lamp, or gas discharge lamp. The light source system may include a power control device to control the light output power. The light source system may include wavelength-selective optical filters to ensure that its output light is within the desired wavelengths. The light source system may include optical devices to focus and / or collimate its output photon flux. In some cases, the excitation light source can generate multiple light beams with different wavelength. In some cases, one of the multiple light beams can switch a photo-switchable moiety to a first conformation, another one of the multiple light beams can switch the photo-switchable moiety to a second conformation, and the first conformation is different from the second conformation. In some embodiment, still another of the multiple light beams can excite a label on the analyte or the probe, for example, a FRET donor fluorophore.

[0067] Temperature controller

[0068]

[0050] A temperature controller can establish a specific temperature for the aqueous solution in the reaction chamber, and / or create a temperature profile that requires heating and / or cooling. A temperature controller can include a feedback control system that measures the temperature, using temperature sensors (such as a thermistor or a thermocouple), and, based on the measured temperature, add or remove heat from the reaction chamber using thermal devices (such as WSGR Docket No: 63452-705.601

[0069] Peltier devices or resistive heaters). Temperature controllers can comprise heat sinks for removing heat.

[0070]

[0051] Temperature controllers can change the temperature of a substrate or reaction chamber. The rate of temperature change (increase or decrease) can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C / second. The rate of temperature change (increase or decrease) can be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C / second. The rate of temperature change (increase or decrease) can be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C / second.

[0071] Temperature controllers can change temperature (increase or decrease) at a linear rate (e.g., 5°C / second). Alternatively, temperature controllers can change temperature (increase or decrease) at a non-linear rate. Temperature controllers can increase or decrease temperature.

[0072]

[0052] In some embodiments, the temperature controller is optional.

[0073] Detection system

[0074]

[0053] Also provided herein is a detection system having at least one detector that is configured to capture, detect and / or monitor signals from an array or a reaction chamber. Various signals may be produced, such as optical or electromagnetic signals. The signals may be correlated with the presence, amount, concentration, and / or binding characteristics of one or more species (e.g., primers, amplicons, nucleic acid sequences, reporter molecules, polymerases, dNTPs, antigens, peptides, proteins, or any other analytes and reagents). The signals can be reflective or indicative of the progress of one or more reactions (e.g., PCR amplification or ligase chain reaction). The signals can be detected at a single time point or multiple time points. The signals can be detected in real-time.

[0075]

[0054] The detection system may comprise a single detector or a plurality of detectors (e.g., an array of detectors). The detector(s) may be fixed or movable. The detectors may be comprised in the integrated sensors of the sensor array. Depending upon the type of signals to be detected, various types of detectors may be used, for example, optical detectors, electrical detectors, electrochemical detectors, or electrostatic detectors. Examples of optical detectors may include but not limited to charge-coupled device (CCDs) arrays (including cooled CCDs), complementary metal-oxide-semiconductor (CMOS) imagers, n-type metal-oxide semiconductor (NMOS), active-pixel sensors (APS), or photomultiplier tubes (PMTs). The detectors can also include wavelength-selective components such as optical filters to allow measurement of selective wavelengths. Examples of other detectors may include electrodes.

[0076]

[0055] The detector can sample (e.g., acquire measurements) at a rate of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, WSGR Docket No: 63452-705.601

[0077] 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, 300, 400, 500, 1000, 10,000, or 100,000 times per minute. The detector can sample at a rate of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 Hz.

[0078]

[0056] The detection system can comprise a light source. The light source can comprise at least one lamp, such as an incandescent, halogen, fluorescent, gas-discharge, arc, or light emitting diode (LED). The light source can comprise a laser. The light source can produce a specific wavelength or range of wavelengths, such as UV. The light source can comprise filters for controlling the output wavelength or wavelengths. The light source can comprise multiple light sources, of the same or of different types, which can be used separately or in combination.

[0079]

[0057] The detector can comprise various optical elements, including but not limited to filters, lenses, collimators, mirrors, reflectors, beam splitters, and diffusers. The detector can comprise a filter or a plurality of filters, including but not limited to wavelength filters (e.g., color filters, UV filters, IR filters), dichroic filters, and polarizing filters. The filters can comprise multiple filters, of the same or of different types, which can be used separately or in combination. The detector can comprise elements (e.g., signal processing unit) for removing image distortion or aberration, such as barrel or fisheye distortion, pincushion distortion, mustache distortion, monochromatic aberrations (e.g., piston, tilt, defocus, spherical aberration, coma, astigmatism, field curvature, image distortion), or chromatic aberrations (e.g., axial, longitudinal, lateral, transverse). Such elements can comprise computer systems programmed to implement instructions for partially or fully correcting image distortion. For example, Brown’s distortion model or the Brown-Conrady model can be used to correct for radial distortion and tangential distortion. In some examples, the detector can measure emitted photons coming from individual addressable locations. These photons can be correlated to the presence and / or activity of reporter molecules in that location.

[0080]

[0058] Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorimeter. Excitation to initiate energy transfer, or to allow direct detection of a fluorophore, can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range. WSGR Docket No: 63452-705.601

[0081] Computer Systems

[0082]

[0059] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. The computer systems can be the controls for the disclosed system for sequence specific nucleic acid amplification. FIG. 8 shows a computer system 801 that is programmed or otherwise configured to implement the methods described herein, e.g., methods for detecting a presence or absence of an analyte or a target nucleic acid. The computer system 801 can regulate various aspects of excitation light generation, acquisition of output signal, and signal processing of the present disclosure, such as, for example, generating pulsed excitation light for the detection of a presence or absence of an analyte or for the ligated product of oligonucleotide probes. The computer system 801 part of a system configured for detection of an analyte. The computer system may be integrated with the detection system. Alternatively, or in addition to, the computer system may be an external computer system coupled to the detection system via wired connection or wireless connection (e.g., WiFi or Bluetooth connection).

[0083]

[0060] The computer system 801 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 805, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 801 also includes memory or memory location 810 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 815 (e.g., hard disk), communication interface 820 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 825, such as cache, other memory, data storage and / or electronic display adapters. The memory 810, storage unit 815, interface 820 and peripheral devices 825 are in communication with the CPU 805 through a communication bus (solid lines), such as a motherboard. The storage unit 815 can be a data storage unit (or data repository) for storing data. The computer system 801 can be operatively coupled to a computer network (“network”) 830 with the aid of the communication interface 820. The network 830 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 830 in some cases is a telecommunication and / or data network. The network 830 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 830, in some cases with the aid of the computer system 801, can implement a peer-to-peer network, which may enable devices coupled to the computer system 801 to behave as a client or a server.

[0084]

[0061] The CPU 805 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 810. The instructions can be directed to the CPU 805, which can subsequently program or otherwise configure the CPU 805 to implement methods of the present disclosure. WSGR Docket No: 63452-705.601

[0085] Examples of operations performed by the CPU 805 can include fetch, decode, execute, and writeback.

[0086]

[0062] The CPU 805 can be part of a circuit, such as an integrated circuit. One or more other components of the system 801 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0087]

[0063] The storage unit 815 can store files, such as drivers, libraries and saved programs. The storage unit 815 can store user data, e.g., user preferences and user programs. The computer system 801 in some cases can include one or more additional data storage units that are external to the computer system 801, such as located on a remote server that is in communication with the computer system 801 through an intranet or the Internet.

[0088]

[0064] The computer system 801 can communicate with one or more remote computer systems through the network 830. For instance, the computer system 801 can communicate with a remote computer system of a user (e.g., laboratory technician, researcher, etc.). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 801 via the network 830.

[0089]

[0065] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 801, such as, for example, on the memory 810 or electronic storage unit 815. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 805. In some cases, the code can be retrieved from the storage unit 815 and stored on the memory 810 for ready access by the processor 805. In some situations, the electronic storage unit 815 can be precluded, and machine-executable instructions are stored on memory 810.

[0090]

[0066] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0091]

[0067] Aspects of the systems and methods provided herein, such as the computer system 801, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. WSGR Docket No: 63452-705.601

[0092] “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0093]

[0068] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0094]

[0069] The computer system 801 can include or be in communication with an electronic display 835 that comprises a user interface (UI) 840 for providing, for example, operating parameters of WSGR Docket No: 63452-705.601

[0095] the system, system status, or outputs of methods described elsewhere herein. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0096]

[0070] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 805. The algorithm can, for example, process output signals for determination of a presence or absence of an analyte.

[0097] Nucleic acid construct

[0098]

[0071] The term “nucleic acid construct,” or “NA construct,” as used herein, generally refers to nucleic acid (NA) molecules that comprise 1) one or more photo-switchable moieties that can reside in a first confirmation (e.g., confirmation A) upon exposure to an excitation beam of a first wavelength, and can reside in a second confirmation (e.g., confirmation B) upon exposure to an excitation beam of a second wavelength; and 2) one or more DNA or RNA molecules covalently linked to the one or more photo-switchable moieties. When photons of light of the first wavelength are applied to the one or more photo-switchable moieties in the nucleic acid construct, the photo-switchable moieties may switch to conformation A, which in turn changes the biochemical properties of the NA construct. For example, the photons of light of the first wavelength can cause conformational changes of the photo-switchable moieties in the NA construct by rotating or moving or flipping or changing relative positions of the photo-switchable moieties with regard to the nucleic acid to which the photo-switchable moieties are bound. Such conformational change may enable or disable hybridization (binding) of the affected nucleic acid construct to its hybridization target / partner (e.g., template nucleic acid). The NA construct can be any of the plurality of oligonucleotide constructs, or a ligated product of two or more of the plurality of oligonucleotide constructs. The NA constructs typically reside in a reaction chamber to which photons of light can be applied to by an excitation light source.

[0099]

[0072] The NA construct can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 NA molecules. The NA construct can comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 NA molecules. The NA construct can comprise more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 NA molecules.

[0100] Photo-switchable moieties

[0101]

[0073] In some embodiments, the present disclosure provides NA constructs with one or more photo-switchable moieties. In some embodiments, a photo-switchable moiety can undergo photoinduced isomerization reactions (e.g., transacts, denoted as E^Z henceforth) which exhibit efficient relaxation mechanisms mediated via internal conversion (IC), yielding either WSGR Docket No: 63452-705.601

[0102] the starting isomer or its photoisomer counterpart depending on the wavelength of the excitation light. In some embodiments, a photo-switchable moiety can undergo photoinduced isomerization reactions (e.g., cis trans, denoted as Z^E henceforth).

[0103]

[0074] Due to the efficient relaxation mechanism, photoisomerization can be used as a targeted photoinduced reaction mechanism with efficient and safe dissipation of absorbed energy in many applications including photo-switchable moieties. The two isomers may have different confirmations: one conformation may enable efficient binding of two partners (e.g., hybridization of two complementary nucleic acids); the other confirmation may disrupt efficient binding of two partners, thereby dissociating one partner from the other (e.g., breaking up the hybridization complex of two complementary nucleic acids to produce two single-stranded partners) or impeding the functions of an enzyme catalyzing biochemical reactions between two reactants. In the latter case, different conformation of the photo-switchable moiety may increase or decrease the binding affinity of the NA construct to the enzyme. The movement of the photo-switchable moiety may push the NA construct away from the catalytic site of the enzyme due to steric hinderance introduced by the new conformation of the photo-switchable moiety.

[0104] Examples of photo-switchable moieties

[0105]

[0075] For example, azobenzene derivatives may be suitable photo-switchable molecules that are also readily available and chemically stable. A planar trans form can be obtained upon photo-irradiation at wavelength X > 400 nm and a nonplanar cis form is obtained by photoirradiation at X from 300 nm to 400 nm. Accordingly, azobenzene can reversibly photoisomerize between trans and cis forms upon irradiation with the appropriate spectra. See Scheme 1 below. In other words, the co-planar conformation can be photo- switched to a nonplanar conformation. Depending on the binding interaction between an enzyme and a nucleic acid (NA) construct, these two different conformations can enable or disable the binding interaction between the enzyme and the NA construct.

[0106]

[0076] Scheme 1: Photo-isomerization of azobenzene between the planar trans form and nonplanar cis form:

[0107] > >

[0108] >

[0109]

[0110]

[0077] It has been shown that azobenzene can be utilized as a photo-switch in a DNA-based duplex formation [references 14-16], In this method, phosphoramidite monomer are synthesized bearing an azobenzene group covalently connected through an amide bond to D-threoninol as WSGR Docket No: 63452-705.601

[0111] the scaffold. Use of this monomer allows the simple introduction of an azobenzene construct into DNA.

[0112]

[0078] To provide the functionality of azobenzene in DNA, the azobenzene cartridge (indicated as “X”) may be introduced between base pairs of the DNA to replace a phosphate diester linkage. In some embodiments, the azobenzene cartridge X may be an azobenzene moiety attached to a prochiral or chiral 2,2-bis(hydroxymethyl)propionic acid via an amide linker. Then X can be inserted between the base pairs of an oligonucleotide and replace a phosphate diester linkage, thus providing an oligonucleotide construct. See Scheme 2. For example, one way to provide 5’-ACGTGTCG-3’ with photo-switching capability is to introduce the X residue and obtain an oligonucleotide construct, e.g., 5’-ACGTXGTCG-3’. The modified DNA strand can still form a duplex with its complementary strand, 3’-TGCACAGC-5’ and all the base pairs may be maintained in the duplex thus formed. It was reported that replacing a natural nucleotide with X is not recommended as this may cause destabilization of the duplex. Planar / ra / / .s-azobenzene stabilizes the double-stranded DNA as it intercalates in the duplex whereas the non-planar cisazobenzene destabilizes the duplex due to steric hindrance [reference 17],

[0113]

[0079] Scheme 2: Examples of an azobenzene moiety linked to 2,2-bis(hydroxymethyl)propionic acid (X)

[0114]

[0115]

[0080] In order to attain efficient photo-switching, introduction of multiple azobenzene residues may be more effective. In some embodiments, a complex such as 5’-ACXGTXGTXCG-373’-TGCACACGC-5’ may have multiple azobenzene residues in one strand (FIG. 1). In some embodiments, if both strands can be modified, sequence design such as 5’-ACXGTXGTXCG-373’-TXGCXACXAGXC-5’ may be particularly effective. Using these design strategies, repetitive and efficient regulation of hybridization and dissociation of the DNA duplex can be promoted by alternating between two wavelengths to assess to the two different conformations, one enabling the DNA duplex formation, the other disrupting the DNA duplex. In addition to controlling the formation or dissociation of DNA duplexes, photo-control of the hybridization of DNA / RNA and RNA / RNA duplexes and triplex DNA can also be achieved by introducing azobenzene as a photo-responsive switch [references 18-19], As shown in FIG. 1, photo- WSGR Docket No: 63452-705.601

[0116] isomerization of azobenzene can be used as a mechanism for photo-switching of DNA hybridization (duplex formation). To form the duplex, radiating the reaction chamber and the aqueous solution comprising the photo-switchable oligonucleotide construct (5’-ACXGTXGTXCG-3’) in the Cis conformation with a light beam having a wavelength (X) > 400 nm may change the photo-switchable oligonucleotide construct (5’-ACXGTXGTXCG-3’) from the Cis conformation to the Trans conformation. The Cis conformation in this case may prevent the duplex to be formed (mitigated base-pairing with the hybridization partner due to steric effect) while the Trans confirmation may enable the formation of the duplex with the hybridization partner.

[0117]

[0081] Examples of photo-switchable moieties include, but are not limited to, azobenzene [reference 1], spiropyran [reference 2], merocyanine [reference 3], diarylethene [reference 4], spirooxazine [reference 5], fulgide [reference 6], hydrazone [reference 7], nobormadiene [reference 8], thioindigo [reference 9], acrylamide-azobenzene-quaternary ammonia [reference 10], donor-acceptor Stenhouse adducts [reference 11-12], stilbene [reference 13], etc. Some examples are shown in Scheme 3.

[0118]

[0082] Scheme 3: Examples of additional photo-switchable moieties (X) replacing the phosphate diester moiety

[0119]

[0120] WSGR Docket No: 63452-705.601

[0121] Photo-triggered ligase chain reaction

[0122]

[0083] The hybridization and dissociation of the DNA duplex with the help from photoswitches, promoted by alternating between spectra of light, can be used to amplify nucleic acids.

[0123]

[0084] Ligase chain reaction (LCR) is an amplification method. In a known example, two ligatable pairs of oligonucleotides known as LCR primers or probes, are employed in excess over the target nucleotide sequence, where one pair of the LCR primers are hybridizable to the other. Template containing the target nucleotide sequence is first denatured if the template is double-stranded. Then the LCR primers are allowed to hybridize to their respective complementary strands, and the hybridized primers are then ligated by DNA ligase to form LCR products. The LCR products are then dissociated thermally from the template (i.e., by temporary heating the sample) that can consequently function as target nucleotide sequences themselves. By repeated cycles of hybridization and ligation, amplification of the target nucleotide sequence is achieved.

[0124]

[0085] In one embodiment of the present disclosure, DNA amplification using ligase chain reaction (LCR) is enabled through photo-regulation of photo-switchable moieties. The minimum building blocks that are required are four (4) DNA oligonucleotide probes with sequences X, Y, X and Y (X is complementary to X, Y is complementary to Y), each of which may comprises one or more photo-switchable moieties; a template nucleic acid that comprises one strand comprising the sequence 5’-X + F-3’ and another strand comprising the complementary sequence 3’-X + F-5’ (see FIG. 2); and a DNA ligase enzymatic system (e.g., one or more ligase enzymes). In this embodiment, the light source may facilitate hybridization and disassociation of the oligonucleotide probes from the template and / or the amplicons based on the sequential radiation of two light beams with different wavelength upon the aqueous solution of the reaction chamber. In some embodiments, only one strand of the template nucleic acid is present in the aqueous sample. In some embodiments, both strands of the template nucleic acid are present in the aqueous sample. In some embodiments, the photo-switchable moieties on oligonucleotide probes with sequences X and Y are the same. In some embodiments, the photo-switchable moieties on oligonucleotide probes with sequences X and Y are the same. In some embodiments, the photo-switchable moieties on oligonucleotide probes with sequences X and Y are the same as those on oligonucleotide probes with sequences X and Y. In some embodiments, the photo-switchable moieties on oligonucleotide probes with sequences X and Y are different from those on oligonucleotide probes with sequences X and Y such that the photo-switchable moieties on oligonucleotide probes with sequences X and Y can be selectively photoisomerized without affecting the conformations of the photo-switchable moieties on oligonucleotide probes with sequences X and Y. Alternatively, In some embodiments, the photo-switchable moieties on WSGR Docket No: 63452-705.601

[0125] oligonucleotide probes with sequences X and Y are different from those on oligonucleotide probes with sequences X and Y such that the photo-switchable moieties on oligonucleotide probes with sequences X and Y can be selectively photoisomerized without affecting the conformations of the photo-switchable moieties on oligonucleotide probes with sequences X and Y. In some embodiments, ligase chain reactions using the oligonucleotide probes with sequences X and Y can be independently performed in the presence of oligonucleotide probes with sequences X and Y. In some embodiments, ligase chain reactions using the oligonucleotide probes with sequences X and Y can be independently performed in the presence of oligonucleotide probes with sequences X and Y.

[0126]

[0086] In FIG. 3, the photo-triggered ligation of oligonucleotide probes with sequences X and Y using the template sequence is shown. The generated ligated product of this process, i.e., amplicon, is 5’-X + Y. At the start of this process, the photo-switchable moieties of both oligonucleotide probes with sequences X and Y stay at the Cis conformation preventing duplex formation with the target single-stranded nucleic acid template. Upon radiating with an excitation light beam with a first wavelength (e.g., > 400 nm), the photo-switchable moieties of both oligonucleotide probes with sequences X and Y switch to the Trans confirmation enabling duplex formation between both oligonucleotide probes and the single-stranded nucleic acid template, thereby allowing ligation of the two oligonucleotide probes bound to the nucleic acid template by the ligase enzyme. Radiating with another excitation light beam at a second wavelength (e.g., with 400 nm > wavelength > 300 nm) switches the photo-switchable moieties on the ligated product (amplicon, 5’- + F) comprising sequences X and Y to the Cis conformation, thereby dissociating the ligated product 5'-X + Y from the single-stranded nucleic acid template.

[0127]

[0087] In FIG. 4, the photo-triggered ligation of oligonucleotide probes with sequences X and Y using the template sequence is shown. The generated product of this process, i.e., amplicon is 3’-X + Y. At the start of this process, the photo-switchable moieties of both oligonucleotide probes with sequences X and Y stay at the Cis conformation preventing duplex formation with the target single-stranded nucleic acid template. Upon radiating with an excitation light beam with a first wavelength (e.g., > 400 nm), the photo-switchable moieties of both oligonucleotide probes with sequences X and Y switch to the Trans confirmation enabling duplex formation between both oligonucleotide probes and the single-stranded nucleic acid template, thereby allowing ligation of the two oligonucleotide probes bound to the nucleic acid template by the ligase enzyme. Radiating with another excitation light beam with 400 nm > wavelength > 300 nm switches the photo-switchable moieties on the ligated product (amplicon, 3’-X + Y ) WSGR Docket No: 63452-705.601

[0128] comprising sequences X and Y to the Cis conformation, thereby dissociating the ligated product from the single-stranded nucleic acid template.

[0129]

[0088] In some embodiment, the amplicons (ligated product or ligated oligonucleotide constructs) thus made using the target template sequence can become the templates for additional rounds of amplification via the ligase chain reaction. In FIG. 5 and FIG. 6, the phototriggered ligation of oligonucleotide probes and amplicons are shown.

[0130]

[0089] In FIG. 5, the photo-triggered ligation of oligonucleotide probes with sequences X and Y using the amplicon 3’-X + Y is shown. The generated ligated product of this process, i.e., amplicon, is amplicon, 5'-X + Y ). At the start of this process, the photo-switchable moi eties of both oligonucleotide probes with sequences X and Y and the amplicon 3’-X + Y stay at the Cis conformation preventing duplex formation with the amplicon 3’-X + Y. Upon radiating with an excitation light beam e.g., with a wavelength > 400 nm, the photo-switchable moieties of both oligonucleotide probes with sequences X and Y and the amplicon 3’-X + Y switch to the Trans confirmation enabling duplex formation between both oligonucleotide probes and the amplicon 3’-X + Y, thereby allowing ligation of the two oligonucleotide probes bound to the amplicon 3’-X + Y by the ligase enzyme. Radiating with another excitation light beam e.g., with 400 nm > wavelength > 300 nm switches the photo-switchable moieties on the ligated product (amplicon, 5’-X + Y ) comprising sequences X and Y and amplicon 3’-X + Y to the Cis conformation, thereby dissociating the ligated product 5’-X + Y from the amplicon 3’-X + Y.

[0131]

[0090] In FIG. 6, the photo-triggered ligation of oligonucleotide probes with sequences X and Y using the amplicon, 5’-X + Y is shown. The generated ligated product of this process, i.e., amplicon, is 3’-X + Y. At the start of this process, the photo-switchable moieties of both oligonucleotide probes with sequences X and Fand the amplicon 5’-X + Y stay at the Cis conformation preventing duplex formation with the amplicon 5’-X + Y. Upon radiating with an excitation light beam e.g., with a wavelength > 400 nm, the photo-switchable moieties of both oligonucleotide probes with sequences X and Y and the amplicon 5’-X + Y switch to the Trans confirmation enabling duplex formation between both oligonucleotide probes and the amplicon 5’-X + Y, thereby allowing ligation of the two oligonucleotide probes bound to the amplicon 5’-X + Y by the ligase enzyme. Radiating with another excitation light beam e.g., with 400 nm > wavelength > 300 nm switches the photo-switchable moieties on the ligated product (amplicon, 3’-X + F.) comprising sequences X and Y and amplicon 5’-X + Y to the Cis conformation, thereby dissociating the ligated product 3’-X + Y from the amplicon 5’-X + Y. WSGR Docket No: 63452-705.601

[0132] Methods of detection

[0133]

[0091] In some embodiments, the concentration of the amplicons during the amplification process is measured over time. This data can then be used to evaluate the presence and / or concentration of selected target template. One approach to implement that is to use a minimum threshold level for the measured amplicons and use the time (or cycles) of the amplification to reach that as an indicator of the initial concentration. This method is generally referred to as the “threshold cycle method” which is widely used in qPCR reaction that use fluorogenic probes [references 20-23],

[0134]

[0092] For example, in real-time PCR the amount of product formed can be monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules can be registered. Assuming a certain amplification efficiency, for example, about a doubling of the number of molecules per amplification cycle, it may be possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays the number of DNA molecules of a particular sequence in a complex sample can be determined with accuracy and sensitivity sufficient to detect a single molecule.

[0135]

[0093] Regarding dyes, there are a variety of dyes available for detection. For example, two intercalating dyes SYTO-13 and SYTO-82 have the following attributes: they do not inhibit PCR. They show no preferential binding to GC rich sequences and do not influence melting temperature, Tm, even at high concentrations. In addition, SYTO-82 demonstrated a 50-fold lower detection limit in a dilution series assay. The properties of SYTO-82 and SYTO-13 simplify the development of multiplex assays and increase the sensitivity of real-time PCR.

[0136]

[0094] The 5’ to 3’ exonuclease activity of the thermostable enzyme Thermus aquaticus (T. aquaticus) DNA polymerase may be employed in a ligase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. In this case, an oligonucleotide probe, nonextendable at the 3’ end, labeled at the 5’ end, and designed to hybridize within the target sequence, is introduced into the ligase chain reaction assay.

[0137] Annealing of probe to one of the ligase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5’ to 3’ exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. This assay is sensitive and specific enough to identify the target nucleic acid. WSGR Docket No: 63452-705.601

[0138]

[0095] In an aspect, the present disclosure provides a method for sequence-specific nucleic acid amplification. The method comprises radiating an aqueous sample enclosed in a reaction chamber with a first excitation light beam having a first wavelength. In some embodiments, the aqueous sample may comprise a nucleic acid template. In some embodiments, the aqueous sample may comprise a plurality of oligonucleotide constructs. In some embodiments, an oligonucleotide construct of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, each oligonucleotide construct of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, the aqueous sample may comprise one or more enzymes configured to interact with the nucleic acid temple and / or the plurality of oligonucleotide constructs.

[0139]

[0096] In some embodiments, radiating with the first excitation light beam may change a confirmation of an oligonucleotide construct. In some embodiments, the method may comprise, via the radiating with a first excitation light beam, changing one or more photo-switchable moieties of a first oligonucleotide construct of the one or more oligonucleotide constructs into a first conformation. In some embodiments, the method may comprise, via the radiating with the first excitation light beam, changing one or more photo-switchable moieties of a second oligonucleotide construct of the one or more oligonucleotide constructs into a first conformation. In some embodiments, changing the one or more photo-switchable moieties of the first oligonucleotide construct and the second oligonucleotide construct may facilitate a binding or hybridization of the first oligonucleotide and / or the second oligonucleotide construct with the nucleic acid template.

[0140]

[0097] In some embodiments, the method may comprise ligating the first oligonucleotide construct with the second oligonucleotide construct. In some embodiments, the one or more enzymes may comprise a ligation enzyme configured to ligate the first oligonucleotide construct and the second oligonucleotide construct under a ligation condition, thereby producing the ligated construct. In some embodiments, the ligation condition may comprise binding of the first oligonucleotide construct and the second oligonucleotide construct to the nucleic acid template. In some embodiments, the binding may bring the first oligonucleotide construct and the second oligonucleotide construct within each other’s vicinity for the ligation enzyme to ligate the first oligonucleotide construct and the second oligonucleotide construct. In some embodiments, the ligation may create a ligated construct. In some embodiments, the ligated construct may remain bound to the nucleic acid template. In some embodiments, the method may further comprise ligating a third oligonucleotide construct of the plurality of oligonucleotide constructs with the WSGR Docket No: 63452-705.601

[0141] ligated construct. In some embodiments, the third oligonucleotide construct may comprise one or more photo-switchable moieties in the first confirmation. In some embodiments, the ligation of the third oligonucleotide construct and the ligated construct may generate an additional ligated construct. The additional ligated construct may comprise the ligated construct and the third oligonucleotide construct. In some embodiments, the method may further comprise repeating the ligation one or more times (e.g., ligating one or more additional oligonucleotide constructs to the additional ligated construct), thereby generating a complementary copy of at least a portion of the nucleic acid template. In some embodiments, the method may further comprise radiating the aqueous sample with a second excitation light beam having a second wavelength. In some embodiments, the method may further comprise switching the one or more photo-switchable moieties of the first oligonucleotide construct, the second oligonucleotide construct, and / or the third oligonucleotide construct to a second conformation. In some embodiments, the second confirmation of the one or more photo-switchable moieties of the first oligonucleotide construct, the second oligonucleotide construct, and / or the third oligonucleotide construct may facilitate a dissociation of the first oligonucleotide construct, the second oligonucleotide construct, and / or the third oligonucleotide construct from the nucleic acid template. In some embodiments, the second confirmation of the one or more photo-switchable moieties of the first oligonucleotide construct, the second oligonucleotide construct, and / or the third oligonucleotide construct may facilitate a dissociation of the ligated construct, the additional ligated construct, or the complementary copy from the nucleic acid template. In some embodiments, the method may further comprise repeating the above processes to generate a plurality of complementary copies of at least a portion of the nucleic acid template.

[0142]

[0098] In some embodiments, the method may further comprise controlling a temperature of the aqueous sample substantially the same. In some embodiments, the method may further comprise controlling a temperature of the reaction chamber substantially the same. In some embodiments, the method may further comprise a heating step within or immediately after the ligated construct, the additional ligated construct, or the complementary copy is generated to facilitate the dissociation from the nucleic acid template. In some embodiments, the heating step may raise a temperature of the reaction chamber or the aqueous sample up to a specific temperature at which at least about half, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the ligated construct, the additional ligated construct, or the complementary copy dissociates from the nucleic acid template in the absence of the one or more photo-switchable moieties. In some embodiments, the method may further comprise a cooling step after the heating step. In some embodiments, the cooling step may facilitate duplex WSGR Docket No: 63452-705.601

[0143] formation between the nucleic acid template and one or more oligonucleotide constructs of the plurality of oligonucleotide constructs, the ligated construct, and the additional ligated construct.

[0144]

[0099] In some embodiments, the method may further comprise using one or more of the ligated construct, the additional ligated construct, and the complementary copy as a template in a first additional ligation catalyzed by the one or more enzymes.

[0145]

[0100] In some embodiments, the method may further comprise detecting a presence or absence of the ligated construct, the additional ligated construct, and / or the complementary copy. In some embodiments, the first oligonucleotide construct may comprise a first moiety of a fluorescent resonance energy transfer (FRET) pair and the second oligonucleotide construct may comprise a second moiety of the FRET pair. The first moiety of the FRET pair may be a donor or acceptor. The second moiety of the FRET pair may be an acceptor or donor. In some embodiments, when the first oligonucleotide construct and the second oligonucleotide construct are within each other’s vicinity, the FRET pair may emit a FRET signal. In some embodiments, the method may comprise detecting a presence or absence of the FRET signal.

[0146]

[0101] In an aspect, the present disclosure provides a method of detecting a presence or absence of at least one analyte in a sample. The method may comprise radiating an aqueous sample with a first excitation light beam. In some embodiments, the first excitation light beam may comprise a first wavelength. In some embodiments, the aqueous sample may comprise the sample suspected to comprise at least one analyte. In some embodiments, the aqueous sample may comprise a plurality of oligonucleotide constructs disclosed herein. In some embodiments, an oligonucleotide construct of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties. In some embodiments, each of the plurality of oligonucleotide constructs may comprise one or more photo-switchable moieties as disclosed herein. In some embodiments, the aqueous sample may comprise a ligase enzyme configured to interact with the at least one analyte and / or the plurality of oligonucleotide constructs.

[0147]

[0102] In some embodiments, upon the radiation of the first excitation light beam, the one or more photo-switchable moieties may transform to a first confirmation. In some embodiments, the first confirmation of the one or more photo-switchable moieties may allow two or more oligonucleotide constructs bind or hybridize to the analyte if present. In some embodiments, after hybridizing to the analyte, the two or more oligonucleotide constructs may be ligated to form a ligated construct.

[0148]

[0103] In some embodiments, the method may comprise radiating the aqueous sample with a second excitation light beam having a second wavelength. In some embodiments, the second WSGR Docket No: 63452-705.601

[0149] confirmation may facilitate dissociation of the two or more oligonucleotide constructs from the analyte.

[0150]

[0104] In some embodiments, a first population of the plurality of oligonucleotide constructs may comprise a first moiety of a FRET pair. In some embodiments, a second population of the plurality of oligonucleotide constructs may comprise a second moiety of the FRET pair. In some embodiments, a first oligonucleotide construct comprising the first moiety of the FRET pair and a second oligonucleotide construct comprising the second moiety of the FRET pair may have sequence complementary to a first section and a second section of the at least one analyte, respectively. In some embodiments, the first section and the second section may be in tandem on the at least one analyte. In some embodiments, the first oligonucleotide construct and the second oligonucleotide construct may be hybridized to the at least one analyte. In some embodiments, the first excitation light beam may enable duplex formation between the first oligonucleotide construct and / or the second oligonucleotide construct, and the at least one analyte, thereby allowing a ligated oligonucleotide construct to be formed. In some embodiments, the first oligonucleotide construct comprising the first moiety of the FRET pair and the second oligonucleotide construct comprising the second moiety of the FRET pair may be in each other’s vicinity when hybridized to the analyte. In some embodiments, the method may comprise radiating the aqueous sample with a third excitation light beam having a third wavelength. In some embodiments, the FRET pair may be activated by the third excitation light beam having a third wavelength. In some embodiments, the third excitation light beam may excite the first moiety of the FRET pair (e.g., donor) on the first or second oligonucleotide construct. In some embodiments, the FRET pair may emit a FRET signal upon radiation by the third excitation light beam having a third wavelength. In some embodiments, radiating with the second excitation beam may be performed prior to radiating with the third excitation beam. In some embodiments, radiating with the second excitation beam may be performed subsequent to radiating with the third excitation beam. In some embodiments, radiating with the second excitation beam may be performed substantially concurrent with radiating with the third excitation beam. In some embodiments, radiating with the second excitation beam may transform the one or more photo-switchable moieties to a second confirmation.

[0151]

[0105] In some embodiments, the FRET signal may be configured to be generated when the first oligonucleotide construct and the second oligonucleotide construct of the one or more oligonucleotide constructs are ligated in the presence of the at least one analyte. In some embodiments, the method may comprise detecting the presence or absence of the FRET signal. In some embodiments, a presence of the FRET signal may indicate a presence of the at least one analyte. WSGR Docket No: 63452-705.601

[0152] Labels and dyes

[0153]

[0106] A label or detectable label that is associated with nucleic acid constructs ad described herein may be any moiety that comprises one or more appropriate chemical substances or enzymes that directly or indirectly generate a detectable signal in a chemical, physical or enzymatic reaction.

[0154]

[0107] A wide variety of fluorescent molecules (e.g., small molecules, fluorescent proteins and quantum dots) can be utilized in the present disclosure. Non-limiting examples of fluorescent molecules (or fluorophores) may include: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs — AutoFluorescent Protein — (Quantum Biotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexion; Alizarin Red;

[0155] Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;

[0156] Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; Aminomethylcoumarin (AMCA); APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG;

[0157] Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™- 1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-SN; Calcein; CalceinBlue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca.sup.2+Dye; Calcium Green-2 Ca.sup.2+; Calcium Green-SN Ca.sup.2+; Calcium Green-C18 Ca.sup.2.sup.+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; WSGR Docket No: 63452-705.601

[0158] CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF;

[0159] CMFDA; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD — Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxystilbamidine (FluoroGold);

[0160] Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin;

[0161] Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; WSGR Docket No: 63452-705.601

[0162] Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PL); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2;

[0163] Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL- 1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectrum Aqua;

[0164] Spectrum Green; SpectrumOrange; Spectrum Red; SPQ (6-m ethoxy -N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOXBlue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5;

[0165] Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), Alexa Fluor dye series (e.g., Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750), Cy Dye fluorophore series (e.g., Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), Oyster dye fluorophores (e.g., Oyster-500, -550, -556, 645, 650, 656), DY-Labels series ( e.g., DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL), ATTO fluorescent labels (e.g., ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 61 IX, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740), CAL Fluor and Quasar dyes (e.g., CAL Fluor Gold 540, CAL Fluor Orange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 670), quantum dots (e.g., Qdot 525, Qdot565, Qdot585, Qdot605, Qdot655, Qdot705, Qdot 800), fluorescein, rhodamine, phycoerythrin, or combinations thereof. WSGR Docket No: 63452-705.601

[0166]

[0108] A variety of different report molecules or label moieties are readily employed in forming a FRET pair (donor and acceptor pair for FRET). The FRET pair can comprise a first moiety and a second moiety. One of the first moiety and the second moiety may be a donor. One of the first moiety and the second moiety may be an acceptor. Such groups may include fluorescein labels, rhodamine labels, cyanine labels (i.e., Cy3, Cy5, and the like, generally available from the Amersham Biosciences division of GE Healthcare), the Alexa family of fluorescent dyes and other fluorescent and fluorogenic dyes available from Molecular Probes / Invitrogen / Life Technologies, Inc., and described in ‘The Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Eleventh Edition’ (2010) (available from ThermoFisher), semiconductor nanocrystals and other nanoparticle labels (e.g., Qdot® nanocrystals available from Invitrogen, Inc. (Life Technologies)). A variety of other labeling moieties for use with labeled compounds (e.g., nucleoside polyphosphates and other biomolecules and reaction components), and which would be applicable to the compounds of the present invention are described in, e.g., Published U.S. Patent Application No. 2003 / 0124576, the full disclosure of which is incorporated herein in its entirety for all purposes. Additional examples of useful FRET labels include, e.g., those described in U.S. Pat. Nos. 5,654,419, 5,688,648, 5,853,992, 5,863,727, 5,945,526, 6,008,373, 6,150,107, 6,335,440, 6,348,596, 6,479,303, 6,545,164, 6,849,745 and 6,696,255, and Published U.S. Patent Application No. 2003 / 0143594, the disclosures of which are incorporated herein by reference for all purposes. In some embodiments, the chromophores in a FRET pair are fluorophores. Molecules that can be used in FRET may include fluorophores as described above, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL), and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). For example, a FRET pair may comprise a Cy3 donor dye and Cy3.5 acceptor dye, a Cy5 donor dye and a Cy5.5 acceptor dye. ALEXA Fluors (Molecular Probes / Invitrogen) and DYLIGHT Fluors (Thermo Fisher Scientific). These fluorophores have emission spectra that span a wide range, including ultraviolet, near-ultraviolet, visible, near-infrared, and infrared ranges. Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B -phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1 -pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2-,2'-disulfonic acid derivatives, chelates of Lanthanide ions (e.g., Europium, Dysprosium, Samarium or Terbium). Representative acceptor fluorescent moieties, depending upon the donor fluorescent moiety used, include LC-Red 640, LC-Red 705, Cy5, WSGR Docket No: 63452-705.601

[0167] Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, allophycocyanin, XL665, d2. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0168]

[0109] In some cases, the acceptor of the FRET pair may be used to quench the fluorescence of the donor. The acceptor may have little to no fluorescence. In some cases, the FRET acceptors that are useful for quenching may be referred to as quenchers. Non-limiting examples of quenchers may include Black Hole Quencher Dyes (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10), QSY Dye fluorescent quenchers (e.g., QSY7, QSY9, QSY21, QSY35), Dabcyl and Dabsyl, Cy5Q, Cy7Q, Dark Cyanine dyes (which can be used, for example, in conjunction with donor fluorophores such as Cy3B, Cy3, or Cy5), DY-Quenchers (e.g., DYQ-660 and DYQ-661), ATTO fluorescent quenchers (e.g., ATTO 540Q, 580Q, 612Q), or combinations thereof.

[0169] [HO] FRET between two different fluorophores can be assayed by several methods: looking at the change in color of the fluorescence, measuring the fluorescence lifetime of the donor, examining the changes upon photobleaching either the donor or acceptor or both donor / acceptor fluorophore. Regardless of the approach, most of these assays share common features of the instrumentation. Examples of such are the EnVision Plate Reader (Molecular Devices), ViewLux ultraHTS Microplate Imager (PerkinElmer), OPTIMA Microplate Readers, FLUOstar and POLARstar (BMG Labtech). Preferred measurement is by time-resolved fluorimetry.

[0170] [Hl] FRET between two different fluorophores can be assayed by high-content cell screening using an instrument that detects changes in fluorescence in cells or in particular subcellular localizations. Examples of such instrumentation are the INCell Analyzer (GE Healthcare), ImageXpress Micro High Content Screening System (Molecular Devices), Opera, Operetta (PerkinElmer), Cellomics ArrayScan VTI HCS Reader (Thermo Scientific).

[0171]

[0112] A large variety of labels are well known in the art. (See, for instance, PCT / GB2007 / 001770).

[0172]

[0113] For instance, one class of such labels is fluorescent labels. Fluorescent labels have the advantage of coming in several different wavelengths (colors) allowing distinguishably labeling each different terminator molecule. (See, for example, Welch etal., Chem. Eur. J.,5(3):951-960, 1999). One example of such labels is dansyl-functionalized fluorescent moieties. Another example is the fluorescent cyanine-based labels Cy3 and Cy5, which can also be used in the present disclosure. (See, Zhu et al., Cytometry, 28:206-211, 1997). Labels suitable for use are also disclosed in Prober etal., Science, 238:336-341, 1987; Connell et

[0173] al., BioTechniques, 5(4):342-384, 1987; Ansorge et al., NucL Acids Res., 15(11):4593-4602, WSGR Docket No: 63452-705.601

[0174] 1987; and Smith et al., Nature, 321:674, 1986. Other commercially available fluorescent labels include, but are not limited to, fluorescein and related derivatives such as isothiocyanate derivatives, e.g. FITC and TRITC, rhodamine, including TMR, texas red and Rox, bodipy, acridine, coumarin, pyrene, benzanthracene, the cyanins, succinimidyl esters such as NHS-fluorescein, maleimide activated fluorophores such as fluorescein-5-mal eimide, phosphoramidite reagents containing protected fluorescein, boron-dipyrromethene (BODIPY) dyes, and other fluorophores, e.g. 6-FAM phosphoramidite 2. All of these types of fluorescent labels may be used in combination, in mixtures and in groups, as desired and depending on the application.

[0175]

[0114] Various commercially available fluorescent labels are known in the art, such as Alexa Fluor Dyes, e.g., Alexa 488, 555, 568, 660, 532, 647, and 700 (Invitrogen-Life Technologies, Inc., California, USA, available in a wide variety of wavelengths, see for instance, Panchuk, et al., J. Hist. Cyto., 47:1179-1188, 1999). Also commercially available are a large group of fluorescent labels called ATTO dyes (available from ATTO-TEC GmbH in Siegen, Germany). These fluorescent labels may be used in combinations or mixtures to provide distinguishable emission patterns for all terminator molecules used in the assay since so many different absorbance and emission spectra are commercially available.

[0176]

[0115] In various exemplary embodiments, a label comprises a fluorescent dye, such as, but not limited to, a rhodamine dye, e.g., R6G, R1 10, TAMRA, and ROX, a fluorescein dye, e.g., JOE, VIC, TET, HEX, FAM, etc., a halo-fluorescein dye, a cyanine dye. e.g., CY3, CY3.5, CY5, CY5.5, etc., a BODIPY® dye, e.g., FL, 530 / 550, TR, TMR, etc., a dichloro-rhodamine dye, an energy transfer dye, e.g., BIGD YE™ v 1 dyes, BIGD YE™ v 2 dyes, BIGD YE™ v 3 dyes, etc., Lucifer dyes, e.g., Lucifer yellow, etc., CASCADE BLUE®, Oregon Green, and the like. Other exemplary dyes are provided in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Products, Ninth Ed. (2003) and the updates thereto. Non-limiting exemplary labels also include, e.g., biotin, weakly fluorescent labels (see, for instance, Yin et al., Appl Environ Microbiol., 69(7): 3938, 2003; Babendure et A . Anal. Biochem., 317(1): 1, 2003; and Jankowiak et al., Chem. Res. Toxicol., 16(3):304, 2003), non-fluorescent labels, colorimetric labels, chemiluminescent labels (see, Wilson et al., Analyst, 128(5):480, 2003; Roda etal., Luminescence ,18(2):72, 2003), Raman labels, electrochemical labels, bioluminescent labels (Kitayama et al., Photochem. Photobiol., 77(3):333, 2003; Arakawa et al., Anal. Biochem., 314(2):206, 2003; and Maeda, J. Pharm. Biomed. Anal., 30(6): 1725, 2003), and the like.

[0177]

[0116] Multiple labels can also be used in the disclosure. For example, bi-fluorophore FRET cassettes Tet. Letts., 46:8867-8871, 2000) are well known in the art and can be utilized in the WSGR Docket No: 63452-705.601

[0178] disclosed methods. Multi-fluor dendrimeric systems (J. Amer. Chem. Soc., 123:8101-8108, 2001) can also be used. Other forms of detectable labels are also available. For example, microparticles, including quantum dots (Empodocles, et al., Nature, 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem., 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Set. USA, 97(17):9461-9466, 2000), and tags detectable by mass spectrometry can all be used.

[0179]

[0117] Multi-component labels can also be used in the disclosure. A multi-component label is one which is dependent on the interaction with a further compound for detection. The most common multi-component label used in biology is the biotin-streptavidin system. Biotin is used as the label attached to the nucleotide base. Streptavidin is then added separately to enable detection to occur. Other multi-component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection.

[0180]

[0118] Thus, a “label” as presently defined is a moiety that facilitates detection of a molecule. Common labels in the context of the present disclosure include fluorescent, luminescent, lightscattering, and / or colorimetric labels. Suitable labels may also include radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350;

[0181] 3,996,345; 4,277,437; 4,275,149; and 4,366,241. As other non-limiting examples, the label can be a luminescent label, a light-scattering label (e.g., colloidal gold particles), or an enzyme (e.g., Horse Radish Peroxidase (HRP)).

[0182]

[0119] Fluorescence energy transfer (FRET) dyes may also be employed, such as DY-630 / DY-675 from Dyomics GmbH of Germany, which also commercially supplies many different types of dyes including enzyme-based labels, fluorescent labels, etc. (See, for instance, Dohm etal., “Substantial biases in ultra-short read data sets from high-throughput DNA sequencing,” Nucleic Acids Res., 36:el05, 2008). Other donor / acceptor FRET labels include, but are not limited to:

[0183] Donor Acceptor Ro (A)

[0184] Fluorescein Tetramethylrhodamine 55

[0185] IAEDANS Fluorescein 46

[0186] EDANS Dabcyl 33

[0187] Fluorescein Fluorescein 44

[0188] BODIPY FL BODIPY FL 57

[0189] Fluorescein QSY 7 and QSY 9 dyes 61 WSGR Docket No: 63452-705.601

[0190] (See also, Johansen, M. K., “Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers,” Methods in Molecular Biology, vol. 335:

[0191] Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, Edited by: V. V. Didenko, Humana Press Inc., Totowa, N. J.). Other dye quenchers are commercially available, including dabcyl, QSY quenchers and the like. (See also, Black Hole Quencher Dyes from Biosearch Technologies, Inc., Novato, Calif.; Iowa Black Dark Quenchers from Integrated DNA Technologies, Inc. of Coralville, Iowa; and other dye quenchers sold by Santa Cruz Biotechnology, Inc. of Dallas, Tex.).

[0192]

[0120] General synthetic routes

[0193]

[0121] The phosphate diester moiety as the photo-switchable moiety can be synthesized based on phosphoramidite-based DNA synthesis. The key step in this synthetic approach for DNA synthesis is the reaction of the phosphoramidite reacts with the terminal 5 ’-OH of another phosphoramidite of an oligonucleotide. Scheme 4 shows examples of the phosphoramidite reagents to be used in the synthesis of oligonucleosides.

[0194]

[0122] Scheme 4: Examples of selected phosphoramidite reagents of the present disclosure.

[0195]

[0196]

[0123] The label, such as, for example, FRET labels, are attached to the terminal nucleotide or nucleotides adjacent to the terminal nucleotide of some specific oligonucleotide construct.

[0197]

[0124] The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiment or aspect described herein. Indeed, many modifications and variations may be apparent to those WSGR Docket No: 63452-705.601

[0198] skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

[0199] EXAMPLES

[0200]

[0125] The following examples are set forth to illustrate more clearly the principle and practice of instances disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed instances. Unless otherwise stated, all parts and percentages are on a weight basis.

[0201] EXAMPLE 1. Using FRET Moiety in Solution

[0202]

[0126] To enable real time detection, a fluorescence energy transfer system (FRET) [references 24-26] is used to include a pair of fluorophores to quantify the generation of amplicons during the amplification.

[0203]

[0127] In one embodiment, the donor fluorophore and the acceptor fluorophore are placed on two of the four participating probes which do not have the reverse complementary sequence. Once the ligation happens and the two probes modified with the donor fluorophore and the acceptor fluorophore, respectively, are ligated, the donor fluorophore and acceptor fluorophore remain in the molecular proximity (within excitable distance). This established the FRET system which can be detected by exciting the donor and monitoring the emission of the acceptor. The emission signal in this case is proportional to the amount of amplicon formed. See FIG. 7A.

[0204]

[0128] FIG. 7A and FIG. 7B show two exemplary embodiments of this system. The difference is that in FIG. 7A, the donor and acceptor are on the same allele. However, in FIG. 7B, they are placed on different alleles. In both cases, the emission from the acceptor fluorophore can be detected only when the probes are brought within proximity (within excitable distance) after at least one round of successful ligation of two of the probes. In one embodiment according to FIG. 7A, the donor fluorophore and acceptor fluorophore are on the same allele. The probes can be ligated in the presence of the template nucleic acid and the ligase enzyme. When the two probes modified with the donor fluorophore and the acceptor fluorophore, respectively, are ligated, the donor fluorophore and acceptor fluorophore remain in the molecular proximity (within excitable distance) to enable FRET detection. This ligation can happen when the target nucleic acid template is present and after the photo-switchable moieties are switched to the conformations enabling duplex formation. Thus, the detection of FRET signal suggests that the template nucleic acid is present in the aqueous sample. WSGR Docket No: 63452-705.601

[0205]

[0129] In another embodiment according to FIG. 7B, the donor fluorophore and the acceptor fluorophore are placed on two of the four participating probes which have complementary sequences to form two pairs. The probes can be ligated in the presence of the template nucleic acid and the ligase enzyme. In this case, the donor fluorophore and the acceptor fluorophore are placed on different alleles. Thus, even after the two amplicons are formed, no FRET signal is detected when the two amplicons with complementary sequences remain single stranded.

[0206] Radiating the two amplicons with a selected wavelength to switch the conformations of all photo-switchable moiety to the conformation enabling duplex formation leads to the formation of duplex, thereby bringing the two participating probe sequences with the donor fluorophore and the acceptor fluorophore into proximity (within excitable distance) of FRET detection.

[0207] Thus, the detection of FRET signal suggests that the template nucleic acid is present in the aqueous sample.

[0208] Numerated Embodiments

[0209]

[0130] Embodiment 1. A system for sequence-specific nucleic acid amplification, comprising:

[0210] (i) a reaction chamber;

[0211] (ii) an aqueous sample enclosed in the reaction chamber, comprising:

[0212] (a) a nucleic acid template;

[0213] (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; the one or more photo-switchable moieties being configured to (1) switch to a first conformation when radiated by a first excitation light beam with a first wavelength; and (2) switch to a second conformation when radiated by a second excitation light beam with a second wavelength; and (c) one or more enzymes configured to interact with the nucleic acid temple and / or the plurality of oligonucleotide constructs; and

[0214] (iii) an excitation light source configured to emit at least the first excitation light beam with the first wavelength and the second excitation light beam with the second wavelength.

[0215]

[0131] Embodiment 2. The system of Embodiment 1, further comprising a temperature control configured to control temperature of the aqueous sample and / or the reaction chamber.

[0216]

[0132] Embodiment 3. The system of Embodiment 2, further comprising a control, wherein the control is configured to operate the temperature control and / or the excitation light source.

[0217]

[0133] Embodiment 4. The system of any one of Embodiments 1-3, wherein the one or more enzymes comprise a ligation enzyme configured to ligate a first oligonucleotide construct and a second oligonucleotide construct of the one or more oligonucleotide constructs under a ligation condition, thereby producing a ligated construct. WSGR Docket No: 63452-705.601

[0218]

[0134] Embodiment 5. The system of Embodiment 4, where the ligation condition comprises binding of the first oligonucleotide construct and the second oligonucleotide construct to the nucleic acid template, thereby bringing the first oligonucleotide construct and the second oligonucleotide construct within each other’s vicinity for the ligation enzyme to ligate the first oligonucleotide construct and the second oligonucleotide construct.

[0219]

[0135] Embodiment 6. The system of any one of Embodiments 1-5, wherein the first conformation of the one or more photo-switchable moieties of the first oligonucleotide construct allows the first oligonucleotide construct to bind to the nucleic acid template; and wherein the first conformation of the one or more photo-switchable moieties of the second oligonucleotide construct allows the second oligonucleotide construct to bind to the nucleic acid template.

[0220]

[0136] Embodiment 7. The system of Embodiment 6, wherein the second conformation of the one or more photo-switchable moieties of the first oligonucleotide construct enables the first oligonucleotide construct to dissociate from the nucleic acid template; and wherein the second conformation of the one or more photo-switchable moieties of the second oligonucleotide construct enables the second oligonucleotide construct to dissociate from the nucleic acid template.

[0221]

[0137] Embodiment 8. The system of any one of Embodiments 4-7, wherein the aqueous solution further comprises a third oligonucleotide construct configured to be ligated with the ligated construct under an additional ligation condition by the one or more enzymes, thereby producing an additional ligated construct.

[0222]

[0138] Embodiment 9. The system of Embodiment 8, wherein the additional ligation condition comprises binding of the third oligonucleotide construct and the ligated construct to the nucleic acid template, thereby bringing the third oligonucleotide construct and the ligated construct within each other’s vicinity for the ligation enzyme to ligate the third oligonucleotide construct and the ligated construct.

[0223]

[0139] Embodiment 10. The system of any one of Embodiments 4-9, wherein the ligation enzyme is configured to bind to the ligated construct and use the ligated construct as a template in a first additional ligation, thereby producing a complimentary copy of the ligated construct.

[0224]

[0140] Embodiment 11. The system of any one of Embodiments 8-10, wherein the ligation enzyme is configured to bind to the additional ligated construct and use the additional ligated construct as a template in a second additional ligation, thereby producing a complimentary copy of the additional ligated construct.

[0225]

[0141] Embodiment 12. The system of any one of Embodiments 1-11, wherein a first population of the plurality of oligonucleotide constructs comprises a first moiety of a fluorescent resonance WSGR Docket No: 63452-705.601

[0226] energy transfer (FRET) pair and a second population of the plurality of oligonucleotide constructs comprises a second moiety of the FRET pair.

[0227]

[0142] Embodiment 13. A method for sequence-specific nucleic acid amplification, comprising:

[0228] (i) radiating an aqueous sample enclosed in a reaction chamber with a first excitation light beam having a first wavelength, wherein the aqueous sample comprises:

[0229] (a) a nucleic acid template;

[0230] (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; and (c) one or more enzymes configured to interact with the nucleic acid temple and / or the plurality of oligonucleotide constructs;

[0231] (ii) changing, via the radiating in (i), one or more photo-switchable moieties of a first oligonucleotide construct of the one or more oligonucleotide constructs into a first conformation; changing one or more photo-switchable moieties of a second oligonucleotide construct of the one or more oligonucleotide constructs into the first conformation; and

[0232] (iii) subsequent to (ii), ligating, via the one or more enzymes, the first oligonucleotide construct with the second oligonucleotide construct; thereby providing a ligated construct, wherein the ligated construct remains bound to the nucleic acid template.

[0233]

[0143] Embodiment 14. The method of Embodiment 13, further comprising:

[0234] (iv) ligating a third oligonucleotide construct of the plurality of oligonucleotide constructs with the ligated construct from (iii), thereby producing an additional ligated construct comprising the ligated construct and the third oligonucleotide construct, wherein the third oligonucleotide construct comprises one or more photo-switchable moieties in the first confirmation.

[0235]

[0144] Embodiment 15. The method of Embodiment 14, further comprising: repeating (iv) one or more times, thereby producing a complementary copy of at least a portion of the nucleic acid template.

[0236]

[0145] Embodiment 16. The method of any one of Embodiments 13 to 15, further comprising:

[0237] (v) after (iii) or after (iv), radiating the aqueous sample with a second excitation light beam having a second wavelength, thereby switching the one or more photo-switchable moieties of the first oligonucleotide construct, the second oligonucleotide construct, and / or the third oligonucleotide construct to a second conformation.

[0238]

[0146] Embodiment 17. The method of Embodiment 16, wherein: during (v) or after (v), dissociating the ligated construct, the additional ligated construct, or the complementary copy from the nucleic acid template. WSGR Docket No: 63452-705.601

[0239]

[0147] Embodiment 18. The method of Embodiment 17, further comprising: repeating (i), (ii), (iii), and (v), thereby producing a plurality of complementary copies of at least a portion of the nucleic acid template.

[0240]

[0148] Embodiment 19. The method of Embodiment 17, further comprising: repeating (i), (ii), (iii), (iv), and (v), thereby producing a plurality of complementary copies of at least a portion of the nucleic acid template.

[0241]

[0149] Embodiment 20. The method of any one of Embodiments 16-19, further comprising controlling a temperature of the aqueous sample substantially the same.

[0242]

[0150] Embodiment 21. The method of any one of Embodiments 16-19, further comprising controlling a temperature of the reaction chamber substantially the same.

[0243]

[0151] Embodiment 22. The method of any one of Embodiments 16-19, further comprising a heating step within (v) or immediately after (v), thereby facilitating the dissociating.

[0244]

[0152] Embodiment 23. The method of Embodiment 22, wherein the heating step raises a temperature of the reaction chamber or the aqueous sample up to a specific temperature at which at least half of the ligated construct, the additional ligated construct, or the complementary copy dissociates from the nucleic acid template in the absence of the one or more photo-switchable moieties.

[0245]

[0153] Embodiment 24. The method of Embodiment 22, wherein the heating step raises a temperature of the reaction chamber or the aqueous sample up to a specific temperature at which at least 75% of the ligated construct, the additional ligated construct, or the complementary copy dissociates from the nucleic acid template in the absence of the one or more photo-switchable moieties.

[0246]

[0154] Embodiment 25. The method of any one of Embodiments 22-24, further comprising a cooling step after the heating step, thereby facilitating duplex formation between the nucleic acid template and one or more of the plurality of oligonucleotide constructs, the ligated construct, and the additional ligated construct.

[0247]

[0155] Embodiment 26. The method of Embodiment 25, wherein the cooling step is prior to (i).

[0248]

[0156] Embodiment 27. The method of any one of Embodiments 13-26, further comprising: using one or more of the ligated construct, the additional ligated construct, and the complementary copy as a template in a first additional ligation catalyzed by the one or more enzymes.

[0249]

[0157] Embodiment 28. The method of Embodiment 27, wherein the one or more enzymes comprise a ligation enzyme configured to ligate the first oligonucleotide construct and the second oligonucleotide construct under a ligation condition, thereby producing the ligated construct. WSGR Docket No: 63452-705.601

[0250]

[0158] Embodiment 29. The method of Embodiment 28, wherein the ligation condition comprises binding of the first oligonucleotide construct and the second oligonucleotide construct to the nucleic acid template, thereby bringing the first oligonucleotide construct and the second oligonucleotide construct within each other’s vicinity for the ligation enzyme to ligate the first oligonucleotide construct and the second oligonucleotide construct.

[0251]

[0159] Embodiment 30. The method of any one of Embodiments 13-29, wherein the first conformation of the one or more photo-switchable moieties of the first oligonucleotide construct allows the first oligonucleotide construct to bind to the nucleic acid template; and wherein the first conformation of the one or more photo-switchable moieties of the second oligonucleotide construct allows the second oligonucleotide construct to bind to the nucleic acid template.

[0252]

[0160] Embodiment 31. The method of any one of Embodiments 13-30, wherein the second conformation of the one or more photo-switchable moieties of the first oligonucleotide construct enables the first oligonucleotide construct to dissociate from the nucleic acid template.

[0253]

[0161] Embodiment 32. The method of any one of Embodiments 13-31, wherein the second conformation of the one or more photo-switchable moieties of the second oligonucleotide construct enables the second oligonucleotide construct to dissociate from the nucleic acid template.

[0254]

[0162] Embodiment 33. The method of any one of Embodiments 13-31, further comprising: detecting a presence or absence of the ligated construct or the additional ligated construct.

[0255]

[0163] Embodiment 34. The method of any one of Embodiments 13-31, further comprising: detecting a presence or absence of the ligated construct and the additional ligated construct.

[0256]

[0164] Embodiment 35. The method of Embodiment 33 or 34, wherein the first oligonucleotide construct comprises a first moiety of a fluorescent resonance energy transfer (FRET) pair and the second oligonucleotide construct comprises a second moiety of the FRET pair, and wherein the detecting comprises detecting a presence or absence of a FRET signal.

[0257]

[0165] Embodiment 36. A method of detecting a presence or absence of at least one analyte in a sample, comprising:

[0258] (i) radiating an aqueous sample with a first excitation light beam having a first wavelength, wherein the aqueous sample comprises:

[0259] (a) a sample suspected to comprise at least one analyte;

[0260] (b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of the plurality of oligonucleotide constructs comprising one or more photo-switchable moieties;

[0261] (c) a ligase enzyme configured to interact with the at least one analyte and the plurality of oligonucleotide constructs; WSGR Docket No: 63452-705.601

[0262] (ii) radiating the aqueous sample with a second excitation light beam having a second wavelength;

[0263] (iii) radiating the aqueous sample with a third excitation light beam having a third wavelength; and

[0264] (iv) detecting the presence or absence of a fluorescent resonance energy transfer (FRET) signal, the FRET signal is configured to be generated when a first oligonucleotide construct and a second oligonucleotide construct of the plurality of oligonucleotide constructs are ligated in the presence of the at least one analyte.

[0265]

[0166] Embodiment 37. The method of Embodiment 36, wherein the first oligonucleotide construct comprises a FRET donor; and wherein the second oligonucleotide construct comprises a FRET acceptor.

[0266]

[0167] Embodiment 38. The method of Embodiment 36 or 37, wherein the first oligonucleotide construct and the second oligonucleotide construct have sequence complementary to a first section and a second section of the at least one analyte, respectively, and wherein the first section and the second section are in tandem on the at least one analyte.

[0267]

[0168] Embodiment 39. The method of any one of Embodiments 36-38, wherein the first excitation light beam enables duplex formation between the first oligonucleotide construct / the second oligonucleotide construct and the at least one analyte, thereby allowing a ligated oligonucleotide construct to be formed.

[0268]

[0169] Embodiment 40. The method of Embodiment 39, wherein the second excitation light beam facilitates dissociating the ligated oligonucleotide construct from the at least one analyte.

[0269]

[0170] Embodiment 41. The method of Embodiment 37, wherein the third excitation light beam excites the FRET donor on the first oligonucleotide construct.

[0270] OTHER EMBODIMENTS

[0271]

[0171] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0272]

[0172] List of References (each of which is incorporated by reference in its entirety):

[0273] 1. Bandara HM, Burdette SC (March 2012). "Photoisomerization in different classes of azobenzene". Chemical Society Reviews. 41 (5): 1809-25.

[0274] 2. Kortekaas L, Browne WR (June 2019). "The evolution of spiropyran: fundamentals and progress of an extraordinarily versatile photochrome". Chemical Society Reviews. 48 (12): 3406-3424. WSGR Docket No: 63452-705.601

[0275] 3. Klajn R (January 2014). "Spiropyran-based dynamic materials". Chemical Society Reviews.

[0276] 43 (1): 148-84.

[0277] 4. Pu SZ, Sun Q, Fan CB, Wang RJ, Liu G (2016-04-14). "Recent advances in diarylethene-based multi-responsive molecular switches". Journal of Materials Chemistry C. 4 (15): 3075-3093.

[0278] 5. Berkovic G, Krongauz V, Weiss V (May 2000). "Spiropyrans and Spirooxazines for Memories and Switches". Chemical Reviews. 100 (5): 1741-1754.

[0279] 6. Yokoyama Y (May 2000). "Fulgides for Memories and Switches". Chemical Reviews. 100 (5): 1717-1740.

[0280] 7. Su X, Aprahamian I (March 2014). "Hydrazone-based switches, metallo-assemblies and sensors". Chemical Society Reviews. 43 (6): 1963-81.

[0281] 8. Orrego-Hemandez J, Dreos A, Moth-Poulsen K (August 2020). "Engineering of

[0282] Norb ornadiene / Quadri cyclane Photoswitches for Molecular Solar Thermal Energy Storage Applications". Accounts of Chemical Research. 53 (8): 1478-1487.

[0283] 9. Navratil R, Wiedbrauk S, Jasik J, Dube H, Roithova J (March 2018). "Transforming hemithioindigo from a two-way to a one-way molecular photoswitch by isolation in the gas phase". Physical Chemistry Chemical Physics. 20 (10): 6868-6876.

[0284] 10. Polosukhina A, Litt J, Tochitsky I, Nemargut J, Sychev Y, De Kouchkovsky I, et al. (July 2012). "Photochemical restoration of visual responses in blind mice". Neuron. 75 (2): 271-82.

[0285] 11. Lerch MM, Szymanski W, Feringa BL (March 2018). "The (photo)chemistry of Stenhouse photoswitches: guiding principles and system design". Chemical Society Reviews. 47 (6): 1910— 1937.

[0286] 12. Helmy, Sameh; Oh, Saemi; Leibfarth, Frank A.; Hawker, Craig J.; Read de Alaniz, Javier (2014-12-05). "Design and Synthesis of Donor-Acceptor Stenhouse Adducts: A Visible Light Photoswitch Derived from Furfural". The Journal of Organic Chemistry. 79 (23): 11316-11329.

[0287] 13. Abourashed EA (2017-02-24). "Review of Stilbenes: Applications in Chemistry, Life Sciences and Materials Science". Journal of Natural Products. 80 (2): 577.

[0288] 14. H. Asanuma,et al., “Enantioselective Incorporation of Azobenzenes into Oligodeoxyribonucleotide for Effective Photoregulation of Duplex Formation”. Angew Chem IntEdEngl. 2001 Jul 16;40(14):2671-2673.

[0289] 15. H. Asanuma, et al., “Synthesis of azobenzene-tethered DNA for reversible photoregulation of DNA functions: hybridization and transcription”. Nat Protoc. 2007;2(l):203-12. 16. Y.Kamiya and H. Asanuma, “Light-driven DNA nanomachine with a photoresponsive molecular engine”. Acc Chem Res. 2014 Jun 17;47(6): 1663-72. WSGR Docket No: 63452-705.601

[0290] 17. X.G. Liang, et al., “NMR Study on the Photoresponsive DNA Tethering an Azobenzene. Assignment of the Absolute Configuration of Two Diastereomers and Structure Determination of Their Duplexes in the trans-Form”, J. Am. Chem. Soc. 2003, 125, 16408-16415.

[0291] 18. H. Ito, et al., “Construction of photoresponsive RNA for photoswitching RNA hybridization”, Org. Biomol. Chem. 2010, 8, 5519-5524.

[0292] 19. X.G. Liang, et al., “Photoregulation of DNA triplex formation by azobenzene”, J Am Chem Soc. 2002 Mar 6;124(9):1877-1883.

[0293] 20. Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonak, J., Lind, K., Sindelka, R., Sjoback, R., Sjbgreen, B., Strombom, L. and Stahlberg, A., 2006. The real-time polymerase chain reaction. Molecular aspects of medicine, 27(2-3), pp.95-125.

[0294] 21. Gudnason, H., Dufva, M., Bang, D.D. and Wolff, A., 2007. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic acids research, 35(19), p.el27.

[0295] 22. Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H., 1991. Detection of specific polymerase chain reaction product by utilizing the 5' — 3'exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences, 88(16), pp.7276-7280.

[0296] 23. Hossain, M.M., Abidin, S.A.S.Z., Bujang, A., Taib, M.N., Sagadevan, S., Johan, M.R. and Nizar, N.N.A., 2023. TaqMan multiplex qPCR for detecting animal species in meat and meat products: Development, recent advances and future prospects. Food Control, 150, p.109761.

[0297] 24. Yuan, L.I.N., Lin, W., Zheng, K. and Zhu, S., 2013. FRET -based small-molecule fluorescent probes: rational design and bioimaging applications. Accounts of chemical research, 46(7), pp.1462-1473.

[0298] 25. Massey, M., Algar, W.R. and Krull, U.J., 2006. Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances for various dye-DNA conjugates. Analytica chimica acta, 565(1-2), pp.181-189.

[0299] 26. Johansson, M.K., 2006. Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers. Fluorescent energy transfer nucleic acid probes: designs and protocols, pp.17-29.

Claims

WSGR Docket No: 63452-705.601CLAIMS WHAT IS CLAIMED IS:

1. A system for sequence-specific nucleic acid amplification, comprising:(i) a reaction chamber;(ii) an aqueous sample enclosed in said reaction chamber, comprising:(a) a nucleic acid template;(b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of said plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; said one or more photo-switchable moieties being configured to (1) switch to a first conformation when radiated by a first excitation light beam with a first wavelength; and (2) switch to a second conformation when radiated by a second excitation light beam with a second wavelength; and(c) one or more enzymes configured to interact with said nucleic acid temple and / or said plurality of oligonucleotide constructs; and(iii) an excitation light source configured to emit at least said first excitation light beam with said first wavelength and said second excitation light beam with said second wavelength.

2. The system of claim 1, further comprising a temperature control configured to control temperature of said aqueous sample and / or said reaction chamber.

3. The system of claim 2, further comprising a control, wherein said control is configured to operate said temperature control and / or said excitation light source.

4. The system of any one of claims 1-3, wherein said one or more enzymes comprise a ligation enzyme configured to ligate a first oligonucleotide construct and a second oligonucleotide construct of said one or more oligonucleotide constructs under a ligation condition, thereby producing a ligated construct.

5. The system of claim 4, where said ligation condition comprises binding of said first oligonucleotide construct and said second oligonucleotide construct to said nucleic acid template, thereby bringing said first oligonucleotide construct and said second oligonucleotide construct within each other’s vicinity for said ligation enzyme to ligate said first oligonucleotide construct and said second oligonucleotide construct.

6. The system of any one of claims 1-5, wherein said first conformation of said one or more photo-switchable moieties of said first oligonucleotide construct allows said first oligonucleotide construct to bind to said nucleic acid template; and wherein said first conformation of said oneWSGR Docket No: 63452-705.601or more photo-switchable moieties of said second oligonucleotide construct allows said second oligonucleotide construct to bind to said nucleic acid template.

7. The system of claim 6, wherein said second conformation of said one or more photo-switchable moieties of said first oligonucleotide construct enables said first oligonucleotide construct to dissociate from said nucleic acid template; and wherein said second conformation of said one or more photo-switchable moieties of said second oligonucleotide construct enables said second oligonucleotide construct to dissociate from said nucleic acid template.

8. The system of any one of claims 4-7, wherein said aqueous solution further comprises a third oligonucleotide construct configured to be ligated with said ligated construct under an additional ligation condition by said one or more enzymes, thereby producing an additional ligated construct.

9. The system of claim 8, wherein said additional ligation condition comprises binding of said third oligonucleotide construct and said ligated construct to said nucleic acid template, thereby bringing said third oligonucleotide construct and said ligated construct within each other’s vicinity for said ligation enzyme to ligate said third oligonucleotide construct and said ligated construct.

10. The system of any one of claims 4-9, wherein said ligation enzyme is configured to bind to said ligated construct and use said ligated construct as a template in a first additional ligation, thereby producing a complimentary copy of said ligated construct.

11. The system of any one of claims 8-10, wherein said ligation enzyme is configured to bind to said additional ligated construct and use said additional ligated construct as a template in a second additional ligation, thereby producing a complimentary copy of said additional ligated construct.

12. The system of any one of claims 1-11, wherein a first population of said plurality of oligonucleotide constructs comprises a first moiety of a fluorescent resonance energy transfer (FRET) pair and a second population of said plurality of oligonucleotide constructs comprises a second moiety of said FRET pair.

13. A method for sequence-specific nucleic acid amplification, comprising:(i) radiating an aqueous sample enclosed in a reaction chamber with a first excitation light beam having a first wavelength, wherein said aqueous sample comprises: (a) a nucleic acid template;(b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of said plurality of oligonucleotide constructs comprising one or more photo-switchable moieties; andWSGR Docket No: 63452-705.601(c) one or more enzymes configured to interact with said nucleic acid temple and / or said plurality of oligonucleotide constructs;(ii) changing, via said radiating in (i), one or more photo-switchable moieties of a first oligonucleotide construct of said one or more oligonucleotide constructs into a first conformation; changing one or more photo-switchable moieties of a second oligonucleotide construct of said one or more oligonucleotide constructs into said first conformation; and(iii) subsequent to (ii), ligating, via said one or more enzymes, said first oligonucleotide construct with said second oligonucleotide construct; thereby providing a ligated construct, wherein said ligated construct remains bound to said nucleic acid template.

14. The method of claim 13, further comprising:(iv) ligating a third oligonucleotide construct of said plurality of oligonucleotide constructs with said ligated construct from (iii), thereby producing an additional ligated construct comprising said ligated construct and said third oligonucleotide construct, wherein said third oligonucleotide construct comprises one or more photo-switchable moieties in said first confirmation.

15. The method of claim 14, further comprising: repeating (iv) one or more times, thereby producing a complementary copy of at least a portion of said nucleic acid template.

16. The method of any one of claims 13 to 15, further comprising:(v) after (iii) or after (iv), radiating said aqueous sample with a second excitation light beam having a second wavelength, thereby switching said one or more photo-switchable moieties of said first oligonucleotide construct, said second oligonucleotide construct, and / or said third oligonucleotide construct to a second conformation.

17. The method of claim 16, wherein: during (v) or after (v), dissociating said ligated construct, said additional ligated construct, or said complementary copy from said nucleic acid template.

18. The method of claim 17, further comprising: repeating (i), (ii), (iii), and (v), thereby producing a plurality of complementary copies of at least a portion of said nucleic acid template.

19. The method of claim 17, further comprising: repeating (i), (ii), (iii), (iv), and (v), thereby producing a plurality of complementary copies of at least a portion of said nucleic acid template.

20. The method of any one of claims 16-19, further comprising controlling a temperature of the aqueous sample substantially the same.WSGR Docket No: 63452-705.60121. The method of any one of claims 16-19, further comprising controlling a temperature of the reaction chamber substantially the same.

22. The method of any one of claims 16-19, further comprising a heating step within (v) or immediately after (v), thereby facilitating said dissociating.

23. The method of claim 22, wherein said heating step raises a temperature of said reaction chamber or said aqueous sample up to a specific temperature at which at least half of said ligated construct, said additional ligated construct, or said complementary copy dissociates from said nucleic acid template in the absence of said one or more photo-switchable moieties.

24. The method of claim 22, wherein said heating step raises a temperature of said reaction chamber or said aqueous sample up to a specific temperature at which at least 75% of said ligated construct, said additional ligated construct, or said complementary copy dissociates from said nucleic acid template in the absence of said one or more photo-switchable moieties.

25. The method of any one of claims 22-24, further comprising a cooling step after said heating step, thereby facilitating duplex formation between said nucleic acid template and one or more of said plurality of oligonucleotide constructs, said ligated construct, and said additional ligated construct.

26. The method of claim 25, wherein said cooling step is prior to (i).

27. The method of any one of claims 13-26, further comprising: using one or more of said ligated construct, said additional ligated construct, and said complementary copy as a template in a first additional ligation catalyzed by said one or more enzymes.

28. The method of claim 27, wherein said one or more enzymes comprise a ligation enzyme configured to ligate said first oligonucleotide construct and said second oligonucleotide construct under a ligation condition, thereby producing said ligated construct.

29. The method of claim 28, wherein said ligation condition comprises binding of said first oligonucleotide construct and said second oligonucleotide construct to said nucleic acid template, thereby bringing said first oligonucleotide construct and said second oligonucleotide construct within each other’s vicinity for said ligation enzyme to ligate said first oligonucleotide construct and said second oligonucleotide construct.

30. The method of any one of claims 13-29, wherein said first conformation of said one or more photo-switchable moieties of said first oligonucleotide construct allows said first oligonucleotide construct to bind to said nucleic acid template; and wherein said first conformation of said one or more photo-switchable moieties of said second oligonucleotide construct allows said second oligonucleotide construct to bind to said nucleic acid template.WSGR Docket No: 63452-705.60131. The method of any one of claims 13-30, wherein said second conformation of said one or more photo-switchable moieties of said first oligonucleotide construct enables said first oligonucleotide construct to dissociate from said nucleic acid template.

32. The method of any one of claims 13-31, wherein said second conformation of said one or more photo-switchable moieties of said second oligonucleotide construct enables said second oligonucleotide construct to dissociate from said nucleic acid template.

33. The method of any one of claims 13-31, further comprising: detecting a presence or absence of said ligated construct or said additional ligated construct.

34. The method of any one of claims 13-31, further comprising: detecting a presence or absence of said ligated construct and said additional ligated construct.

35. The method of claim 33 or 34, wherein said first oligonucleotide construct comprises a first moiety of a fluorescent resonance energy transfer (FRET) pair and said second oligonucleotide construct comprises a second moiety of said FRET pair, and wherein said detecting comprises detecting a presence or absence of a FRET signal.

36. A method of detecting a presence or absence of at least one analyte in a sample, comprising:(i) radiating an aqueous sample with a first excitation light beam having a first wavelength, wherein said aqueous sample comprises:(a) a sample suspected to comprise at least one analyte;(b) a plurality of oligonucleotide constructs, one or more oligonucleotide constructs of said plurality of oligonucleotide constructs comprising one or more photo-switchable moieties;(c) a ligase enzyme configured to interact with said at least one analyte and said plurality of oligonucleotide constructs;(ii) radiating said aqueous sample with a second excitation light beam having a second wavelength;(iii) radiating said aqueous sample with a third excitation light beam having a third wavelength; and(iv) detecting the presence or absence of a fluorescent resonance energy transfer (FRET) signal, said FRET signal is configured to be generated when a first oligonucleotide construct and a second oligonucleotide construct of said plurality of oligonucleotide constructs are ligated in the presence of said at least one analyte.

37. The method of claim 36, wherein said first oligonucleotide construct comprises a FRET donor; and wherein said second oligonucleotide construct comprises a FRET acceptor.WSGR Docket No: 63452-705.60138. The method of claim 36 or 37, wherein said first oligonucleotide construct and said second oligonucleotide construct have sequence complementary to a first section and a second section of said at least one analyte, respectively, and wherein said first section and said second section are in tandem on said at least one analyte.

39. The method of any one of claims 36-38, wherein said first excitation light beam enables duplex formation between said first oligonucleotide construct / said second oligonucleotide construct and said at least one analyte, thereby allowing a ligated oligonucleotide construct to be formed.

40. The method of claim 39, wherein said second excitation light beam facilitates dissociating said ligated oligonucleotide construct from said at least one analyte.

41. The method of claim 37, wherein said third excitation light beam excites said FRET donor on said first oligonucleotide construct.

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