Patent Application
20190032109
Fluorescence microscopy is a technology used for studying a wide range of processes in cell and molecular biology, and is of central importance in both basic and applied biomedical research. This technology has recently been improved by extensions, such as super-resolution microscopy (Huang, B. et al. Annu. Rev. Biochem. 2009, 78 (1), 993), which improves resolution by approximately tenfold compared to a conventional microscope. Nonetheless, only a handful of probes can be distinguished in a visible light microscope due to spectral overlap, thus, it is challenging to image more than a few distinct molecular targets at a time.
The present disclosure provides, in some aspects, technology that can reliably distinguish a large number of fluorescent probes used in a single fluorescence microscopy experiment and, by extension, the molecular species represented by the probes. Thus, provided herein, in some embodiments, are imaging methods and associated molecules, referred to as “single-molecule timers” or “single-molecule clocks” or simply “timers” or “clocks,” that provide, for example: (1) control over the duration of probe-target interactions, permitting discrimination among multiple species on the basis of kinetics; (2) rapid multiplexed imaging (e.g., less than an hour) of thousands of distinct targets via kinetic barcoding, which does not require buffer exchange or photobleaching steps, resulting in an acquisition time of minutes rather than hours or days; and (3) compact “molecular barcodes” (e.g., encoded nucleic acids) that do not require spatial encoding of information, permitting, in some aspects, high-density and super-resolution imaging in crowded samples, such as cells and tissues. Advantageously, the methods and molecules of the present disclosure are compatible with standard fluorescence microscopes, thus avoiding the need for custom robotic mixers, microfluidic cells or more specialized instrumentation.
The methods and molecules of the present disclosure use precise single-molecule timing elements to construct compact, high-density labels for fluorescence microscopy. The methods and molecules rely, in part, on engineered quasi-deterministic (exhibiting a variance significantly lower than for a single-step chemical reaction) kinetic properties as well as spectral and spatial information, which permit the production of physically compact barcodes having elements that: (1) are distinguishable from a small number of binding event observations, even when using the same fluorophore; and (2) can be imaged simultaneously in a one-pot mixture of probes. This bypasses time-consuming probe exchange, chemical modification, photobleaching, and ex situ imaging steps, and permits super-resolution imaging of dozens to thousands of targets on a timescale of tens of minutes, for example.
Some aspects of the present disclosure provide a kinetically encoded imaging system, comprising (a) an unpaired initiator nucleic acid comprising a 3′ nucleotide subdomain and a 5′ nucleotide subdomain, (b) a template probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain, and (c) a primer linked to a detectable molecule.
Some aspects of the present disclosure provide a kinetically encoded imaging system, comprising (a) an unpaired initiator nucleic acid comprising a 3′ nucleotide subdomain and a 5′ nucleotide subdomain, (b) a template probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain, wherein the template probe is linked to a detectable molecule, and (c) a primer. In some embodiments, the detectable molecule is linked to the 3′ of the template probe.
Some aspects of the present disclosure provide a kinetically encoded imaging method, comprising: combining in reaction buffer (a) an unpaired initiator nucleic acid comprising a 3′ nucleotide subdomain and a 5′ nucleotide subdomain, wherein the initiator nucleic acid is associated with a target of interest, (b) a hairpin template probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain, (c) a primer linked to a detectable molecule, (d) a DNA polymerase, and (e) dNTPs, thereby forming a reaction mixture; and incubating the reaction mixture under conditions that result in DNA polymerization.
In some embodiments, the 3′ nucleotide subdomain of the initiator of (a) is complementary to and binds to the unpaired toehold domain of the probe of (b), and the 5′ nucleotide subdomain of the initiator of (a) is complementary to and binds to the 5′ subdomain of the probe of (b). In some embodiments, the primer is complementary to and binds to the 3′ subdomain of the probe of (b).
In some embodiments, the method further comprises imaging the reaction mixture during the incubation step and identifying periods of time during which there is an increase in a level of fluorescence relative to a start time control level of fluorescence, thereby identifying dwell times.
In some embodiments, the method further comprises identifying the presence or absence of a target of interest based on the dwell times.
In some embodiments, dNTPs are present at a concentration of 2.5 μM to 10 mM. For example, dNTPs may be present at a concentration of 100 μM.
In some embodiments, the template probe further comprises 3′ phosphate (PO42−) group.
In some embodiments, the system further comprises a DNA polymerase. In some embodiments, the DNA polymerase has strand displacement activity. For example, the DNA polymerase may be phi29 or Bst DNA polymerase, large fragment.
In some embodiments, the initiator nucleic acid has a length of 15-50 nucleotides. For example, the initiator nucleic acid may have a length of 20-30 nucleotides.
In some embodiments, the 3′ nucleotide subdomain of the initiator nucleic acid has a length of 5-15 nucleotides. In some embodiments, the 5′ nucleotide subdomain of the initiator nucleic acid has a length of 10-20 nucleotides.
In some embodiments, the template probe has a length of 30-200 nucleotides. For example, the template probe may have a length of 30-50 nucleotides.
In some embodiments, the toehold domain has a length of 2-15 nucleotides.
In some embodiments, the hairpin stem domain has a length of 10-20 nucleotides.
In some embodiments, the hairpin loop domain has a length of 4-100 nucleotides. For example, the hairpin loop domain may have a length of 4-20 nucleotides.
In some embodiments, the primer has length of 10-20 nucleotides.
Some aspects of the present disclosure provide a nucleic acid molecule comprising a 5′ paired domain, an internal unpaired domain, and a 3′ paired domain linked to a detectable molecule.
Also provided herein, in some aspects, is a kinetically encoded imaging system, comprising (a) a target nucleic acid, (b) a 5′-phosphorylated nucleic acid probe linked to a 3′ detectable molecule, and (c) a 5′-phosphate-specific exonuclease.
In some embodiments, the probe is complementary to and binds to the target.
Also provided herein, in some aspects, is a kinetically encoded imaging method, comprising: combining in reaction buffer (a) a target nucleic acid, (b) a 5′-phosphorylated nucleic acid probe linked to a 3′ detectable molecule, and (c) a 5′-phosphate-specific exonuclease; and incubating the reaction mixture under conditions that result in exonuclease-mediated degradation of the probe.
Some aspects of the present disclosure provide a kinetically encoded imaging system, comprising: (a) an unpaired initiator nucleic acid; and (b) a first hairpin probe, a second hairpin probe, a third hairpin probe and a fourth hairpin probe, each hairpin probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by intramolecular base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain located between the 5′ subdomain and the 3′ subdomain, wherein the first hairpin probe is linked to a detectable molecule.
In some embodiments, (i) the toehold domain and the 5′ subdomain of the first probe are complementary to and bind to the initiator nucleic acid, (ii) the toehold domain and the 5′ subdomain of the second probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the first probe bound to the initiator sequence, (iii) the toehold domain and the 5′ subdomain of the third probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the second probe bound to the first probe, and (iv) the toehold domain and the 5′ subdomain of the fourth probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the third probe bound to the second probe, and wherein the hairpin loop and 3′ subdomain of the fourth probe are complementary to and bind to the toehold domain and the 5′ subdomain of the first probe. Some aspects of the present disclosure provide a kinetically encoded imaging method, comprising: combining in reaction buffer (a) an unpaired initiator nucleic acid, and (b) a first hairpin probe, a second hairpin probe, a third hairpin probe and a fourth hairpin probe, each hairpin probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by intramolecular base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain located between the 5′ subdomain and the 3′ subdomain, wherein the first hairpin probe is linked to a detectable molecule; and incubating the reaction mixture under conditions that result in DNA hybridization.
Additionally provided herein, in some embodiments, are compositions, comprising (a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash, (b) a nucleic acid primer comprising a sequence complementary to the primer binding sequence, and (c) a labeled nucleic acid imager strand. In some embodiments, the compositions further comprise polymerase. In some embodiments, component (c) comprises multiple imager strands, each with different sequences relative to each other and with distinguishable labels. The use of multiple imager strands enables the generation of an ordered series of distinguishable signal ‘pulses’ (e.g., red, red, blue . . . red, red, blue . . . etc.), which provides additional multiplexing capabilities. In some embodiments, the imager strand is bound to a quencher strand that comprises a quencher molecule. In further embodiments, the quencher strand is shorter than the imager strand. In some embodiments, the imager stand is fluorescently labeled on its 3′ end, and the quencher strand comprises a quencher molecule on its 5′ end. In other embodiments, the imager stand is fluorescently labeled on its 5′ end, and the quencher strand comprises a quencher molecule on its 3′ end. In some embodiments, a composition further comprises an endonuclease.
In some embodiments, the present disclosure provides methods that comprise combining in reaction buffer (a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash, (b) a nucleic acid primer comprising a sequence complementary to the primer binding sequence, (c) a labeled nucleic acid imager strand, (d) a polymerase (e.g., DNA polymerase or RNA polymerase), and (e) deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (NTPs) (depending on whether DNA polymerase or RNA polymerase is used), thereby forming a reaction mixture, and incubating the reaction mixture under conditions that result in nucleic acid polymerization and nucleic acid hybridization.
In some embodiments, the present disclosure further provides compositions, comprising (a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash, (b) a primer comprising a sequence complementary to the primer binding sequence, and (c) a mixture of nucleoside triphosphates (NTPs or dNTPs) comprising subsets of ATPs (or dATPs), TTPs (or dTTPs), CTPs (or dCTPs) and GTPs (or dGTPs), wherein NTPs of at least one of the subsets comprise a label.
In some embodiments, the present disclosure also provides methods that comprise combining in reaction buffer (a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash strand, (b) a primer comprising a sequence complementary to the primer binding sequence, (c) a mixture of nucleoside triphosphates (NTPs or dNTPs) comprising subsets of ATPs (or dATPs), TTPs (or dTTPs), CTPs (or dCTPs)and GTPs (or dGTPs), wherein NTPs of at least one of the subsets comprise a label, and (d) a polymerase, thereby forming a reaction mixture, and incubating the reaction mixture under conditions that result in nucleic acid polymerization and nucleic acid hybridization.
The ability to rapidly image a large fraction of the proteome and transcriptome of intact cells and tissues (e.g., from a tumor biopsy) at super-resolution yields critical insights for the personalized diagnosis and treatment of cancer and other diseases. In addition to information about tumor heterogeneity and drug resistance, spatially resolved proteomics reveals nontrivial subcellular organization of surface antigens that can guide the development of more effective targeted therapies, especially those involving multivalent recognition, for example. The limited capacity of light microscopy for multiplexing (3-4 colors) limits the number of targets that can be simultaneously imaged. Provided herein are compact barcodes that encode information in the time domain as a series of deterministic fluorescence pulses, akin to a “molecular Morse code” with high information content. These compact barcodes, referred to as “single-molecule timers,” may be used to achieve temporal encoding by coupling the dissociation of a fluorescent probe to a series of irreversible reactions. “Single-molecule timers” and “single-molecule clocks” permit one to readily distinguish among multiple fluorescent probes based on the temporal pattern of fluorescence intensity, even when they are not separable by color or position. The timers and clocks permits the spatially resolved detection of thousands of distinct molecular targets in a single imaging experiment lasting only ˜10 minutes, for example. Following validation in well-studied mammalian cell lines, these kinetic barcodes may be used to rapidly profile RNA and/or protein expression within intact cancer cells and tissue samples, with single-molecule sensitivity and super-resolution, thus guiding the development of more effective personalized treatments for cancer and other diseases.
Single-molecule timers, as provided herein, in some aspects, utilize a cascade of several irreversible reactions to establish a well-defined time delay between binding of a fluorescent primer (ON State) to a target and subsequent displacement of the fluorescent primer (in the form of an elongated waste complex, as described below) from the target by a DNA polymerase (OFF state) (see, e.g.,
where N is the number or irreversible reactions between binding and dissociation. This quasi-deterministic behavior permits the unambiguous identification of a target by observing the duration of a single binding event.
To provide robust timer behavior, the lifetime of fluorescent primer binding to the target is controlled primarily by the series of irreversible reactions—this irreversible cascade is rate-limiting. The reactions may also have partly reversible character as long as there is a forward bias in the equilibrium governing each step; in such cases, the degree of randomness (or width relative to the mean value) of the dwell time distribution increases with increasing reversibility. The present disclosure provides polymerase-based single-molecule timers, exonuclease-based single-molecule timers, and single-molecule timers constructed from hybridization cascades. For polymerase-based single-molecule timers, the polymerase concentration may be kept high and/or the polymerization rate may kept low. The polymerization rate may be kept low by modifying, for example, buffer conditions, temperature, and dNTP concentrations. For exonuclease-based single-molecule timers, the exonuclease concentration may be kept high and/or the degradation rate may be kept low.
The binding equilibrium of a bimolecular complex can usually be approximated as a two-state system characterized by a bimolecular association rate constant k0 and a unimolecular dissociation rate constant k1 (
obeys a gamma distribution, with standard deviation proportional to 1/√{square root over (N)} (
Although the single-molecule timer principle may be implemented in a number of different ways, one approach is to use a processive enzyme such as a DNA polymerase (DNAP). Described herein is a system in which a DNAP with strand displacement activity controls the delay between the binding and dissociation of a fluorescent probe (
With polymerase-based single-molecule timers, extension of a DNA primer by a polymerase supplies a series of irreversible reactions that constitute the timer (see, e.g.,
An “initiator (I)” refers to a contiguous sequence of nucleotides to which a template probe binds (hybridize). An initiator may form part of the sequence of a nucleic acid (e.g., DNA or RNA) target of interest, or an initiator may be an independent molecule associated with (e.g., directly or indirectly linked to) a target of interest (e.g., a protein or other biomolecule). For example, an initiator may be an oligonucleotide linked to protein (or other biomolecule) of interest. An initiator (see
In some embodiments, a target of interest is associated with more than one initiator. For example, more than one template probe may bind to the same target. Thus, in some embodiments, a target of interest includes, or is linked to an oligonucleotide that includes, a series of initiators (each including a 3′ and 5′ subdomain) such that multiple different template probes are capable of binding to a single target of interest. In some embodiments, a target of interest is associated with 2-50 different single-molecule timers. For example, a target of interest may be associated with 2-5, 2-10, 2-20, 2-15, 2-20, 2-25, 2-30, 2-35, 2-40, 2-45, 2-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45 or 10-50 different single-molecule timers. In some embodiments, a target of interest is associated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different single-molecule timers.
The length of each subdomain of an initiator may vary and depends, in part, on the lengths of the domains of a corresponding template probe to which the initiator binds. In particular, the length of the 5′ subdomain of the initiator typically depends on the length of the hairpin stem domain of the template probe, while the 3′ subdomain of the initiator typically depends on the length of the 5′ single-stranded domain of the template probe.
In some embodiments, a 5′ subdomain of an initiator has a length of 5-40 nucleotides. For example, a 5′ subdomain of an initiator may have a length of 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a 5′ subdomain of an initiator has a length of 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides. In some embodiments, a 5′ subdomain of an initiator has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A 5′ subdomain of an initiator, in some embodiments, is longer than 40 nucleotides, or shorter than 5 nucleotides.
In some embodiments, a 3′ subdomain of an initiator has a length of 5-40 nucleotides. For example, a 3′ subdomain of an initiator may have a length of 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a 3′ subdomain of an initiator has a length of 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides. In some embodiments, a 3′ subdomain of an initiator has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A 5′ subdomain of an initiator, in some embodiments, is longer than 40 nucleotides, or shorter than 5 nucleotides.
A “template probe (T)” refers to a nucleic acid hairpin molecule that binds to an initiator and a fluorescent primer. For simplicity, the domains of a template probe are described in the context of a single strand of nucleic acid. For example, a nucleic acid hairpin molecule occurs when two domains of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix (hairpin stem) that ends in an unpaired loop (hairpin loop). It should be understood, however, that a single strand of nucleic acid may be made up of a contiguous sequence of nucleotides, or a single strand of nucleic acid may be made up of two or more domains of contiguous sequences of nucleotides, each domain joined by a linker (e.g., nucleic acid or chemical linker).
A template probe (see
A “toehold domain” refers to an unpaired sequence of nucleotides located at the 5′ end of the template probe and is complementary to (and binds to) the 3′ subdomain of an initiator. The length of a toehold domain may vary. In some embodiments, a toehold domain has a length of 5-40 nucleotides. For example, a toehold domain may have a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a toehold domain has a length of 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides. In some embodiments, a toehold domain has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A toehold domain, in some embodiments, is longer than 40 nucleotides, or shorter than 5 nucleotides.
A 5′ single-stranded toehold domain of a template probe binds to an initiator via a toehold-mediated strand displacement reaction (Zhang D. & Winfree E. JACS 2009, 131(47, 17303-17314; Zhang D. & Seelig G Nature Chemistry 2011, 3, 103-113). In this reaction (see
A “hairpin stem domain” refers to a paired sequence of nucleotides (e.g., Watson-Crick nucleobase pairing) located adjacent to (and 3′ from) the unpaired toehold domain of a template probe. The hairpin stem domain is formed by intramolecular base pairing of two subdomains of a template probe: e.g., an internal subdomain located 3′ from and adjacent to the toehold domain bound (hybridized) to a subdomain located at the 3′ end of the template probe. The length of a hairpin stem domain may vary. In some embodiments, a hairpin stem domain has a length of 5-40 nucleotides. For example, a hairpin stem domain may have a length of 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a hairpin stem domain has a length of 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides. In some embodiments, a hairpin stem domain has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A hairpin stem domain, in some embodiments, is longer than 40 nucleotides, or shorter than 5 nucleotides.
While a hairpin stem domain is generally formed by intramolecular base pairing of two subdomains of a template probe, it should be understood that this paired domain may contain at least one mismatch pair (e.g., pairing of A with C or G, or pairing of T with C or G), as shown in the example template probes depicted in
A “hairpin loop domain” refers to a primarily unpaired sequence of nucleotides that form a loop-like structure at the end of the hairpin stem domain. The length of a hairpin loop domain may vary. In some embodiments, an hairpin loop domain has a length 3-200 nucleotides. For example, a hairpin loop domain may have a length of 3-175, 3-150, 3-125, 3-100, 3-75, 3-50, 3-25, 4-175, 4-150, 4-125, 4-100, 4-75, 4-50, 4-25, 5-175, 5-150, 5-125, 5-100, 5-75, 5-50 or 5-25 nucleotides. In some embodiments, a hairpin loop domain has a length of 3-10, 3-15, 32-10, 3-25, 3-30, 3-35, 3-40, 3-35, 3-40, 3-45, 3-50, 4-10, 4-15, 4-10, 4-25, 4-30, 4-35, 4-40, 4-35, 4-40, 4-45 or 4-50 nucleotides. In some embodiments, a hairpin stem domain has a length of 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49 or 50 nucleotides. A hairpin stem domain, in some embodiments, is longer than 300 nucleotides. It should be understood that while hairpin loop in generally described as an unpaired domain, it may have subdomains of intramolecular nucleotide binding. For example, the single-molecule timer depicted in
“Dwell time” refers to the period of time that a primer-template probe complex (e.g., formed by binding of the fluorescently-labeled primer to the template probe) remains bound to the initiator. Binding of a fluorescent-primer-template probe complex to initiator, for example, results in emission (a “pulse”) of a fluorescent signal. The duration of fluorescent signal corresponds with, or is indicative of, dwell time. In some embodiments, dwell time is controlled (varied) by changing the length of the hairpin loop region of the template, as the “clock” graphic indicates in
Dwell time may also be controlled by varying buffer conditions, temperature, and deoxynucleotides (dNTPs) concentrations in a kinetically encoded imaging reaction, as DNA polymerases, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions). For example, increasing the temperature of a reaction using phi29 polymerase from 15° C. to 30° C. decreases dwell time Likewise, increasing the temperature of a reaction using Bst DNA polymerase, large fragment from room temperature (˜25° C.) to 65° C. (the optimal temperature for polymerization by this enzyme) decreases dwell time. Thus, the temperature at which a kinetically encoded imaging reaction is performed may vary from, for example, 4° C. to 65° C. (e.g., 4° C., 25° C., 37° C., 42° C. or 65° C.). In some embodiments, a kinetically encoded imaging reaction is performed at room temperature, while in other embodiments, a kinetically encoded imaging reaction is performed at 37° C. As another example, increasing salt concentration (e.g., increasing [NaCl] from 40 mM to 200 mM) results in slower DNA polymerization and longer dwell times (e.g., for Bst DNA polymerase, large fragment).
As shown in Example 3 (
A “primer (P)” refers to an unpaired (single-stranded) nucleic acid that binds to the 3′ subdomain of the hairpin stem domain of a template probe (following binding of the probe to the initiator and dissociation of the stem subdomains). Thus, the length of a primer depends, in part, on the length of the hairpin stem domain. In some embodiments, a primer has a length of 5-40 nucleotides. For example, a primer may have a length of 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a primer has a length of 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides. In some embodiments, a primer has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A primer, in some embodiments, is longer than 40 nucleotides, or shorter than 5 nucleotides.
A primer, as provided herein, may be linked to (labeled with) a detectable molecule (e.g., a molecule that emits a detectable signal, such as a fluorescent or chemiluminescent signal). In some embodiments, the label is a fluorophore. A primer linked to a fluorophore or other fluorescent/chemiluminescent molecule is referred to simply as a “fluorescent primer.” Examples of fluorophores that may be used herein include, without limitation, Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 405, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 647, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7 and Cy7.5. Other fluorophores and molecules that emit a detectable signal are encompassed by the present disclosure.
In some embodiments, a detectable molecule is linked to the template probe rather than the primer. In such embodiments, the primer concentration in a kinetically encoded imaging reaction is high enough that the primer binds immediately after the template probe binds to the initiator nucleic acid. In some embodiments, the concentration of the primer is 100 nM-10 μM. For example, the concentration of the primer may be 100-500 nM, 100-1000 nM, or 100-1500 nM. In some embodiments, the concentration of the primer is 1 μM or at least 1 μM. In some embodiments, the concentration of the primer is 1 μM to 5 μM, or 1 μM to 10 μM.
In some embodiments, a kinetically encoded imaging reaction comprises a fluorescent primer associated with a nucleic acid quencher strand by base pairing (when the fluorescent primer is not bound to the template probe). The proximity of the quencher and the fluorophore in this primer-quencher complex results in reduced fluorescence before the primer binds to the template. This association reduces background fluorescence at a given concentration of primer, which makes it practical to use higher concentrations of the primer, resulting in faster binding kinetics of the primer to the template. To facilitate binding of the primer to the template even when a quencher strand is present, the design includes at least one overhang (or toehold) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides within the primer that do not form base pairs with the quencher strand but which do form base pairs with the template. A “waste complex (W)” refers to the primer-probe complex that results following polymerization (elongation) and which dissociates from the initiator during the final phase of a polymerization reaction. An example of a waste complex is depicted in
Kinetically encoded imaging reactions that use polymerase-based single-molecule timers require the use of a polymerase. In some embodiments, the polymerase is a DNA polymerase (DNAP), such as a DNA polymerase having DNA strand displacement activity. “Strand displacement” describes the ability to displace downstream DNA encountered during synthesis. Examples of polymerases having DNA strand displacement activity that may be used as provided herein include, without limitation, phi29 (e.g., NEB #M0269) and Bst DNA polymerase (e.g., NEB #M0275). Phi29 polymerase is most active at moderate temperatures (e.g., 20-37° C.), while Bst polymerase is most active at elevated temperatures (e.g., 65° C.). In some embodiments, the polymerase is an RNA polymerase, reverse transcriptase or a polymerase engineered to incorporate non-natural nucleotides.
With exonuclease-based timers, an exonuclease that selectively degrades one strand of a nucleic acid duplex is used to generate timer behavior (see, e.g.,
Thus, a kinetically encoded imaging system, in some embodiments, comprises (a) a target nucleic acid, (b) a 5′-phosphorylated nucleic acid probe linked to a 3′ detectable molecule, and (c) a 5′-phosphate-specific exonuclease.
A “5′-phosphorylated nucleic acid probe” refers to an unpaired (single-stranded) nucleic acid that is complementary to and binds to a target sequence of interest. The length of a probe may vary and depends, in part, on the length of the target sequence. In some embodiments, a probe has a length of 5-200 nucleotides. For example, a probe may have a length of 5-190, 5-180, 5-170, 5-160, 5-150, 5-140, 5-130, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a probe has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. In some embodiments, a probe has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A probe, in some embodiments, is longer than 200 nucleotides, or shorter than 5 nucleotides.
A 5′-phosphorylated nucleic acid probe, as provided herein, may be linked to (labeled with) a detectable molecule. In some embodiments, the label is a fluorophore. A probe linked to a fluorophore or other fluorescent/chemiluminescent molecule is referred to simply as a “fluorescent probe.” Examples of fluorophores that may be used herein include, without limitation, Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 405, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 647, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7 and Cy7.5. Other fluorophores and molecules that emit a detectable signal are encompassed by the present disclosure.
Exonuclease-based single-molecule timers may be used in a kinetically encoded imaging method. In some embodiments, the method comprises combining in reaction buffer (a) a target nucleic acid, (b) a 5′-phosphorylated nucleic acid probe linked to a 3′ detectable molecule, and (c) a 5′-phosphate-specific exonuclease; and incubating the reaction mixture under conditions that result in exonuclease-mediated degradation of the probe.
With hybridization cascade single-molecule timers, a series of nucleic acids react with each other in a specific order, resulting in the production of a branched Waste Complex (see, e.g.,
Thus, a kinetically encoded imaging system, in some embodiments, comprises (a) an unpaired initiator nucleic acid, and (b) a first hairpin probe, a second hairpin probe, a third hairpin probe and a fourth hairpin probe, each hairpin probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by intramolecular base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain located between the 5′ subdomain and the 3′ subdomain, wherein the first hairpin probe is linked to a detectable molecule.
In some embodiments, (i) the toehold domain and the 5′ subdomain of the first probe are complementary to and bind to the initiator nucleic acid, (ii) the toehold domain and the 5′ subdomain of the second probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the first probe bound to the initiator sequence, (iii) the toehold domain and the 5′ subdomain of the third probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the second probe bound to the first probe, and (iv) the toehold domain and the 5′ subdomain of the fourth probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the third probe bound to the second probe, and wherein the hairpin loop and 3′ subdomain of the fourth probe are complementary to and bind to the toehold domain and the 5′ subdomain of the first probe.
Hybridization cascade single-molecule timers may be used in a kinetically encoded imaging method. In some embodiments, the method comprises combining in reaction buffer (a) an unpaired initiator nucleic acid, and (b) a first hairpin probe, a second hairpin probe, a third hairpin probe and a fourth hairpin probe, each hairpin probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by intramolecular base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain located between the 5′ subdomain and the 3′ subdomain, wherein the first hairpin probe is linked to a detectable molecule, and incubating the reaction mixture under conditions that result in DNA hybridization.
It should be understood that the nucleic acids of the present disclosure do not occur in nature. Thus, the nucleic acids may be referred to as “engineered nucleic acids.” An “engineered nucleic acid” is a nucleic acid (e.g., at least two nucleotides covalently linked together, and in some instances, containing phosphodiester bonds, referred to as a phosphodiester “backbone”) that does not occur in nature. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. A “recombinant nucleic acid” is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) and, in some embodiments, can replicate in a living cell. A “synthetic nucleic acid” is a molecule that is amplified or chemically, or by other means, synthesized. A synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with (also referred to as “binding to,” e.g., transiently or stably) naturally-occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
While an engineered nucleic acid, as a whole, is not naturally-occurring, it may include wild-type nucleotide sequences. In some embodiments, an engineered nucleic acid comprises nucleotide sequences obtained from different organisms (e.g., obtained from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, or a combination of any two or more of the foregoing sequences.
In some embodiments, an engineered nucleic acid of the present disclosure may comprise a backbone other than a phosphodiester backbone. For example, an engineered nucleic acid, in some embodiments, may comprise phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, peptide nucleic acids or a combination of any two or more of the foregoing linkages. An engineered nucleic acid may be single-stranded (ss) or double-stranded (ds), as specified, or an engineered nucleic acid may contain portions of both single-stranded and double-stranded sequence. In some embodiments, an engineered nucleic acid contains portions of triple-stranded sequence, or other non-Watson-Crick base pairing such as G-quartets, G-quadruplexes, and i-motifs. An engineered nucleic acid may comprise DNA (e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA), RNA or a hybrid molecule, for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.
Engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5′ exonuclease, the 3′ extension activity of a DNA polymerase and DNA ligase activity. The 5′ exonuclease activity chews back the 5′ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed domains. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. Other methods of producing engineered nucleic acids are known in the art and may be used in accordance with the present disclosure.
An individual single-molecule timer exhibits characteristic changes in fluorescence over time, with intensity spikes (“ON”) that occur at random intervals amid a low-fluorescence background (“OFF”) but have a precisely controlled duration (see, e.g.,
Unlike existing approaches to multiplexing in fluorescence microscopy, barcoding with single-molecule timers does not require multiple buffer exchanges, photobleaching steps, or ex situ imaging. Rather, the sample can be imaged directly in the presence of the mixture of timers, because they all can be distinguished by a combination of dwell time and wavelength. This imaging may take place over minutes or tens of minutes, rather than the several hours or days required for competing approaches.
Single-molecule timers may be used, in some embodiments, for multiplexed super-resolution microscopy of cells and tissues (see, e.g.,
Single-molecule clocks, as provided herein, in some aspects, utilize a cascade of several irreversible reactions to establish a well-defined time delay between repeat binding, dissociation, or any combination thereof, of a fluorescent imager strand or strands to a nucleic acid generated or displaced from a complementary template; the summation of several exponentially distributed variables yields a sharp gamma distribution of dwell times (see, e.g.,
where N is the number of reversible reactions between binding and dissociation. This quasi-deterministic behavior permits the unambiguous identification of a target by observing the duration of a single binding event.
The binding equilibrium of a bimolecular complex can usually be approximated as a 1-step binding system (or a two-state system) characterized by a bimolecular association rate constant ko and a unimolecular dissociation rate constant k1 (
obeys a gamma distribution, with standard deviation proportional to 1/√{square root over (N)} (
Single-molecule clocks enable the generation of periodic fluorescent signals. A DNA polymerase is used in combination with a circular DNA template to generate delays between signaling events. As shown in
Signal is generated by the binding of a short fluorescent DNA imager strand (e.g., ˜20 nucleotides) to the product DNA sequence as it is liberated by the strand displacement activity of the polymerase. The imager strand, in some embodiments, is present at high concentration (e.g., ˜5 micromolar) so that imager strand binding is fast compared to the lifetime of a period (the time it takes for the polymerase to synthesize one copy from the template sequence). In some embodiments, other signal-generating mechanisms may be used, such as the addition of fluorescent or fluorogenic nucleotides.
The length of delays between these pulses can be used to encode the identity of a molecular target that is bound by the leash. This type of encoding scheme may be useful for multiplexed fluorescence microscopy, for example.
In addition to time delays between fluorescent pulses, target identity can also be encoded in the order of multicolor probe binding events. For instance, by embedding two different probe sequences in the template, each specifying a different color (e.g., red and blue), a variety of permutations of color and order should be possible (see, e.g.,
Further, by combining both time delays and multiple probe colors, an encoding scheme resembling a “multicolor Morse code” may be used (see, e.g.,
Morse Probes. In some embodiments, a “Morse probe” labeling system with a catenated DNA structure may be used. Morse probe systems provide a low signal-to-noise ratio due to at least two features. First, only a single imager strand should bind to a template at any given time. Therefore, only a single fluorophore emits a fluorescent signal (for a given probe) at any given time, which greatly limits sources of noise, such as double (imager) binding events. Second, in order for the polymerase to move forward, it displaces the imager strand, which enables a dark state before the arrival of the next imager strand and obviates a need to photobleach the fluorophore, or to wait for quencher strand binding to achieve the dark state.
A schematic of an example of a Morse probe labeling system is shown in
Imager strands may be present in high concentration in solution, in some embodiments, in order to facilitate favorable kinetics (Zhang et al., J Am Chem Soc., 2009, 131: 17303-14). To minimize background fluorescence resulting from the presence of imager strands at high concentrations, the imager strands may be hybridized with a complementary strand containing a quencher, in a molecular beacon type configuration. Once the imager strand attaches to the template, the quencher is displaced, resulting in emission of a fluorescent signal (fluorescent pulse). A short time thereafter, the polymerase reaches the site of attachment and displaces the imager strand, bringing the system back to its “dark” state.
The imager strand binds in front of the polymerase and, therefore, the directionality of the polymerase prescribes which end (3′ end or 5′ end) of the probe comprises the label (e.g., fluorophore). In some embodiments, a fluorescent label is located at the 3′ end of the imager strand, while the quencher molecule is located at the 5′ end of a corresponding (complementary) quencher strand. In some embodiments, the imager strand is 5-20 nucleotides longer than its corresponding quencher strand. In such embodiments, this length limits the fluorophore of the imager strand to one end (e.g., the 3′ end) and the fluorophore of the quencher strand to the other end (e.g., 5′ end).
Examples of quencher molecules include, but are not limited to, the following: BHQ 0, BHQ 1, BHQ 2, BHQ 3, Iowa Black FQ, and Iowa Black RQ. Other quencher molecules may be used. Each molecule quenches a fluorescent signal within a characteristic range; however, the system is agnostic to the choice of fluorophore-quencher pair and therefore, as more quenchers become available, these other quenchers may be used. The ratio of imager strand:quencher strand should be sufficient so that there is minimal background fluorescence during TIRF imaging. The imager:quencher ratio can be, for example, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, or 1:14. In some embodiments, the imager:quencher ratio is 1:4. The imager strand:quencher strand ratio may vary depending on the particular reaction conditions used.
In some embodiments, the leash is ssDNA or dsDNA. The outcome is independent of the length of the leash, but the leash is typically long enough such that the lumen of the circular leash allows the passage of polymerase without restriction.
In some embodiments, the template (template strand) is ssDNA. The length of the template in part determines the number of binding sites, which provides the encoding capacity of the Morse probe library. The GC content of the template, salt concentration in buffers, imaging temperature, and concentration of dNTPs in solution are some of the parameters that contribute to the speed of polymerase activity. Thus, these parameters also contribute toward the speed of attachment and detachment of the imager strand and the distribution of wait times.
In some embodiments, an endonuclease may be introduced to cleave the product strand, so that the imager strand has unfettered access to bind to the template. In time-lapse imaging, the large product strand may clump to the point where it limits the imager strand's access to the template strand, which may limit the time a system can be studied. With the inclusion of an endonuclease, little to no binding will occur on the product strand and imaging should not be adversely affected.
In some embodiments, the binding sites for imager strands may partially overlap on the template. This works because the number of nucleotides “ahead” of the polymerase is small enough (3-5 nucleotides) to ensure binding of only one strand at a time. Once one strand is displaced by the polymerase, a separate, partially overlapping binding site can become available. Therefore, the coding capacity for a template of a given length is increased.
Circular Nucleic Acid Template
Single-molecule clocks of the present disclosure are architecturally similar to a catenane: a mechanically-interlocked molecular structure that includes at least two interlocked macrocycles. A single-molecule clock (“clock”) comprises a circular nucleic acid “template strand” (or “template”) interlocked with a circular nucleic acid “leash strand.” A circular nucleic acid may be a single-stranded or double-stranded nucleic acid joined at each end (e.g., the 5′ and the 3′ end of the strand joined to each other via ligation) to form a circular structure. As example of a single-molecule clock catenane is shown in
A template typically includes a primer binding sequence. A primer binding sequence is a sequence of nucleotides (e.g., comprising A, T, C and G) to which a nucleic acid primer can bind. Thus, a nucleic acid primer comprises a nucleotide sequence complementary to a primer binding sequence. The length of a primer binding sequence (and thus the complementary primer) may vary. In some embodiments, a primer binding sequence has a length of 5-50 nucleotides. For example, a primer binding sequence may have a length of 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10, 40, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a primer binding sequence has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. In some embodiments, a primer binding sequence has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A primer binding sequence, in some embodiments, is longer than 50 nucleotides.
A circular nucleic acid template, in some embodiments, comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or a combination thereof. In some embodiments, a template comprises DNA.
The length of a circular template may vary. It should be understood that “length” in the context of a circular structure refers to the number of contiguous nucleotides in the structures (the number of nucleotides that form the circular structure). In some embodiments, the length of a template is 50-10000 nucleotides. For example, a template may have a length of 50-5000, 50-1000, 50-500, 50-200, 50-100, 100-10000, 100-5000, 100-1000, 100-500, 100-200, 200-10000, 200-5000, 200-1000, 200-500, 500-10000, 500-5000, or 500-1000 nucleotides. In some embodiments, a template has a length of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides.
Product Strand
A template may also include a nucleotide sequence that “specifies” or “encodes” an imager strand binding sequence. It should be understood that in the context of the present disclosure, a nucleotide sequence of a template is considered to “specify” or “encode” its complementary strand, e.g., produced via a rolling circle replication reaction. This complementary strand is referred to herein as the “product strand.” Thus, as shown, for example, in
In some embodiments, a primer binding sequence “specifies” or “encodes” an imager strand binding sequence. That is, an imager strand, in some embodiments, may bind to a sequence on a product strand that is specified by the primer binding sequence (is complementary to the primer binding sequence).
The length of a product strand may also vary. In some embodiments, the length of a product strand is 50-10000 nucleotides. For example, a product strand may have a length of 50-5000, 50-1000, 50-500, 50-200, 50-100, 100-10000, 100-5000, 100-1000, 100-500, 100-200, 200-10000, 200-5000, 200-1000, 200-500, 500-10000, 500-5000, or 500-1000 nucleotides. In some embodiments, a product strand has a length of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides. In some embodiments, a product strand is longer than 10000 nucleotides.
Circular Nucleic Acid Leash
A circular nucleic acid leash (“leash”) enables the template to be physically linked to an affinity tag while not interfering with access of a polymerase to the template. Thus, in some embodiments, a circular nucleic acid leash is linked to (bound to) a binding partner molecule, such as biotin, a ligand, a receptor, an antibody or a nucleic acid (e.g., hybridization probe). Other binding partner molecules (molecules that specifically bind to other molecules) are encompassed herein.
The length of a circular nucleic acid leash may also vary. In some embodiments, the length of a leash is 50-10000 nucleotides. For example, a leash may have a length of 50-5000, 50-1000, 50-500, 50-200, 50-100, 100-10000, 100-5000, 100-1000, 100-500, 100-200, 200-10000, 200-5000, 200-1000, 200-500, 500-10000, 500-5000, or 500-1000 nucleotides. In some embodiments, a leash has a length of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides. In some embodiments, a leash is shorter than a template. In some embodiments, a leash has a length of less than 50 nucleotides. For example, a leash may have a length of 10, 15, 20, 25, 30, 35, 40 or 45 nucleotides. In some embodiments, a leash has a length of 5-100 nucleotides, 10-100 nucleotides, or 20-100 nucleotides.
A circular nucleic acid leash, in some embodiments, comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or a combination thereof. In some embodiments, a circular nucleic acid leash comprises DNA. In some embodiments, a circular nucleic acid leash comprises RNA.
In some embodiments, a non-nucleic acid leash is used to enable the template to be physically linked to an affinity tag while not interfering with access of a polymerase to the template. For example, a leash may be a polypeptide or other cyclic organic molecule, provided (1) the leash can be mechanically interlocked with the template, (2) the leash is large enough to permit passage of the polymerase (e.g., diameter greater than ˜5 nanometers), and (3) the leash can be chemically linked to an antibody or other affinity reagent.
Primer
Typically, a primer is an unpaired (single-stranded) nucleic acid (e.g., DNA), although, in some instances, a primer may be partially paired (partially double-stranded) (containing a paired domain and an unpaired domain). A primer comprises a nucleotide sequence that is complementary to a primer binding sequence of a template and can bind to a template to initiate polymerization (in the presence of polymerase and dNTPs). In some embodiments, a primer is a single strand of DNA. Thus, the length of a primer depends, in part, on the length of the primer binding sequence on the template. In some embodiments, a primer has a length of 5-50 nucleotides. For example, a primer may have a length of 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10, 40, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a primer has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. In some embodiments, a primer has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A primer, in some embodiments, is longer than 50 nucleotides.
Polymerase
Compositions and methods of the present disclosure, in some embodiments, include a polymerase. In some embodiments, the polymerase is a DNA polymerase, a RNA polymerase or reverse transcriptase. Polymerases used herein should have nucleic acid (e.g., DNA) strand displacement activity. “Strand displacement” describes the ability to displace downstream DNA encountered during synthesis. Examples of polymerases having DNA strand displacement activity that may be used as provided herein include, without limitation, phi29 (e.g., NEB #M0269) and Bst DNA polymerase (e.g., NEB #M0275). Phi29 polymerase is most active at moderate temperatures (e.g., 20-37° C.), while Bst polymerase is most active at elevated temperatures (e.g., 65° C.). In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the polymerase is an RNA polymerase, reverse transcriptase or a polymerase engineered to incorporate non-natural nucleotides.
Imager Strand
In some embodiments, an imager strand is used to generate a detectable signal (a pulse). An imager strand is a single nucleic acid (e.g., DNA) strand that is linked to a detectable molecule (e.g., a molecule that emits a detectable signal, such as a fluorescent or chemiluminescent signal), referred to as a label. An imager strand comprises a sequence that is complementary to and can bind to a sequence on the product strand (specified by the template). In some embodiments, a imager strand has a length of 5-50 nucleotides. For example, a imager strand may have a length of 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10, 40, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35 or 35-40 nucleotides. In some embodiments, a imager strand has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. In some embodiments, a imager strand has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. A imager strand, in some embodiments, is longer than 50 nucleotides, or shorter than 5 nucleotides.
In some embodiments, the label is a fluorophore. An imager strand linked to a fluorophore or other fluorescent/chemiluminescent molecule is referred to simply as a “fluorescent imager strand.” Examples of fluorophores that may be used herein include, without limitation, Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 405, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 647, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7 and Cy7.5. Other fluorophores and molecules that emit a detectable signal are encompassed by the present disclosure.
In some embodiments, a template specifies more than one imager strand binding sequence on a product strand (each imager strand binding sequence having a different sequence relative to one another, thus, each “distinct” on the product strand). Thus, some compositions comprise more than one type of imager strand. “Types” of imager strands differ by their sequence composition (number, type and arrangement of nucleotides). Different types of imager strands may also have different types of labels. For example, one imager strand in a composition may have a red fluorophore, while another imager strand in the same composition may have a blue fluorophore (see, e.g.,
Binding (stable binding) of an imager strand to a product strand results in emission (a “pulse”) of a fluorescent signal. In some embodiments, the duration of the fluorescent pulse is limited by photobleaching of the label associated with the imager strand. For example, a bound imager strand label may photobleach 0.01-100 seconds after binding. In some embodiments, the duration of the fluorescent pulse is controlled by subsequent binding of a quencher-labeled oligonucleotide that is complementary to a sequence adjacent to the imager binding site on the product strand (i.e., quencher binding site). In such a case, the duration of the pulse may be controlled in part by the rate of the multi-step addition of nucleotides comprising the adjacent quencher binding site to the product. For example, a quencher-labeled strand may bind 0.01-100 seconds after the imager strand binds. In some embodiments, the duration of the fluorescent pulse is controlled by subsequent degradation of the imager strand by an exonuclease (e.g., lambda exonuclease) that selectively degrades the imager strand upon binding to the template. For example, lambda exonuclease may degrade an imager strand 0.1-100 seconds after imager strand binding to the product strand. In some embodiments, the fluorescent signal of an imager strand lasts for ˜0.1-100 seconds, depending, in part, on the photostability of the imager strand label, the intensity of illumination during imaging, the number of nucleotides that must be added before quencher binding can occur, and the reaction conditions (e.g., buffer, temperature, dNTP concentration, oxygen concentration, concentration of quencher strand, concentration of exonuclease).
The amount of time between pulses may be controlled, in some embodiments, by varying the distance between imager strand binding sequences along the product or template, buffer conditions, temperature, and/or deoxynucleotides (dNTPs) concentrations in a reaction, as DNA polymerases, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions). For example, increasing the temperature of a reaction using phi29 polymerase from 15° C. to 30° C. decreases dwell time. Likewise, increasing the temperature of a reaction using Bst DNA polymerase, large fragment from room temperature (˜25° C.) to 65° C. (the optimal temperature for polymerization by this enzyme) decreases dwell time. Thus, the temperature at which a reaction is performed may vary from, for example, 4° C. to 65° C. (e.g., 4° C., 25° C., 37° C., 42° C. or 65° C.). In some embodiments, a reaction is performed at room temperature, while in other embodiments, a reaction is performed at 37° C. As another example, increasing salt concentration (e.g., increasing [NaCl] from 40 mM to 200 mM) results in slower DNA polymerization and longer times between pulses (e.g., for Bst DNA polymerase, large fragment).
In some embodiments, the concentration of dNTPs in a reaction is 100 nM-100 μM. For example, the concentration of dNTPs in a reaction may be 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nM (μM). In some embodiments, the concentration of dNTPs in a reaction is 2.5 μM-100 μM. For example, the concentration of dNTPs in a reaction may be 2.5-75, 2.5-50, 2.5-25, 2.5-20, 2.5-5, 5-100, 5-75, 5-50, 5-25, 5-20, 10-100, 10-75, 10-50, 105-25, 10-20, 25-100, 25-75, 25-50, 50-100, 50-75 or 75-100 μM. In some embodiments, the concentration of dNTPs in a reaction is 0.1, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 90 or 100 μM. In some embodiments, the concentration of dNTPs in a reaction is 2.5 μM to 1 mM, or 2.5 μM to 10 mM. For example, the concentration of dNTPs in a reaction may be 0.5, 1 mM, 5 mM or 10 mM.
Labeled dNTPs
In some embodiments, the incorporation of one or more labeled species of nucleotide triphosphate monomer (for example, fluorescently labeled ribonucleoside triphosphates (NTPs), deoxyribonucleoside triphosphates (dNTPs), or a non-natural nucleic acid monomer) is used to generate a detectable signal (pulse). In some embodiments, the labeled oligonucleotide comprises both a fluorescent label and a quencher that is removed upon incorporation into the product strand (i.e., an internally quenched nucleotide), resulting in an enhancement of fluorescent signal upon incorporation. In some embodiments, the template comprises one or more contiguous nucleotide sequences (pulse zones) that template the addition of at least one fluorescent nucleotides. For example, a pulse zone may template the incorporation of 1-100 (e.g., 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100) fluorescent nucleotides. In some embodiments, the pulse zones are separated by contiguous sequences that template the addition of only non-labeled nucleotides (non-pulse zones). In some embodiments, the duration of a pulse is determined by the amount of time required to incorporate all labeled nucleotides within a pulse zone. In some embodiments, the duration of a pulse is determined by the rate of photobleaching of a fluorescent label. In some embodiments, the duration of a pulse is determined by the rate of degradation of the product strand by an exonuclease. In some embodiments, the delay between fluorescent pulses is controlled by the number of nucleotides in a non-pulse zone. For example, 1-1000 (e.g., 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000) non-labeled nucleotides may be incorporated between consecutive labeled nucleotides, resulting in a delay of ˜0.1-1000 seconds between pulses.
It should be understood that the nucleic acids of the present disclosure do not occur in nature. Thus, the nucleic acids may be referred to as “engineered nucleic acids.” An “engineered nucleic acid” is a nucleic acid (e.g., at least two nucleotides covalently linked together, and in some instances, containing phosphodiester bonds, referred to as a phosphodiester “backbone”) that does not occur in nature. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. A “recombinant nucleic acid” is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) and, in some embodiments, can replicate in a living cell. A “synthetic nucleic acid” is a molecule that is amplified or chemically, or by other means, synthesized. A synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with (also referred to as “binding to,” e.g., transiently or stably) naturally-occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
While an engineered nucleic acid, as a whole, is not naturally-occurring, it may include wild-type nucleotide sequences. In some embodiments, an engineered nucleic acid comprises nucleotide sequences obtained from different organisms (e.g., obtained from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, or a combination of any two or more of the foregoing sequences.
In some embodiments, an engineered nucleic acid of the present disclosure may comprise a backbone other than a phosphodiester backbone. For example, an engineered nucleic acid, in some embodiments, may comprise phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, peptide nucleic acids or a combination of any two or more of the foregoing linkages. An engineered nucleic acid may be single-stranded (ss) or double-stranded (ds), as specified, or an engineered nucleic acid may contain portions of both single-stranded and double-stranded sequence. In some embodiments, an engineered nucleic acid contains portions of triple-stranded sequence, or other non-Watson-Crick base pairing such as G-quartets, G-quadruplexes, and i-motifs. An engineered nucleic acid may comprise DNA (e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA), RNA or a hybrid molecule, for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.
Nucleic acids (e.g., circular template, circular leash, imager strands and/or primers) of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5′ exonuclease, the 3′ extension activity of a DNA polymerase and DNA ligase activity. The 5′ exonuclease activity chews back the 5′ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed domains. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. Other methods of producing nucleic acids are known in the art and may be used in accordance with the present disclosure.
An individual single-molecule clock exhibits characteristic changes in fluorescence over time, with intensity spikes (“ON”) that occur amid a low-fluorescence background (“OFF”), with the duration between consecutive ON states, OFF states, or any combination thereof precisely controlled. Due to the use of a circular template, the series of ON and OFF states may occur and be observed several times in succession for greater confidence in the signal. By varying the characteristic dwell times in the ON and OFF states of a clock system (e.g., by varying the number of nucleotides between imager binding sites on the product, or the number of nucleotides between an imager binding site and a quencher binding site on the product), multiple clocks that are distinguishable by kinetics alone due to their well-separated dwell time distributions can be constructed. In a polymerase-based clock system, the mean dwell time between two events is linearly dependent on the length of the intervening template sequence, allowing the systematic design of multiple distinguishable clocks. For multiplexed detection, a single-molecule clock system may comprise a template that, though the action of a polymerase, generates an ordered series of ON and OFF states of defined length and other distinguishable characteristics (e.g., color of fluorescence) to create kinetic barcodes that can be read out in minutes. The number of possible distinguishable kinetic barcodes is dictated by the number of pulses per cycle encoded by the template; the number of distinguishable delays between consecutive pulses, OFF states, or any combination thereof; and the number of distinguishable labels (e.g., fluorophores of different emission or excitation wavelength). For example, a clock with four pulses, two distinguishable delay times, and three distinguishable labels can distinguish (2×3)4=1,296 targets. This high degree of multiplexing does not require any kind of strand exchange or spatially resolved (i.e. by direct imaging) encoding of information. In the embodiments discussed above, each barcode may be only tens of nanometers in size, making super-resolution imaging within intact cells and tissues a possibility.
Unlike existing approaches to multiplexing in fluorescence microscopy, barcoding with single-molecule clocks does not require multiple buffer exchanges, photobleaching steps, or ex situ imaging. Rather, the sample can be imaged directly in the presence of the mixture of clocks, because they all can be distinguished by a combination of dwell time and wavelength. This imaging may take place over minutes or tens of minutes, rather than the several hours or days required for competing approaches.
Single-molecule clocks may be used, in some embodiments, for multiplexed super-resolution microscopy of cells and tissues. Kinetic barcodes constructed from single-molecule clocks can used to detect and spatially resolve thousands of distinct targets in a single imaging experiment lasting only 1-10 minutes. The target biomolecules may be proteins, nucleic acids, or any other biomolecule for which selective affinity reagents (e.g., antibodies or hybridization probes) are available and may be conjugated to appropriate barcode sequences. In one embodiment, a DNA-barcoded antibody library is constructed; that is, each antibody species is associated with a unique DNA barcode specifying an ordered combination of pulse delays and colors. Each barcode, and hence, each antibody, is identifiable based on the combination of clock probes that bind to it. Furthermore, because single pulses are detected and can be localized using various existing fitting algorithms, super-resolution microscopy is possible. This technique can be used, for instance, in the high-content imaging of heterogeneous tissues, such as tumors, where the expression levels and localization of biomarkers within the tissue may help to characterize and respond to drug resistance.
The present disclosure further provides embodiments encompassed by the following numbered paragraphs:
(a) an unpaired initiator nucleic acid comprising a 3′ nucleotide subdomain and a 5′ nucleotide subdomain;
(b) a template probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain; and
(c) a primer.
18. The system of any one of paragraphs 1-17,wherein the hairpin stem domain has a length of 10-20 nucleotides.
combining in reaction buffer
incubating the reaction mixture under conditions that result in DNA polymerization.
(a) a target nucleic acid;
(b) a 5′-phosphorylated nucleic acid probe linked to a 3′ detectable molecule; and
(c) a 5′-phosphate-specific exonuclease.
combining in reaction buffer
incubating the reaction mixture under conditions that result in exonuclease-mediated degradation of the probe.
(a) an unpaired initiator nucleic acid; and
(b) a first hairpin probe, a second hairpin probe, a third hairpin probe and a fourth hairpin probe, each hairpin probe comprising (i) an unpaired 5′ toehold domain, (ii) a hairpin stem domain formed by intramolecular base pairing between nucleotides located in a 5′ subdomain of the probe and nucleotides located in a 3′ subdomain of the probe, and a hairpin loop domain located between the 5′ subdomain and the 3′ subdomain, wherein the first hairpin probe is linked to a detectable molecule.
(i) the toehold domain and the 5′ subdomain of the first probe are complementary to and bind to the initiator nucleic acid;
(ii) the toehold domain and the 5′ subdomain of the second probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the first probe bound to the initiator sequence;
(iii) the toehold domain and the 5′ subdomain of the third probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the second probe bound to the first probe; and
(iv) the toehold domain and the 5′ subdomain of the fourth probe are complementary to and bind to the hairpin stem domain and the 3′ subdomain of the third probe bound to the second probe, and wherein the hairpin loop and 3′ subdomain of the fourth probe are complementary to and bind to the toehold domain and the 5′ subdomain of the first probe.
combining in reaction buffer
incubating the reaction mixture under conditions that result in DNA hybridization.
(a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash;
(b) a nucleic acid primer comprising a sequence complementary to the primer binding sequence; and
(c) a labeled nucleic acid imager strand.
combining in reaction buffer
incubating the reaction mixture under conditions that result in nucleic acid polymerization and nucleic acid hybridization.
(a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash;
(b) a primer comprising a sequence complementary to the primer binding sequence; and
(c) a mixture of deoxynucleoside triphosphates (dNTPs) comprising subsets of dATPs, dTTPs, dCTPs and dGTPs, wherein dNTPs of at least one of the subsets comprise a label; or a mixture of nucleoside triphosphates (NTPs) comprising subsets of ATPs, TTPs, CTPs and GTPs, wherein NTPs of at least one of the subsets comprise a label.
combining in reaction buffer
incubating the reaction mixture under conditions that result in nucleic acid polymerization and nucleic acid hybridization.
(a) a circular nucleic acid template comprising a primer binding sequence and interlocked with a circular nucleic acid leash;
(b) a nucleic acid primer comprising a sequence complementary to the primer binding sequence; and
(c) a labeled nucleic acid imager strand bound to a quencher strand comprising a quencher molecule, wherein the quencher strand is shorter than the imager strand.
combining in reaction buffer
incubating the reaction mixture under conditions that result in nucleic acid polymerization and nucleic acid hybridization.
The present disclosure is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teachings that are referenced herein.
As the number of irreversible steps between binding and dissociation increases, two trends are expected: (1)
is inversely proportional to template length, indicating more deterministic behavior as the number of steps increases. However, contrary to naïve predictions, Napp is not equal to the number of nucleotides added, but is related by a proportionality constant of ˜0.43, suggesting that not all nucleotide addition steps are equally rate-limiting. On the other hand,
This Example provides data for characterizing parameters for gamma fit versus template length. When the gamma distribution function is fit to the experimental dwell time distributions (
This Examples provides data for characterizing variation of the dwell time of single-molecule timers. Using a single template design (41 nucleotides), it is possible to vary the dwell time of single-molecule timers by varying the concentration of free nucleotides (dNTPs). This demonstrates that it is primarily the rate of DNA polymerization that controls the duration of timer binding events.
This Example provides data from a kinetic barcoding experiment using polymerase-based single-molecule timers. Multiple timers were used in the same experiment to construct barcodes for multiplexed imaging. In such a scheme, each barcode includes one or more initiator sequences, each of which binds to a specific timer. Each barcode's identity is determined by measuring the kinetics of timer binding to that barcode; the presence or absence of each timer lifetime is interpreted as a binary “bit” (1 or 0). Because the distributions of dwell times used for different “bits” do not overlap substantially, the presence or absence of each initiator sequence in the barcode can be determined with high confidence, particularly if many timer binding events are observed for each barcode. Two potential schemes for kinetic barcoding are shown.
This Example shows that a short DNA-PAINT probe (Jungmann R et al. Nature Methods, 2014, 11, 313-318) may be used alongside single-molecule clocks to provide another well-resolved dwell time distribution (“bit”) in a kinetic barcode. A representative fluorescence vs. time trace is shown (
This Example provides data representative of the simultaneous use of multiple timers. The mixture gives rise to a dwell time distribution that resembles the sum of the three underlying timer distributions. A representative single-molecule fluorescence trace is also shown. Dwell time distributions focus on binding events that last 30 seconds or less. There were not enough binding events per trace to classify each barcode, so it was necessary to increase Cy5 primer concentration, add quencher-labeled protector strand, and increase acquisition time.
This Example provides data of dwell time distributions with binding events lasting longer than 30 seconds. Rarely, binding events can have much longer dwell times. This gives rise to a background that appears exponential, rather than gamma-distributed. This portion of the dwell time distributions is shown above for three templates: 41 nt (
This Example provides additional data from a kinetic barcoding experiment using polymerase-based single-molecule timers. For a single-molecule timer of the type described in
To demonstrate deterministic timer behavior at the single-molecule level, a total internal reflection fluorescence (TIRF) assay was used in which biotinylated initiator strand I was immobilized on a passivated coverslip (
This Example provides additional data from a kinetic barcoding experiment using polymerase-based single-molecule timers.
A catenane with an 84 nucleotide leash and a 179 nucleotide template was used. DNA replication was achieved by phi29 polymerase. Due to the direction of DNA replication, the toehold on the imager-quencher beacon was located on the 5′ end. Therefore, the fluorophore, Cy5 was available at the 3′-end. Iowa Black RQ was used as a quencher. The system is agnostic to the choice of fluorophore-quencher pairs. A generic imager-quencher pair is shown in
Catenanes were immobilized on a DDS passivated glass surface, using a biotin-streptavidin sandwich. Total Internal Reflection Fluorescence (TIRF) microscopy was performed to visualize the fluorescently labeled catenanes. TIRF videos were collected under 63× objective. The fluorescent blinking pattern observed and the wait times between individual pulses, shown in
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of and “consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/287,816, filed Jan. 27, 2016, and U.S. provisional application No. 62/409,305, filed Oct. 17, 2016, each of which is incorporated by reference herein it its entirety.
This invention was made with government support under 1435964 awarded by National Science Foundation and under N000141510073 awarded by Office of Naval Research. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/15057 | 1/26/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62287816 | Jan 2016 | US | |
| 62409305 | Oct 2016 | US |
