Reaction compositions and methods for intensely labeled sequencing

Abstract

Provided is a composition that is useful for detecting single molecules in sample.In some embodiments, the present disclosure provides a nucleotide linked to nucleic acid, which comprises FRET label comprising at least three luminescent molecules.In some embodiments, the nucleic acid described herein comprises one or more structural features that provide enhanced fluorescence intensity.In some embodiments, provided is a sequencing method using the labeled nucleotide of the present disclosure.

Description

[Technical field]

[0001] This application relates generally to brightly labeled reaction compositions and methods for the detection of single molecules using same. [Background technology]

[0002] Advances in next generation sequencing technologies have made it possible to perform massively parallel analyses of single molecules, fundamentally changing the landscape of life science research. Some of these techniques involve monitoring biological reactions in real time using luminescently labeled reaction components. The labels are made to luminesce upon illumination with a light source, and the luminescent emissions are detected with a photodetector. These events can be recorded and analyzed to identify individual reaction components based on their corresponding light-emitting properties. Summary of the Invention [Problem to be solved by the invention]

[0003] In identifying a particular type of labeled molecule among multiple types, it is important that each type has unique and easily identifiable emission characteristics. Furthermore, these parameters may dictate instrument requirements such as excitation source and overall instrument size. [Means for solving the problem]

[0004] Aspects of the technology disclosed herein relate to labeled reaction components comprising a FRET label comprising three or more luminescent light-emitting molecules, comprising at least one donor molecule and at least one acceptor molecule. In some embodiments, the application relates to a labeled nucleotide comprising a nucleoside polyphosphate linked to a nucleic acid comprising a FRET label comprising at least three luminescent light-emitting molecules, comprising at least one donor molecule and at least one acceptor molecule. Other aspects of the technology disclosed herein relate to labeled reaction components comprising two or more luminescent light-emitting labels separated by a linker (e.g., a constrained linker). In some embodiments, the application relates to the separation of luminescent light-emitting labels to prevent attenuation of detectable signals due to label-label interactions. In some embodiments, the application provides labeled nucleotides comprising a nucleotide (e.g., a nucleoside polyphosphate) linked to two or more luminescent light-emitting labels via a linker. In some embodiments, the application provides compositions, methods, and kits for sequencing template nucleic acids.

[0005] In some embodiments, the present application provides a labeled nucleotide comprising a nucleotide (e.g., a nucleoside polyphosphate) linked to a FRET label comprising at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule. In some embodiments, the linker of the present disclosure is useful for providing a nucleotide (e.g., a nucleoside polyphosphate) with a FRET label. In some embodiments, the label of the labeled nucleotide is a FRET label that can be linked to the nucleoside polyphosphate via a linker or as a part of the linker. In some embodiments, the linker is a nucleic acid linker.

[0006] In some embodiments, the nucleotides are configured for use as substrates in a polymerization reaction. In some embodiments, the labeled nucleotides of the present application include a FRET label that includes a donor molecule to acceptor molecule ratio of 1:1. In some embodiments, the donor molecule to acceptor molecule ratio is greater than 1:1 (e.g., 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or more). In some embodiments, the acceptor molecule to donor molecule ratio is greater than 1:1 (e.g., 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or more). In some embodiments, the FRET label includes at least two donor molecules and at least one acceptor molecule. In some embodiments, the FRET label includes two donor molecules and one acceptor molecule. In some embodiments, the FRET label includes three donor molecules and one acceptor molecule. In some embodiments, the FRET label includes four donor molecules and one acceptor molecule. In some embodiments, the FRET label comprises four donor molecules and two acceptor molecules. In some embodiments, the FRET label comprises at least one donor molecule and at least two acceptor molecules. In some embodiments, the FRET label comprises two acceptor molecules and one donor molecule. In some embodiments, the FRET label comprises three acceptor molecules and one donor molecule. In some embodiments, the FRET label comprises four acceptor molecules and one donor molecule.

[0007] In some embodiments, the labeled nucleotide of the present application comprises a FRET label comprising at least three luminescent emission labels separated from each other by a minimum distance. For example, in some embodiments, each donor molecule and acceptor molecule is at least 1 nm apart. In some embodiments, each donor molecule and acceptor molecule is about 1-3 nm apart. In some embodiments, each donor molecule and acceptor molecule is separated by 3-13 bases. In some embodiments, each donor molecule and acceptor molecule is separated by 3-6 bases. In some embodiments, each donor molecule and acceptor molecule is separated by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 bases.

[0008] In some embodiments, each donor and acceptor molecule pair of a FRET label has a FRET efficiency of at least 80% (eg, at least 85%, at least 90%, or at least 95%).

[0009] In some embodiments, the present application provides a labeled nucleotide comprising a nucleotide (e.g., a nucleoside polyphosphate) linked to two or more luminescent labels via a linker. In some embodiments, the nucleotide is configured to be used as a substrate in a polymerization reaction. In some embodiments, the labeled nucleotide of the present application comprises two or more luminescent labels separated from each other by a minimum distance. In some embodiments, each luminescent label is at least 5 angstroms away from any other luminescent label. For example, in some embodiments, each luminescent label is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50 angstroms away from any other luminescent label. In some embodiments, each luminescent label comprises a center of gravity that is at least 5 angstroms away from the center of gravity of any other luminescent label.

[0010] In some embodiments, the labeled nucleotides of the present application include one or more luminescent labels attached to the linker via a spacer molecule. In some embodiments, the spacer molecule links the luminescent label to an attachment site on the linker. In some embodiments, the luminescent label is attached to the linker via a spacer molecule that includes at least 8 consecutive atoms between the luminescent label and the attachment site on the linker. In some embodiments, the spacer molecule includes fewer than 50, fewer than 40, fewer than 30, or fewer than 20 consecutive atoms between the luminescent label and the attachment site on the linker. In some embodiments, the luminescent label is incorporated within the linker.

[0011] In some embodiments, the linker is an oligomer (e.g., an oligomeric linker, or a polymeric linker). In some embodiments, the oligomer comprises monomeric units. In some embodiments, the oligomer comprises two or more different types of monomeric units. In some embodiments, the oligomer comprises a plurality of monomeric units of the same type (e.g., the oligomer is a polymer of one type of monomeric unit). In some embodiments, the oligomer comprises a first region having a plurality of monomeric units of a first type and a second region having a plurality of monomeric units of a second type. In some embodiments, the oligomer comprises a plurality of different regions (e.g., 2, 3, 4, 5, or more), each of which comprises a plurality of different types of monomeric units. In some embodiments, the oligomer comprises at least 5 monomeric units. In some embodiments, the oligomer comprises at least 10 monomeric units. In some embodiments, the oligomer contains fewer than 150, fewer than 100, or fewer than 50 monomer units (e.g., at least 5 monomer units and fewer than 200, 150, 100, 75, 50, or 25 monomer units; at least 10 monomer units and fewer than 200, 150, 100, 75, 50, or 25 monomer units).

[0012] In some embodiments, when the linker is an oligomer (e.g., an oligomeric linker, a polymeric linker), each luminescent light-emitting label or luminescent light-emitting molecule is separated from each other label by at least 5 monomer units of the oligomer. In some embodiments, a first luminescent light-emitting label or luminescent light-emitting molecule is incorporated into the linker at a first position that is at least 5 monomer units away from the second position, and a second luminescent light-emitting label or luminescent light-emitting molecule is incorporated into or attached to the linker at a second position. In some embodiments, when adjacent luminescent light-emitting labels or luminescent light-emitting molecules are incorporated into the linker, the labels may be separated by fewer than 5 monomer units. In some embodiments, each luminescent light-emitting label or luminescent light-emitting molecule is attached to the linker at an attachment site that is at least 5 monomer units (e.g., at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or more monomer units) away from any other attachment site. In some embodiments, each luminescent light-emitting label or molecule is attached to an attachment site that is at least 5 monomer units and fewer than 40 monomer units (e.g., fewer than 38, fewer than 36, fewer than 34, fewer than 32, fewer than 30, fewer than 28, fewer than 26, fewer than 24, fewer than 22, or fewer than 20 monomer units) away from any other attachment site. In some embodiments, the luminescent light-emitting label or molecule is incorporated into a linker between two consecutive monomer units of the oligomer (e.g., covalently links two adjacent monomer units of the oligomer).

[0013] In some embodiments, the linker is sufficiently rigid to prevent interactions between two or more luminescent labels or molecules attached to the linker. In some embodiments, the rigidity of the linker is sufficient to preserve the intensity of each label by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, compared to its intensity when present as a single label attached to the same linker.

[0014] In some embodiments, the linker is a peptide. In some embodiments, the amino acid composition of the peptide provides structural rigidity (e.g., by the presence of one or more polyproline segments in the peptide linker). In some embodiments, 90% or more (e.g., all) of the peptide linker is composed of polyproline polymers. In some embodiments, peptide rigidity is provided by constraining the peptide by chemical modification. For example, in some embodiments, the peptide includes one or more cyclization segments. In some embodiments, the peptide is a cyclized peptide (e.g., stapled peptide, end-to-end cyclized peptide, etc.). In some embodiments, sufficient peptide rigidity can be provided by incorporating a combination of one or more rigid amino acid polymer segments and one or more chemically modified amino acid polymer segments.

[0015] In some embodiments, the linker is a polysaccharide (eg, heparin, heparin sulfate, polyglucose, polylactose, aminoglycosides, N-acetylaminoglycosides, and combinations thereof).

[0016] In some embodiments, the linker is a nucleic acid. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or derivatives thereof. In some embodiments, sufficient rigidity is provided by using one or more double-stranded nucleic acid segments (e.g., separation of two or more different labels). In some embodiments, sufficient rigidity is provided by one or more chemical modifications of the nucleic acid (e.g., of a single-stranded or double-stranded nucleic acid, or of a nucleic acid comprising one or more single strands and one or more double-stranded segments). In some embodiments, the nucleic acid comprises a combination of one or more double-stranded segments and one or more chemically modified segments.

[0017] In some embodiments, the nucleic acid comprises a combination of one or more single-stranded segments and one or more double-stranded segments. The single-stranded segments may, in some embodiments, exist in the form of loops (e.g., in stem-loop secondary structures as described elsewhere herein). In some embodiments, the single-stranded segments exist in the form of unpaired regions in the double-stranded segments. For example, internal loops may form in double-stranded segments where one or more bases of one strand do not form base-pair interactions with one or more adjacent bases of the other strand. A further example of an unpaired region includes a bulge loop, which may form in a double-stranded segment where one strand contains one or more additional bases relative to the other strand. In some embodiments, the single-stranded and double-stranded regions provide structural rigidity.

[0018] In some embodiments, the single-stranded region (e.g., the unpaired region) is at least 2 bases long (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bases long). In some embodiments, the single-stranded region is 2-10 bases long (e.g., 2-8, 4-10, or 4-8 bases long). In some embodiments, the double-stranded region is 2-40 bases long (e.g., 2-20, 2-10, 10-40, 10-30, 10-20, 20-40, or 20-30 bases long).

[0019] In some embodiments, the nucleic acid comprises one or more stem-loop structures (e.g., 2, 3, 4, 5, 6, or more stem-loop structures). In some embodiments, at least one donor molecule of the FRET label is disposed within the loop of the stem-loop structure. In some embodiments, at least one acceptor molecule of the FRET label is disposed within the loop of the stem-loop structure. In some embodiments, the loop region of each stem-loop comprises at least 4 unpaired bases (e.g., 4, 5, 6, 7, 8, or more unpaired bases). In some embodiments, the loop region of each stem-loop comprises a sequence with a G / C content of less than 33%.

[0020] In some embodiments, at least one acceptor molecule of the FRET label is disposed 5' to one or more donor molecules of the FRET label. For example, in some embodiments, the at least one acceptor molecule is disposed at an attachment site that is one or more bases upstream (5') from the one or more donor molecules. In some embodiments, at least one acceptor molecule of the FRET label is disposed 3' to one or more donor molecules of the FRET label. For example, in some embodiments, the at least one acceptor molecule is disposed at an attachment site that is one or more bases downstream (3') from the one or more donor molecules.

[0021] In some embodiments, the nucleic acid comprises a first oligonucleotide strand attached to two or more luminescent light-emitting labels. In some embodiments, the two or more luminescent light-emitting labels are attached to two or more attachment sites on the first oligonucleotide strand. In some embodiments, the three or more luminescent light-emitting molecules of the FRET label are attached to three or more attachment sites on the first oligonucleotide strand. In some embodiments, each luminescent light-emitting label or luminescent light-emitting molecule comprises a steric volume having a central point at least 5 angstroms away from the central point of any other luminescent light-emitting label or luminescent light-emitting molecule. For example, in some embodiments, each luminescent label or molecule comprises a steric volume having a central point at least 6 angstroms, about 5-10 angstroms, about 6-10 angstroms, about 10-15 angstroms, about 15-20 angstroms, about 20-25 angstroms, or about 25-50 angstroms away from the central point of any other luminescent label or molecule.

[0022] In some embodiments, the nucleic acid further comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand. In some embodiments, the first oligonucleotide strand is attached to a nucleotide (e.g., a nucleoside polyphosphate). In some embodiments, the second oligonucleotide strand is attached to a nucleotide (e.g., a nucleoside polyphosphate). In some embodiments, at least one luminescent molecule of the FRET label is on a different strand than the other luminescent molecules. In some embodiments, the acceptor molecule is on a different strand than one or more donor molecules. In some embodiments, at least one donor molecule and at least one acceptor molecule are on the same strand of the nucleic acid.

[0023] In some embodiments, the two or more attachment sites are separated from one another on the first oligonucleotide strand by at least 5 bases (e.g., at least 5 nucleotides). In some embodiments, the two or more attachment sites are separated from one another on the first oligonucleotide strand by at least 5 and fewer than 40 bases (e.g., about 5-30 bases, about 5-20 bases, about 5-10 bases, about 10-40 bases, about 20-40 bases, or about 30-40 bases). In some embodiments, each attachment site is at least 2 bases away from a guanine or cytosine on the first oligonucleotide strand. In some embodiments, each attachment site occurs at an abasic site on the first oligonucleotide strand. In some embodiments, each attachment site occurs at a nucleobase of a nucleotide on the first oligonucleotide strand. In some embodiments, the nucleobase is selected from an A, T, or U nucleobase.

[0024] In some embodiments, the first oligonucleotide strand forms one or more (e.g., 1, 2, 3, 4, or more) stem loops. In some embodiments, the loop region of each stem loop comprises attachment sites for two or more attachment sites. In some embodiments, the loop region of each stem loop comprises at least four unpaired bases (e.g., 4, 5, 6, 7, 8, or more unpaired bases). In some embodiments, the loop region comprises a sequence (e.g., a nucleotide sequence) with a G / C content of less than 33%.

[0025] In some embodiments, the labeled nucleotide of the present disclosure comprises a nucleic acid linker comprising a first oligonucleotide strand attached to two or more branched oligonucleotide strands at the end of the first oligonucleotide strand. In some embodiments, the first oligonucleotide strand is attached to two or more branched oligonucleotide strands via a covalent coupling compound. In some embodiments, each branched oligonucleotide strand comprises at least one luminescent light-emitting label. In some embodiments, the first oligonucleotide strand hybridizes to a second oligonucleotide strand. In some embodiments, the second oligonucleotide strand is attached to a nucleotide (e.g., a nucleoside polyphosphate). In some embodiments, each branched oligonucleotide strand further hybridizes to a complementary branched oligonucleotide strand.

[0026] In some embodiments, the covalent coupling compound has the structure:

[0027] [ka] In the formula, N f is the first oligonucleotide strand; N b is a branched oligonucleotide chain; R f and R b are each independently a bond or linking group selected from the group consisting of substituted or unsubstituted alkylene; substituted or unsubstituted alkenylene; substituted or unsubstituted alkynylene; substituted or unsubstituted heteroalkylene; substituted or unsubstituted heteroalkenylene; substituted or unsubstituted heteroalkynylene; substituted or unsubstituted heterocyclylene; substituted or unsubstituted carbocyclylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; and combinations thereof; O in each instance is the oxygen atom of either the 5' phosphate or the 3' hydroxyl group of the adjacent oligonucleotide strand.

[0028] In some embodiments, the labeled nucleotide of the present disclosure comprises a nucleic acid linker comprising a first oligonucleotide component comprising three or more oligonucleotide strands (e.g., three, four, five, six, or more oligonucleotide strands) extending from a covalent coupling compound. In some embodiments, at least one of the three or more oligonucleotide strands is attached to a nucleotide (e.g., a nucleoside polyphosphate). In some embodiments, the first oligonucleotide component hybridizes to a second oligonucleotide component. In some embodiments, the second oligonucleotide component comprises at least one oligonucleotide strand attached to a luminescent light-emitting label.

[0029] In some embodiments, the labeled nucleotides of the present disclosure are luminescently labeled with a fluorescent dye. In some embodiments, the fluorescent dye is a rhodamine dye, a BODIPY dye, or a cyanine dye.

[0030] In some embodiments, the labeled nucleotides of the present disclosure include a nucleotide (e.g., a nucleoside polyphosphate) that is at least 1 nm away from any of the luminescent labels of the two or more luminescent labels. In some embodiments, the nucleotide is approximately 1-10 nm (e.g., approximately 2-10 nm, approximately 4-10 nm, approximately 6-10 nm, or approximately 8-10 nm) away from any of the luminescent labels of the two or more luminescent labels. In some embodiments, the nucleotide is approximately 2-20 nm (e.g., approximately 6-20 nm, approximately 10-20 nm, approximately 12-20 nm, or approximately 16-20 nm) away from any of the luminescent labels of the two or more luminescent labels.

[0031] In some embodiments, the present disclosure provides a method for determining the sequence of a template nucleic acid. In some embodiments, the method includes a step of exposing a complex to a plurality of types of luminescent labeled nucleotides provided by the present application in a target area, the complex including a template nucleic acid, a primer, and a polymerizing enzyme. In some embodiments, one or more of the plurality of types of luminescent labeled nucleotides (e.g., one, two, three, four, or more types of labeled nucleotides) include a nucleotide (e.g., a nucleoside polyphosphate) linked to a FRET label that includes at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule. The FRET label of each type of nucleotide is distinguishable among the plurality of labeled nucleotides (e.g., one type of FRET-labeled nucleotide is distinguishable from all other types of FRET-labeled nucleotides and / or labels that do not undergo FRET). In some embodiments, one or more of the multiple types of luminescently labeled nucleotides include a nucleotide (e.g., a nucleoside polyphosphate) linked to two or more luminescent labels via a linker. In some embodiments, the linker is an oligomer comprising at least 10 monomer units. In some embodiments, each luminescent label is attached to the linker at an attachment site that is at least 5 monomer units away from any other attachment site. For example, in some embodiments, an attachment site is about 5-30 monomer units, about 5-20 monomer units, about 5-10 monomer units, about 10-40 monomer units, about 20-40 monomer units, or about 30-40 monomer units away from any other attachment site. In some embodiments, each luminescent label is at least 5 angstroms away from any other luminescent label.For example, in some embodiments, each luminescent label is separated from any other luminescent label by approximately 5-10 angstroms, approximately 6-10 angstroms, approximately 10-15 angstroms, approximately 15-20 angstroms, approximately 20-25 angstroms, or approximately 25-50 angstroms. Thus, in some aspects, the present disclosure provides methods of nucleic acid sequencing utilizing any of the luminescently labeled nucleotides described herein.

[0032] In some embodiments, the method further comprises directing a series of one or more pulses of excitation energy near the target zone. In some embodiments, the method further comprises detecting a plurality of photons emitted from the luminescently labeled nucleotides during their successive incorporation into the nucleic acid with the primer. In some embodiments, the method further comprises identifying the sequence of the incorporated nucleotides by determining the timing, and optionally the luminescent emission intensity and / or brightness of the emitted photons. In some embodiments, the method further comprises identifying the sequence of the incorporated nucleotides by determining at least one of the luminescent emission intensity and luminescent emission lifetime based on the emitted photons.

[0033] In some embodiments, the four different types of nucleotides (e.g., adenine, guanine, cytosine, thymine / uracil) in the reaction mixture can each be labeled with one or more luminescent molecules (e.g., with two or more luminescent labels as described herein). In some embodiments, each type of nucleotide can be linked to more than one of the same luminescent molecules (e.g., two or more of the same fluorescent dyes linked to the nucleotides). In some embodiments, each luminescent molecule can be linked to more than one nucleotide (e.g., two or more of the same nucleotides). In some embodiments, more than one nucleotide can be linked to more than one luminescent molecule (e.g., via a linker as described herein).

[0034] In some embodiments, the luminescent label in the set of four nucleotides may be selected from dyes including aromatic or heteroaromatic compounds, and may be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluoroscein, rhodamine, or other similar compounds. Examples of dyes include xanthene dyes, such as fluorescein or rhodamine dyes, naphthalene dyes, coumarin dyes, acridine dyes, cyanine dyes, benzoxazole dyes, stilbene dyes, pyrene dyes, phthalocyanine dyes, phycobiliprotein dyes, squaraine dyes, BODIPY dyes, and the like.

[0035] In some embodiments, the present application provides a kit for sequencing a template nucleic acid. In some embodiments, the kit comprises multiple types of luminescently labeled nucleotides as described herein. In some embodiments, each type of labeled nucleotide comprises two or more luminescent labels attached to one or more nucleotides (e.g., one or more nucleoside polyphosphates) via a linker. In some embodiments, the kit further comprises a polymerizing enzyme. In some embodiments, the kit further comprises a primer complementary to the template nucleic acid to be sequenced.

[0036] In some embodiments, the present application provides a nucleic acid sequencing reaction composition. In some embodiments, the composition comprises two or more (e.g., 2, 3, 4, or more) different types of luminescently labeled nucleotides in a reaction mixture. In some embodiments, each type of luminescently labeled nucleotide comprises a labeled nucleotide according to the present application. In some embodiments, the composition further comprises a polymerizing enzyme. In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the composition further comprises a primer complementary to the template nucleic acid.

[0037] In some embodiments, the present application provides a labeled nucleotide according to any one of the structures shown in Figure 11A. In some embodiments, the present application provides a labeled nucleotide according to any one of the structures shown in Figure 11E. In some embodiments, the present application provides a labeled nucleotide according to any one of the structures shown in Figure 12. In some embodiments, the present application provides a labeled nucleotide according to any one of the structures shown in Figures 13A-13C.

[0038] Those skilled in the art will understand that the figures described herein are for illustrative purposes only. It should be understood that in some cases, various aspects of the invention may be shown exaggerated or enlarged to facilitate understanding of the invention. In the drawings, like reference characters generally represent like features, functionally similar and / or structurally similar elements throughout the various views. The drawings are not necessarily to scale, with emphasis instead being placed on illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way.

[0039] The features and advantages of the present invention will become more apparent from the following detailed description when read in conjunction with the drawings. When describing the embodiments with reference to the drawings, directional references (such as "up", "down", "top", "bottom", "left", "right", "horizontal", "vertical", etc.) may be used. Such references are intended merely to aid the reader in viewing the drawings in a normal orientation. These directional references are not intended to describe a preferred or only orientation of an embodied device. Devices may be embodied in other orientations.

[0040] As will be apparent from the detailed description, the examples shown in the drawings (e.g., FIGS. 1-10) and further described for illustrative purposes throughout this application describe non-limiting embodiments, and in some cases may simplify certain processes or omit features or steps for purposes of more clear illustration. [Brief description of the drawings]

[0041] [Figure 1A] Diagram generally showing luminescence label separation of brightly labeled reactants. [Figure 1B] FIG. 1 shows a generic linker structure with different luminescent label attachment configurations. [Figure 2A] Diagram generally showing a nucleic acid linker attached to two luminescent labels. [Figure 2B]Diagram showing universal nucleic acids with different strand configurations (top) and a diagram generally showing the spatial occupancy of luminescent labels (bottom). [Figure 2C] Diagram showing universal nucleic acids with different relative label attachment sites (top) and different reactant linkages (bottom). [Figure 3A] Diagram showing universal nucleic acids that link labels to reactants via identical (left) or opposite (right) oligonucleotide strand linkages. [Figure 3B] FIG. 1 shows universal nucleic acids with examples of approximate size constraints that can be used in designing brightly labeled reactants. [Figure 3C] FIG. 1 is a diagram of an exemplary structure of a nucleic acid linking two luminescent labels to a nucleoside polyphosphate via a covalent linkage. [Figure 3D] FIG. 3C shows an example of a sequencing reaction that confirmed that rod-shaped nucleic acid linkers (e.g., as shown in FIG. 3C) can be used to detect incorporation of labeled nucleoside polyphosphates. [Figure 3E] FIG. 1 shows an exemplary structure of a nucleic acid linking two luminescent labels to a nucleoside polyphosphate via opposite strand linkages. [Figure 3F] FIG. 1 shows exemplary structures of nucleic acid linkers having luminescent labels incorporated into oligonucleotide strands. [Figure 3G] FIG. 1 shows an example of a sequencing reaction performed using the four different nucleic acid linker constructs shown. [Figure 4A] FIG. 1 shows a universal nucleic acid linker having a single stem-loop secondary structure. [Figure 4B] FIG. 1 shows a universal nucleic acid linker having multiple stem-loop secondary structures. [Figure 4C] 1 is an exemplary structure of a nucleic acid linker having a luminescent light-emitting label attached to the loop of a distinct stem-loop secondary structure. [Figure 4D]FIG. 4C shows an example of a sequencing reaction that confirmed that incorporation of nucleoside polyphosphates can be detected using labeled nucleoside polyphosphates having stem-loop nucleic acid linkers (e.g., as shown in FIG. 4C). [Figure 5A] FIG. 1 shows generally a dendritic nucleic acid linker having branched oligonucleotide strands attached to luminescent light-emitting labels. [Figure 5B] FIG. 1 shows an example of a dendritic nucleic acid linker having luminescent labels attached to the ends of separate branched oligonucleotide strands. [Figure 6A] FIG. 1 illustrates generally a star-shaped nucleic acid linker and provides an example of approximate size constraints that may be used in the design of a star-shaped nucleic acid linker. [Figure 6B] FIG. 1 is a diagram of an example of a star-shaped nucleic acid linker structure. [Figure 6C] FIG. 1 is a diagram of an example of a nucleic acid linker structure with a tetrahedral core. [Figure 6D] FIG. 1 shows an example of a reaction scheme that can be used to synthesize a nucleic acid linker having a tetrahedral core. [Figure 6E] FIG. 1 shows generally a tetrahedron-based nucleic acid linker structure with a luminescent label attached at a three-way junction. [Figure 6F] FIG. 1 illustrates generally a star-shaped nucleic acid linker and provides an example of approximate size constraints that may be used in the design of a star-shaped nucleic acid linker. [Figure 7] FIG. 1 shows generally a process for producing a cyclodextrin-based nucleic acid linker having a luminescent label attached at a three-way junction. [Figure 8] FIG. 1 shows examples of nucleic acid linker structures used in a set of experiments to evaluate the effect of spacer length on fluorescence lifetime measurements. [Figure 9] FIG. 1 shows examples of nucleic acid linker structures used in a set of experiments to evaluate the effect of linker constraints on fluorescence lifetime measurements. [Figure 10]FIG. 1 shows examples of nucleic acid linker structures used in a set of experiments to evaluate the effect of spacer length on the fluorescence lifetime of limited linkers. [Figure 11A] FIG. 1 shows an example of a FRET nucleic acid linker structure with a conventional luminescent label (top panel) versus a large Stokes shift luminescent label (bottom panel). As shown, the large Stokes shift is accompanied by a reduction in brightness loss due to the spectral filter used for laser exclusion. Donor molecules are shown as light gray suns and acceptor molecules are shown as black suns. Nucleotides (e.g., nucleoside polyphosphates) are shown as gray circles. [Figure 11B] 1 shows an exemplary sequencing reaction performed using a FRET nucleic acid linker structure. Donor molecules are shown as light gray suns and acceptor molecules are shown as black suns. Nucleotides (e.g., nucleoside polyphosphates) are shown as gray circles. [Figure 11C] FIG. 1 shows an example of luminescence lifetimes and luminescence intensity clusters for FRET nucleic acid linker structures with conventional luminescence labels (top) versus large Stokes shift luminescence labels (bottom). [Figure 11D] FIG. 1 shows an example of the different effects of spectral filters on FRET nucleic acid linker structures. [Figure 11E] Diagram showing different configurations of FRET nucleic acid linker structures. Donor molecules are shown as light grey suns and acceptor molecules are shown as black suns. Nucleotides (e.g., nucleoside polyphosphates) are shown as grey circles. [Figure 11F] 1 shows an exemplary sequencing reaction performed using a FRET nucleic acid linker structure with multiple donor luminescence emission labels. The donor molecule is shown as a light gray sun and the acceptor molecule is shown as a black sun. Nucleotides (e.g., nucleoside polyphosphates) are shown as gray circles. [Figure 12]FIG. 1 shows an example of a FRET nucleic acid linker structure with a linear nucleic acid with an acceptor molecule in the center (A) and two donor molecules on either side (D). [Figure 13A] FIG. 1 shows a FRET nucleic acid linker structure having spacers between the donor molecule (D) and acceptor molecule (A) and around the labels that contain different numbers of AT pairs to separate portions of the nucleic acid and different numbers of GC pairs to stabilize portions of the nucleic acid. [Figure 13B] FIG. 1 shows a FRET nucleic acid linker structure having spacers between the donor molecule (D) and acceptor molecule (A) and around the labels that contain different numbers of AT pairs to separate portions of the nucleic acid and different numbers of GC pairs to stabilize portions of the nucleic acid. [Figure 13C] FIG. 1 shows a FRET nucleic acid linker structure having spacers between the donor molecule (D) and acceptor molecule (A) and around the labels that contain different numbers of AT pairs to separate portions of the nucleic acid and different numbers of GC pairs to stabilize portions of the nucleic acid. [Figure 14] Diagram showing the results of a study to determine the optimal distance between the ATRho6GC3-BODIPY576 pair, with ATRho6GC3 as the donor (light gray circle) and BODIPY576 as the acceptor (dark gray circle). [Figure 15] FIG. 10 shows a comparison of the distance between the luminescent molecule and the nucleotide in a linear DNA nucleic acid configuration and a three-way DNA junction configuration. [Figure 16] FIG. 13 shows the effect of donor:acceptor molecule ratio, with Cy3 as the donor and Cy5 as the acceptor, on FRET efficiency. [Figure 17] FIG. 1 shows an example of the effect of different FRET nucleic acid linker structures on FRET efficiency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Among other aspects, the disclosure provides luminescent labeled reactions that include two or more labels, the labels being configured to provide high intensity and / or consistent luminescence properties (e.g., consistent luminescence lifetime). In some embodiments, the two or more labels are configured to avoid interactions between the labels that may reduce the intensity of the luminescence or other luminescence properties. In some embodiments, the two or more labels are configured a) to avoid interactions with the linker, and / or b) to each have a similar interaction with the linker.

[0043] In some embodiments, the present disclosure provides methods and compositions related to luminescent labeled reactants with high luminescence intensity. In some embodiments, the present disclosure provides methods and compositions related to luminescent labeled reactants with high luminescence brightness. In some embodiments, the present disclosure relates to brightly labeled reactants with consistent luminescence lifetime. As used herein, in some embodiments, "brightness" (and variations thereof, e.g., "bright," "bright," etc.) refers to a parameter that reports the average luminescence intensity per molecule of a labeled reactant. Thus, in some embodiments, "luminescence intensity" may be used generally to describe the brightness of a composition that includes a brightly labeled reactant. In some embodiments, the brightness of a labeled reactant is equal to the product of its quantum yield and extinction coefficient. In some embodiments, the labeled reactants of the present disclosure are designed to maximize quantum yield values ​​and / or minimize extinction coefficient values ​​to promote increased brightness.

[0044] The brightly labeled reactants of the present disclosure are designed in some embodiments to have increased luminescence brightness and consistent luminescence lifetime. In some embodiments, two-labeled reactants may interact with each other and / or the surrounding environment, resulting in one or more luminescence properties being inconsistent between the two labels. In some embodiments, where single molecule detection methods rely on these features to identify specific types of molecules, inconsistent luminescence properties may be problematic. For example, inconsistent luminescence lifetimes in some embodiments may result in lifetime readouts reporting two separate clusters of information, as opposed to a single grouping of information that may be observed with consistent luminescence lifetimes.

[0045] In some embodiments, a consistent luminescence lifetime may involve preservation of luminescence lifetime, e.g., having a luminescence lifetime that is approximately the same as compared to a labeled reaction with one less label. In some embodiments, the luminescence lifetime of a label in a multi-labeled reaction is unchanged as compared to the same label in a single-labeled reaction. As described herein, increasing the number of luminescence-emitting labels on a single construct to increase brightness may result in a reduced luminescence lifetime in some embodiments. In some embodiments, the present disclosure provides compositions designed with specific structural constraints that separate adjacent labels by a certain minimum distance that increases brightness without affecting luminescence lifetime. In some embodiments, the luminescence lifetime of a multi-labeled construct is compared to the luminescence lifetime of a construct with at least one less luminescence-emitting label (e.g., at least one less fluorophore dye of the same type). In some embodiments, the luminescence lifetime changes by approximately 30% or less (e.g., by less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or approximately 0% increase or decrease).

[0046] In some embodiments, the present disclosure relates to the recognition and understanding that detectable compositions in sequencing reactions can be developed to eliminate the need for certain instrumental components, thereby allowing technology to move to more compact systems. For example, sequencing instruments generally require optical filters to filter the excitation light so as not to cause undesired detection events in sensors. The optical filters used to transmit the desired luminescence and sufficiently block the excitation light are thick, bulky, expensive, and cannot withstand changes in the incidence angle of light, which can hinder miniaturization. However, the inventors have recognized and understood that the use of brightly labeled reactants with preserved lifespans can reduce the need for such filtering, or in some cases, completely eliminate the need for such filters. The bright reactants described herein allow for the use of less optical power (e.g., excitation energy), resulting in less scattering and the need for filtering.

[0047] Aspects of the present application provide brightly labeled reactants configured according to the non-limiting diagram shown in FIG. 1A, which shows two luminescent labels linked to a reactant 180 via a linker 100. As shown, each luminescent label is attached to the linker 100 via a spacer. In some embodiments, the spacer forms a covalent bridge between the label and the linker. Thus, in some embodiments, the spacer is neither a luminescent molecule nor a spacer moiety of the linker composition (e.g., the spacer does not contain a monomer unit of a polymeric or oligomeric linker). A first end of the spacer is attached to the linker attachment site 102, and a second end of the spacer is attached to the label attachment site 122. In some embodiments, the linker attachment site 102 can be approximated by the location of a covalent bond junction that connects an atom of the spacer to an atom of the linker 100. In some embodiments, the linker attachment site 102 occurs at an atom within a continuous chain of the linker 100. In some embodiments, the label attachment site 122 can be approximated by the location of a covalent bond junction that connects an atom of the spacer to an atom of the label. In some embodiments, the label attachment site 122 occurs at an atom of the label that is covalently bonded to an atom of the spacer. In some embodiments, the label attachment site 122 occurs at an atom of the spacer that is covalently bonded to an atom of the label.

[0048] As described elsewhere herein, in some embodiments, the linker constructs of the present application are arranged such that adjacent luminescent labels are separated by a minimum distance d A In some embodiments, the luminescent label comprises two or more luminescent labels having attachment sites spaced apart by a minimum distance d. L As shown in FIG. 1A, in some embodiments, Lis the distance between the label attachment sites. In some embodiments, the label-label separation may be further determined by the size of each label molecule. Thus, in some embodiments, the luminescent label is described by an approximated or calculated steric volume 112 of an ellipsoid or spheroid, and a measurement of the steric radius r1 can be obtained. In some embodiments, the luminescent label is described by an approximated or calculated steric circumference of an ellipsoid or circle, and a measurement of the steric radius r1 can be obtained. In some embodiments, the steric radius (e.g., r1, r2) can be calculated or estimated as half the longest dimension of the luminescent label. For example, in some embodiments, the chemical structure of the luminescent label is evaluated as a two-dimensional or three-dimensional structure (e.g., based on a thermodynamically favored molecular conformation) using software or suitable methods known in the art, and the steric radius (e.g., r1, r2) is determined by calculating half the longest dimension of the structure in two-dimensional or three-dimensional space. In some embodiments, the labels are spaced apart by a minimum distance d for the total radius (r1+r2) of the labels such that the labels do not overlap. L Just leave.

[0049] In some embodiments, the linker architecture and / or spacer rigidity is determined by the distance between attachment sites d A d L In some embodiments, the linker configuration and / or spacer rigidity can be such that the distance between attachment sites d A is the minimum distance d L In some embodiments, the linker configuration and / or spacer stiffness may be such that the distance between attachment sites d A is the minimum distance d L It may be larger (eg, like construct 154).

[0050] It should be understood that in some embodiments, the concepts described herein may be implemented using any suitable molecular scaffold as the linker 100. In some embodiments, the linker is an organic compound. Examples of organic compounds suitable for use as the linker 100 include, without limitation, polyphenyls, polyalkynes, alpha-helix mimetics, and peptidomimetics.

[0051] In some embodiments, the linker 100 is an oligomer, such as, for example, an oligomeric linker comprising monomeric units. In some embodiments, the oligomer comprises one or more types of monomeric units. The types of monomeric units may include, by way of example and without limitation, nucleotides (e.g., ribonucleotides, deoxyribonucleotides, and their analogs and derivatives), amino acids (e.g., natural and unnatural amino acids), monosaccharides, and organic compounds such as phenyl- and alkynyl-containing compounds. In some embodiments, the oligomer comprises one or more of the same type of monomeric units. In some embodiments, an oligomer comprising one type of monomeric unit may be referred to as a polymer (e.g., a polymeric linker). In some embodiments, the oligomer comprises two or more different types of monomeric units (e.g., a mixture of monomeric units).

[0052] In some embodiments, the oligomer (e.g., oligomeric or polymeric linker) contains at least 5 monomer units. In some embodiments, the oligomer contains at least 10 monomer units. In some embodiments, the oligomer contains at least 10 and fewer than 200 monomer units (e.g., at least 10 and fewer than 150 monomer units, at least 10 and fewer than 100 monomer units, at least 10 and fewer than 50 monomer units, at least 10 and fewer than 40 monomer units, at least 10 and fewer than 30 monomer units, or at least 10 and fewer than 20 monomer units).

[0053] In some embodiments, the linker (e.g., polymeric linker, oligomeric linker) is a polysaccharide. Examples of polysaccharides suitable for use as linker 100 are known in the art (e.g., as described in Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries, Wiley 2001).

[0054] In some embodiments, the linker (e.g., polymer linker, oligomeric linker) is a peptide. Non-limiting examples of peptides suitable for use as the linker 100 include, without limitation, oligopeptides, cyclic peptides, and small proteins (e.g., the avian pancreatic peptide-based miniature proteins described in Hodges, AM and Schepartz, A. (2007), J. Am. Chem. Soc. 129:11024-11025, etc.). Methods for designing geometric constraints into peptide structures are well known in the art and are envisioned to be particularly useful, for example, to provide rigidity and to facilitate label separation. For example, the proline content of a peptide amino acid sequence can be altered to control peptide shape (see, for example, Kritzer, JA et al. (2006), ChemBioChem 7:29-31). Additional non-limiting examples of peptide design techniques that are useful include peptide cyclization (see, e.g., Maltsev, OV et al. (2016), Angewandte Chemie 55(4):1535-1539), stapling and / or constraining α-helical peptides with H-bond surrogates (see, e.g., Douse, CH et al. (2014), ACS Chem. Biol. 9:2204-2209), and peptide constraining with cyclic β-sheet and β-hairpin mimetics (see, e.g., Gibbs, AC et al. (1998), Nat. Struc. Biol. 5:284-288).

[0055] In some embodiments, the linker (e.g., polymer linker, oligomeric linker) is a nucleic acid. In some embodiments, the brightly labeled reactant may be designed according to the diagram shown in FIG. 2A, which generally shows two luminescent labels attached to a core nucleic acid construct. As shown, in some embodiments, at least one luminescent label 210 is attached to a nucleic acid linker 200 at an attachment site 202 via a spacer 220. In some embodiments, the nucleic acid linker 200 comprises at least two hybridized oligonucleotide strands. In some embodiments, the nucleic acid linker 200 comprises at least two luminescent labels, each luminescent label being separated from its neighbor by a minimum distance d. L In some embodiments, at least two luminescent labels are attached to the same oligonucleotide strand, with each attachment site being separated from its neighbor by a minimum distance d A In some embodiments, the luminescent labels are attached to the oligonucleotide strand via spacers, each spacer separating a given luminescent label from its attachment site by a minimum distance d AL Just leave it alone.

[0056] In some embodiments, the minimum distance d L , minimum distance d A , and the minimum distance d AL can be obtained, for example, using theoretical methods (e.g., computationally or otherwise) known in the art. In some embodiments, theoretical methods can include any approach that incorporates molecular structure such as bond lengths, bond angles and rotations, electrostatics, nucleic acid helical structure, and other physical factors that can describe molecules in solution. In some embodiments, distance measurements can be obtained experimentally, for example, by crystallographic or spectroscopic means.

[0057] Aspects of the present disclosure are based, at least in part, on the fact that brightly labeled reactants (e.g., labeled nucleotides) having nucleic acid linkers

[0058]

number

[0059] In yet other embodiments, the labeled nucleotides of the present disclosure can be designed according to Equation 2.

[0060]

number

[0061]

number

[0062] In some embodiments, the minimum distance d AL is measured from approximately the central atom of the luminescent label 210 to the atom on the oligonucleotide chain to which the spacer 220 is attached. In some embodiments, the minimum distance d AL is measured from the center of the luminescent label 210 (e.g., estimated based on the center of mass of the luminescent molecule, or other methods known in the art or described elsewhere herein) to the atom of the oligonucleotide chain to which the spacer 220 is attached. In some embodiments, the minimum distance d AL The minimum distance d is measured as the length of the spacer 220 (e.g., measured from the atom of the spacer 220 attached to the luminescent label 210 to the atom of the spacer 220 attached to the nucleic acid 200). In some embodiments, the minimum distance d A is measured as the distance between atoms on the oligonucleotide backbone to which the spacer is covalently attached (e.g., between the carbon atoms of the abasic attachment sites). In some embodiments, the minimum distance d A is measured as the distance between labeled bases on an oligonucleotide strand (e.g., between the atoms on the nucleobases of the base attachment sites).

[0063] In some embodiments, the minimum distance d L is measured as the distance between the central atoms of adjacent luminescent labels. In some embodiments, adjacent luminescent labels are spaced apart by a distance d of approximately 6 Angstroms. L In some embodiments, the distance d L is at least 6 angstroms (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 angstroms). In some embodiments, the minimum distance d L is measured as an approximation and may or may not take into account spacer configuration, spacer flexibility, or nucleic acid flexibility.

[0064] Among other aspects, the present disclosure provides a method for determining the distance d between luminescent labels, such that a multi-labeled reaction can be designed for maximum brightness without compromising luminescence lifetime. L The present invention provides a general strategy for developing a luminescent label that is sufficiently close to the luminescent label to interact with one another, leading to reduced and / or inconsistent luminescent lifetimes. In some embodiments, adjacent luminescent labels that are sufficiently close to one another may interact with one another, leading to quenching effects, leading to reduced and / or inconsistent luminescent lifetimes. Thus, the general design strategy provided herein includes, in some embodiments, structural constraints that limit the degree of interaction between adjacent luminescent labels.

[0065] In some embodiments, the luminescent label may interact with the guanine nucleobase of the nucleic acid linker through radioactive and / or non-radioactive decay, resulting in a reduced luminescent emission lifetime and / or inconsistent luminescent emission lifetime. In some embodiments, the luminescent label attachment site is developed by minimizing the G / C content in the region surrounding the attachment site. Thus, in some embodiments, the attachment site is located within the A / T-rich region of the oligonucleotide chain. In some embodiments, each attachment site is at least two nucleotides away from a G or C nucleotide on the oligonucleotide chain (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 nucleotides away from a G or C nucleotide). Thus, in some embodiments, each attachment site is adjacent to at least two consecutive nucleotides selected from A or T.

[0066] In certain embodiments, the distance between attachment sites on an oligonucleotide strand can be described by the number of intervening unlabeled bases (e.g., intervening nucleotides). In some embodiments, attachments on an oligonucleotide strand are separated by at least 5 unlabeled bases (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 unlabeled bases). In some embodiments, attachment sites are separated by 6, 7, 8, or 9 unlabeled bases. In some embodiments, attachment sites are separated by 5-100 unlabeled bases on an oligonucleotide strand (e.g., 5-80, 5-60, 5-40, 5-20, or 5-10 unlabeled bases).

[0067] In some embodiments, the design principles described herein allow for the addition of successive luminescent labels to labeled reaction components to increase the brightness and / or luminescent emission intensity. In some embodiments, the technology of the present application provides a method for the preparation of a compound of formula L n (x) providing a multi-labeled reaction component having brightness and / or luminescence emission intensity according to formula L n (x) Medium, L n where x is equal to the total number of luminescent emission labels on the labeled reactants, and x is equal to the measured brightness or fluorescence intensity of the corresponding singly labeled reactant. Thus, in some embodiments, a reaction component labeled with two dyes has twice the brightness and / or luminescent emission intensity compared to its one-dye labeled analog. In some embodiments, a reaction component labeled with three or four dyes has three or four times the brightness and / or luminescent emission intensity, respectively, compared to its one-dye labeled analog. In some embodiments, the brightly labeled reactions described herein have a L n exhibits a brightness and / or luminescence emission intensity that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of that predicted by (x).

[0068] In some embodiments, the nucleic acid linker provided herein comprises an oligonucleotide strand having at least two luminescent light-emitting labels. FIG. 2B generally illustrates a single-stranded nucleic acid linker 250 attached to two luminescent light-emitting labels. As shown, the single-stranded linker may have a relatively high degree of flexibility that may facilitate interactions between the labels. In some embodiments, brightly labeled reactions with consistent and / or preserved lifetimes can be generated using single-stranded linker constructs by utilizing certain labels, such as cyanine-based dyes, that do not interact to produce quenching effects. However, for some classes of dyes, the luminescent lifetime may be reduced and / or inconsistent due to the promotion of label-label proximity by the flexibility of the linker. Thus, in some embodiments, the labeled oligonucleotide strand is hybridized with one or more oligonucleotide strands to reduce the overall flexibility of the nucleic acid linker.

[0069] In some embodiments, strand hybridization is achieved by a suitable distance d between the luminescent labels on the nucleic acid linker. L In some embodiments, strand hybridization is used to increase the rigidity of a particular region of a nucleic acid (e.g., within the labeled region and / or within the region separating the labeled region from the reactants). Figure 2B generally shows a double-stranded nucleic acid linker 252 that includes hybridized oligonucleotide strands to rigidify the labeled region, thereby reducing strand flexibility that might otherwise facilitate label-label interactions.

[0070] As shown by nucleic acid linker 252, in some embodiments, movement of luminescent labels around the attachment points can promote label-label proximity. Also, as shown, in some embodiments, movement of labels around the attachment sites can bring the labels into closer proximity within the oligonucleotide strands of the nucleic acid linker. In some embodiments, the oligonucleotide strands can interact with the luminescent labels to produce inconsistent and / or reduced luminescence lifetime measurements. Thus, in some embodiments, the nucleic acid linkers described herein include spacers with a certain length, a certain rigidity, a certain site of attachment, and / or a certain configuration that limits the extent of label-label and / or linker-label interactions.

[0071] In some embodiments, the separation distance between luminescent light-emitting labels can be defined as the volume of space in which the labels reside relative to one another. For example, FIG. 2B shows a distance d L 2B shows a diagram 254 illustrating an example of how the radius r of the luminescent light emitting label may be measured. In some embodiments, each luminescent light emitting label may be defined by a cubic volume 212. In some embodiments, the cubic volume 212 is approximated as a sphere of radius 212-r or otherwise as an ellipse. In some embodiments, each luminescent light emitting label includes a cubic volume 212 having a center point that is at least 5 Angstroms away from any other label (e.g., the center points of adjacent labels are at least 5 Angstroms apart). The center point of the luminescent light emitting label may be any suitable center of the label, including, for example, the center of gravity or geometric center of the label. In some embodiments, the center point of the luminescent light emitting label may be determined by calculating or estimating the radius 212-r as shown in FIG. 2B. In some embodiments, the center point of the luminescent light emitting label may be calculated or approximated as half the longest dimension of the luminescent light emitting label (e.g., as described elsewhere herein with respect to r1 and r2 in FIG. 1A).

[0072] In some embodiments, the distance between adjacent luminescent labels (d L ) can be measured as the distance between the centers of mass of adjacent luminescent labels. In some embodiments, the center of mass represents the average position of all atoms in the luminescent label, weighted according to their mass. Methods for calculating the center of mass are known in the art (see, for example, Leach, AR, Molecular Modelling: Principles and Applications (2nd ed.), Prentice-Hall 2001; Guenza, M. (2002), Macromolecules 35(7):2714-2722).

[0073] In some embodiments, the distance between adjacent luminescent labels (d L ) can be measured as the distance between the geometric centers of adjacent luminescent labels. The geometric center of a molecule, in some embodiments, represents the average position of all atoms of that molecule (e.g., all atoms in a luminescent label), and the atoms are unweighted. Thus, in some embodiments, the geometric center of a molecule represents a point in space that is the average of the coordinates of all atoms in the molecule.

[0074] In some embodiments, the cubic volume 212 is more accurately calculated using any suitable method known in the art. For example, the molar refractive index coefficient of a property including polarizability can be calculated according to the following equation:

[0075]

number

[0076] In some embodiments, the movement of the luminescent labels around the attachment site is controlled by a separation distance d L 2B, the range of motion of the labels around each attachment site may be defined in some embodiments based on the spacer length and the steric volume of the labels. This theoretical range of motion is shown in dashed lines. It should be understood that in some embodiments, the various compositions and design strategies described herein may advantageously limit the extent to which the range of motion approaches the overlap region shown. For example, in some embodiments, the spacer stiffness, spacer length, and attachment site separation can each be manipulated in accordance with the present disclosure to limit the extent to which the labels may approach the overlap region. It should also be understood that diagram 254 is intended to be illustrative, since, for example, the labels do not necessarily need to be in physical contact to cause radioactive and / or non-radioactive decay.

[0077] In some embodiments, the range of motion of the labels shown in diagram 254 may be generally represented as a spatial volume, e.g., a spatial region having regions of different probability that a label may be at a point in space at a given time. In some embodiments, each label occupies a spatial volume that does not substantially overlap with the spatial volume of any other luminescent label. In some embodiments, each label occupies a spatial volume that is substantially free of any other label.

[0078] In some embodiments, the relative attachment sites of the luminescent labels can be designed taking into account the helical structure of the double-stranded linker. Figure 2C shows an example of a nucleic acid 260 with two labels separated by a distance, x. Nucleic acid 262 (drawn to scale) is attached at the attachment sites to two luminescent labels separated by roughly half a base of the nucleic acid 260, but the location of the relative attachment sites along the helix results in a label separation distance in space of approximately 2x. As shown, in some embodiments, the nucleic acid linker can further limit the extent of radioactive and / or non-radioactive decay by acting as a steric barrier between the labels.

[0079] In some embodiments, the term "steric barrier" refers to a linker or linker portion (e.g., a nucleic acid linker or portion thereof) that is located between a luminescent label attached to the linker and another attachment of the linker. Without wishing to be bound by theory, it is believed that the steric barrier absorbs, deflects, or otherwise blocks radioactive and / or non-radioactive decay emitted by the luminescent label. In some embodiments, the steric barrier prevents or limits the extent to which one or more luminescent labels interact with one or more other luminescent labels. In some embodiments, the steric barrier prevents or limits the extent to which one or more luminescent labels interact with one or more reactants. In some embodiments, the steric barrier prevents or limits the extent to which one or more luminescent labels interact with one or more molecules associated with a reactant (e.g., a polymerase bound to a reactant). Thus, in some embodiments, the term steric barrier may generally refer to a protective or shielding effect provided by a portion of a nucleic acid linker.

[0080] In some embodiments, one or more structural motifs can function as steric barriers. For example, in some embodiments, the helix formed by the double-stranded nucleic acid linker functions as a steric barrier. In some embodiments, a stem-loop or a portion thereof (e.g., stem, loop) functions as a steric barrier. In some embodiments, a three-way junction (e.g., as in a nucleic acid having two or more stem-loops) functions as a steric barrier. In some embodiments, a hybridized strand that does not have an attachment (e.g., a support strand) functions as a steric barrier. In some embodiments, a spacer functions as a steric barrier.

[0081] In some embodiments, the brightly labeled reactants described herein provide separation between luminescent labels and reactants. In some embodiments, the nucleic acid 264 comprises a nucleoside polyphosphate 280 reactant that serves as a substrate for a polymerase 290 in a synthesis reaction. In some embodiments, a label in close proximity to the polymerase active site may cause polymerase photodamage (e.g., via non-radioactive decay or otherwise), which may be detrimental to polymerase activity. As shown, the nucleic acid 266 is attached to the reactant, and at least a portion of the linker is in an area that is between the label and the reactant. Thus, in some embodiments, the nucleic acid linker can function as a steric barrier to further protect the polymerase from photodamage caused by the label. This effect of polymerase protection may occur in some embodiments through label-reactant spatial separation and / or the presence of a steric barrier between the label and the reactant. Polymerase protection is described in more detail in co-pending U.S. patent application Ser. No. 15 / 600,979, the contents of which are incorporated herein by reference in their entirety.

[0082] In some embodiments, the size and configuration of the nucleic acid linker (e.g., one or more oligonucleotide strands and / or one or more spacers of the nucleic acid linker) determine the distance between the luminescent label and the nucleoside polyphosphate. In some embodiments, the distance is about 1 nm or 2 nm to about 20 nm. For example, more than 2 nm, more than 5 nm, 5-10 nm, more than 10 nm, 10-15 nm, more than 15 nm, 15-20 nm, more than 20 nm. However, the distance between the luminescent label and the nucleoside polyphosphate cannot be too long, since some detection techniques require the luminescent label to be within a defined irradiation range in which it is excited (e.g., when the nucleoside polyphosphate is held within the active site of a polymerase). Thus, in some embodiments, the total distance is less than 30 nm, less than 25 nm, approximately 20 nm, or less than 20 nm.

[0083] In some embodiments, other features of the compositions described herein can be implemented to facilitate label-reactant separation, for example, spacer length, spacer rigidity, location of attachment site, etc., to minimize the possibility of photodamage caused to the labels. In some embodiments, the reactant linkages to the nucleic acid linkers described herein can be modified in conjunction with the luminescent label to facilitate label-reactant separation. FIG. 3A generally illustrates a nucleic acid linker with same-strand or opposite-strand label-reactant linkages. In some embodiments, the nucleic acid 300 includes two or more luminescent labels and reactants attached to the same oligonucleotide strand. In some embodiments, the same-strand linkages result in a covalent linkage between the label and the reactant.

[0084] In some embodiments, the nucleic acid 302 comprises two or more luminescent light-emitting labels and reactants attached to different oligonucleotide strands. In some embodiments, reverse strand linkage results in a non-covalent bond between the labels and reactants. In some embodiments, reverse strand linkage of the labels and reactants occurs by hybridization of the label-attached oligonucleotide strand and the reactant-attached oligonucleotide strand. As shown, in some embodiments, the nucleic acid linkers of the present disclosure provide a label-reactant separation distance d LR In some embodiments, d LR is the distance between the reactant and the nearest luminescent label. In some embodiments, d LR is measured from the label attachment site to the reactant attachment site. In some embodiments, d LR is measured from the luminescent molecule of the label to the reactant. In some embodiments, d LR is at least 1 nm. In some embodiments, d LR is about 1 nm to about 10 nm (e.g., about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, or greater than 10 nm). LR is about 2 nm to about 30 nm (for example, about 2 to 25 nm, about 2 to 20 nm, about 2 to 15 nm, about 2 to 10 nm, about 2 to 5 nm, about 5 to 10 nm, about 10 to 20 nm, about 5 to 30 nm, about 15 to 30 nm, or about 20 to 30 nm).

[0085] 3B shows several examples of non-limiting distance specifications used in the design of brightly labeled reactants. In some embodiments, a nucleic acid linker 304 is attached to two luminescent labels and a nucleoside hexaphosphate (e.g., reactant) via a same-strand linkage. In some embodiments, as shown, d L is approximately 1 nm, and d LRis approximately 7 nm. In some embodiments, the brightly labeled reactants of the present disclosure include more than two luminescent labels. For example, in some embodiments, the nucleic acid linker 306 is attached to three luminescent labels and a nucleoside hexaphosphate (e.g., reactant) by co-stranded linkages. As shown, in some embodiments, constructs having more than two luminescent labels attached to more than two attachment sites necessarily have more than one separation distance d L In some embodiments, each separation distance d on a single construct will have L can be independently designed according to the description herein. In some embodiments, each label separation distance d L can be designed to be approximately the same. For example, in some embodiments, the separation distance d L Each occurrence of is approximately 3.5 nm, and d LR is approximately 7 nm.

[0086] In some embodiments, the brightly labeled reactants of the present application include two or more luminescent labels attached via different oligonucleotide strands. For example, in some embodiments, the nucleic acid linker 308 includes four luminescent labels attached via separate oligonucleotide strands. In some embodiments, the unlabeled oligonucleotide strand of the nucleic acid linker 308 includes two luminescent labels and a first portion that is complementary to a first oligonucleotide strand attached to a nucleoside polyphosphate. The unlabeled oligonucleotide strand further includes a second portion that is complementary to a second oligonucleotide strand attached to the two luminescent labels. As shown, in some embodiments, the luminescent label attachment sites within each labeled strand are 9 nucleotides apart. In some embodiments, the separation between the nearest attachment sites on non-contiguous labeled strands can be the same or different (e.g., as shown by a 10-nucleotide separation).

[0087] A non-limiting generic nucleic acid linker 304 with same-strand label-reactant linkages was used as the basis for generating labeled nucleoside polyphosphates, as shown in FIG. 3C. As shown, the nucleic acid linker of FIG. 3C includes two hybridized oligonucleotide strands. According to certain embodiments of the present disclosure, two luminescent labels are attached to abasic sites in the first oligonucleotide strand of the linker. As a general attachment strategy, click chemistry technology is used to attach nucleoside polyphosphates to the same oligonucleotide strand, as described elsewhere herein. The second oligonucleotide strand is hybridized to the first strand, promoting a constrained spatial conformation of the attached moiety according to the theory described herein. The construct of FIG. 3C was successfully used in a single molecule sequencing run with other labeled nucleoside polyphosphates (FIG. 3D). A similarly designed construct was generated with reverse strand linkages, as shown in FIG. 3E.

[0088] FIG. 3E shows an example of a nucleic acid linker structure that non-covalently links a nucleoside polyphosphate (e.g., a reactant) with a luminescent light-emitting label. As shown, a first oligonucleotide strand attached to two luminescent light-emitting labels at an abasic attachment site hybridizes with a second oligonucleotide strand attached to a nucleoside hexaphosphate at a terminal attachment site. In some embodiments, the design strategies provided in the present disclosure for generating brightly labeled reactants are contemplated to include alternative label conjugation strategies, such as labels incorporated within the linker (e.g., incorporated into an oligomeric or polymeric linker, such as an oligonucleotide strand). For example, FIG. 3F shows an example of a nucleic acid linker with two labels attached within an oligonucleotide strand.

[0089] As shown in FIG. 3F, in some embodiments, the brightly labeled reactant comprises two luminescent light-emitting labels attached within an oligonucleotide strand (e.g., incorporated into the strand). In some embodiments, such conjugation strategies may implement luminescent light-emitting molecules, such as cyanine-based dyes, that do not interact via radioactive and / or non-radioactive decay (e.g., boxed area, as shown in FIG. 3F). In some embodiments, the number of luminescent light-emitting molecules attached within an oligonucleotide strand may be limited only by the size of the oligonucleotide strand. Thus, in some embodiments, the brightly labeled reactant comprises more than two luminescent light-emitting labels attached within an oligonucleotide strand (e.g., 2, 3, 4, 5, 6, or more than six luminescent light-emitting labels). The construct shown in FIG. 3F was successfully utilized in a sequencing run with three other brightly labeled nucleoside polyphosphates (FIG. 3G).

[0090] The present disclosure relates in some embodiments to the discovery that nucleic acid linkers can be designed with structurally constrained conformations that provide a scaffold for brightly labeled reactants with consistent and / or preserved emission lifetimes. As described herein, in some embodiments, oligonucleotide strand hybridization is a common strategy to promote constrained conformations for label separation purposes. In some embodiments, oligonucleotide strand hybridization comprises hybridization of different oligonucleotide strands. In some embodiments, oligonucleotide strand hybridization comprises self-strand hybridization (e.g., self-hybridization within a single strand). In some embodiments, self-strand hybridization can limit the flexibility of the nucleic acid linker, as described elsewhere herein with respect to separate strand hybridization. In some embodiments, self-strand hybridization is used to form one or more stem-loop structures within the nucleic acid linker.

[0091] A stem loop or hairpin loop is an unpaired loop of nucleotides on an oligonucleotide strand that is formed when the oligonucleotide strand folds back and base pairs with another section of the same strand. In some embodiments, the unpaired loop of the stem loop comprises 3-10 nucleotides. Thus, a stem loop can be formed by two regions of an oligonucleotide strand with reverse complementary sequences that hybridize to form a stem, the two regions being separated by 3-10 nucleotides that form an unpaired loop. In some embodiments, the stem can be designed to have one or more G / C nucleotides, which can provide added stability with additional hydrogen bond interactions formed compared to A / T nucleotides. In some embodiments, the stem comprises a G / C nucleotide immediately adjacent to the unpaired loop sequence. In some embodiments, the stem comprises a G / C nucleotide within the first 2, 3, 4, or 5 nucleotides adjacent to the unpaired loop sequence.

[0092] In some embodiments, the unpaired loop of the stem-loop nucleic acid linker comprises one or more luminescent light-emitting labels. Thus, in some embodiments, one or more label attachment sites are present in the unpaired loop. In some embodiments, the attachment site occurs at an abasic site in the unpaired loop. In some embodiments, the attachment site occurs at a base of the unpaired loop. In some embodiments, the loop comprises an A / T / U-rich sequence, e.g., due to the quenching effect observed with guanine as described elsewhere herein. In some embodiments, the attachment site occurs at an A, T, or U nucleotide in the unpaired loop. In some embodiments, at least four consecutive A, T, or U nucleotides occur on either side of the attachment site. In some embodiments, the unpaired loop comprises less than 33% G / C content (e.g., less than 30%, less than 20%, less than 10%, or 0% G / C content).

[0093] In some embodiments, the luminescent light-emitting label is attached to the unpaired loop of the stem-loop (e.g., the label attachment site occurs within the loop). For example, FIG. 4A generally illustrates an example of a labeled reactant having a stem-loop nucleic acid linker 400. As shown, in some embodiments, a first oligonucleotide strand 410 self-hybridizes to form a stem-loop. In some embodiments, the self-hybridized portion of the oligonucleotide strand forms the stem 412 of the stem-loop. In some embodiments, the self-strand hybridization results in the formation of a loop 414 of the stem-loop (e.g., an unpaired loop). In some embodiments, the first oligonucleotide strand 410 further hybridizes to a second oligonucleotide strand 420 that is attached to the reactant. Thus, in some embodiments, the stem-loop nucleic acid linker non-covalently links the luminescent light-emitting label to the reactant via a reverse strand linkage. It should be understood that in some embodiments, a self-hybridized oligonucleotide strand (e.g., a strand having a stem loop) attached to a luminescent label can be attached to one or more reactants via same-strand ligation.

[0094] In some embodiments, two or more luminescent light-emitting labels are attached to the stem-loop nucleic acid linker. For example, in some embodiments, the stem-loop nucleic acid linker 402 is attached to two luminescent light-emitting labels. As shown, in some embodiments, the attachment sites for the two luminescent light-emitting labels occur within the loop of the stem-loop. In some embodiments, the stability provided by the self-hybridized stem region results in a loop region that has a relatively low level of flexibility. In some embodiments, the luminescent light-emitting label attachment sites within the unpaired loop can be designed to promote favorable label-label spatial separation and / or favorable label-reactant spatial separation. The example provided by the non-limiting stem-loop nucleic acid linker 402 shows label attachment sites that are separated from each other by a distance that is approximately the same as the diameter of the loop. In some embodiments, this design can maximize label-label separation through space. In some embodiments, this design further limits the extent of label-label interaction through the steric barrier effect provided by the loop, as described elsewhere herein. In some embodiments, the stem-loop nucleic acid linker comprises two or more stem-loops (Figure 4B).

[0095] As shown in FIG. 4B, in some embodiments, the stem-loop nucleic acid linker 404 comprises two stem loops. In some embodiments, the formation of the two stem loops results in a nucleic acid linker having a Y-shape, and a three-way junction 410 is formed. In some embodiments, the three-way junction is contemplated as a general design strategy due to the relative stability and geometrically constrained conformation resulting from these features. In some embodiments, the stem-loop nucleic acid linker 404 comprises one luminescent label attached to each loop. In some embodiments, the stem-loop nucleic acid 404 non-covalently links the luminescent label and the reactant via a reverse strand linkage, e.g., one or more labels attached to a first oligonucleotide strand that hybridizes with a second oligonucleotide strand attached to one or more reactants. In some embodiments, the stem-loop nucleic acid 406 links the luminescent label and the reactant via a same strand linkage (e.g., one or more labels and one or more reactants attached to the same oligonucleotide strand). Stem-loop nucleic acid 406 shows an example of non-limiting distance specifications used in the design of brightly labeled reactants with three-way junctions. For example, these approximate distances are used as the basis for the generation of the labeled nucleoside polyphosphate shown in Figure 4C. The construct in Figure 4C was successfully used in a single molecule sequencing run along with other labeled nucleoside polyphosphates (Figure 4D).

[0096] In some embodiments, one or more luminescent labels are attached to the stem of the stem loop. In some embodiments, the stem loop of the nucleic acid linker does not include a luminescent label. In some embodiments, the stem loop of the nucleic acid linker includes an unpaired region (e.g., a "bulge loop") within the stem. In some embodiments, one or more unlabeled structural motifs, such as a stem loop, are included within the nucleic acid linker (e.g., at a position on the nucleic acid linker such that it is attached between one or more luminescent labels and one or more nucleoside polyphosphates). In such embodiments, the one or more unlabeled structural motifs can provide a steric barrier effect, as described elsewhere herein.

[0097] In some embodiments, the stem-loop nucleic acid linker comprises a first oligonucleotide strand attached to a luminescent light-emitting label. In some embodiments, the luminescent light-emitting label is attached to the attachment site of the first oligonucleotide strand via a spacer. In some embodiments, the first oligonucleotide strand forms a stem-loop secondary structure having a stem and a loop (e.g., an unpaired loop). In some embodiments, the loop comprises the attachment site. In some embodiments, the nucleic acid linker comprises a second oligonucleotide strand that hybridizes to the first oligonucleotide strand. In some embodiments, the second oligonucleotide strand is attached to a nucleoside polyphosphate. In some embodiments, the nucleoside polyphosphate is attached to the second oligonucleotide strand via a spacer. In some embodiments, the first oligonucleotide strand has the following structure:

[0098] [ka] In the formula, N A and N Bare consecutive sequences of 5 to 40 nucleotides independently selected from A, U, T, G, and C, and the second oligonucleotide strand is N A or N B N1 and N2 are contiguous sequences of 5 to 20 nucleotides each independently selected from A, U, T, G, and C, and the 5' portion of N1 (S1) and the 3' portion of N2 (S2) are reverse complementary or partially reverse complementary and can hybridize to each other to form a stem motif; when S1 and S2 hybridize to form a stem motif, the 3' portion of N1 (L1), the 5' portion of N2 (L2), and the region in between form a loop motif; (A / T) are nucleotides selected from A, T, and U; X1 is an attachment site on the second oligonucleotide strand; Y1 is a first linker; and Z1 is a luminescent light-emitting label.

[0099] As described herein, several aspects of the disclosure relate to geometrically constrained linker configurations for linking one or more luminescent labels to one or more reactants (e.g., one or more nucleoside polyphosphates). In some embodiments, the geometrically constrained configurations represent nucleic acid linkers having two or more luminescent labels, where the luminescent labels are spatially separated by a symmetrical configuration. In some embodiments, the brightly labeled reactants of the disclosure include nucleic acid linkers resembling a dendritic configuration, as shown in FIG. 5A. In some embodiments, the dendritic linker 500 includes a tris-labeled nucleic acid linker linked to a reactant (e.g., a nucleoside polyphosphate). As shown, in some embodiments, the dendritic linker 500 includes three main oligonucleotide components. In some embodiments, the first oligonucleotide component 510 includes four oligonucleotide strands covalently attached via a branched linker. In some embodiments, three of the oligonucleotide strands of component 510 are each attached to a luminescent light-emitting label (e.g., via end attachment or other conjugation strategies described herein). Thus, in some embodiments, the three labeled oligonucleotide strands of component 510 are generally referred to as the labeled portion of component 510. In some embodiments, the fourth oligonucleotide strand of component 510 is unlabeled. In some embodiments, said fourth oligonucleotide strand hybridizes to a second oligonucleotide component 520. In some embodiments, the second oligonucleotide component 520 of the nucleic acid linker 500 is attached to the reactant (e.g., via end attachment or other conjugation strategies described herein). In some embodiments, the third oligonucleotide component 530 hybridizes to the three oligonucleotide strands of the labeled portion of component 510. In some embodiments, the third oligonucleotide component 530 is referred to as the supporting strand, since its hybridization with component 510 provides structural rigidity within the labeled region to facilitate spatial separation of the luminescent light-emitting labels.

[0100] As should be understood, any linker design strategy described herein can be applied to dendritic nucleic acid linkers or any other nucleic acid linker configurations contemplated by this disclosure (e.g., chain linkages, spacer properties and spacer configurations, label-label separations, label-reactant separations, three-way junctions, etc.). Dendritic nucleic acid 502 shows one example of non-limiting distance specifications used to design brightly labeled reactants with dendritic nucleic acid linkers. As an illustrative example, these approximate distances are used as the basis to generate the labeled nucleoside polyphosphates shown in FIG. 5B. As shown, in some embodiments, dendritic nucleic acids can include more than one reactant (e.g., two nucleoside polyphosphates, or more than two nucleoside polyphosphates).

[0101] In some embodiments, the dendritic nucleic acid linker is described in terms of a first and a second oligonucleotide strand comprising a hybridized portion with a reactant. For example, in some embodiments, the dendritic nucleic acid linker comprises a first oligonucleotide strand attached to two or more branched oligonucleotide strands at the terminus of the first oligonucleotide via a coupling compound (e.g., a branched coupler). In some embodiments, each branched oligonucleotide strand is attached to a luminescent light-emitting label. In some embodiments, the first oligonucleotide strand is hybridized to a second oligonucleotide strand attached to a reactant (e.g., a nucleoside polyphosphate). In some embodiments, a third oligonucleotide strand (e.g., a third oligonucleotide component) is hybridized to two or more branched oligonucleotide strands. In some embodiments, the coupling compound is of the following structure:

[0102] [ka] In the formula, N f is the first oligonucleotide strand; N b is a branched oligonucleotide chain; R f and R bare each independent of the other, and the bond or linking group is selected from the group consisting of substituted or unsubstituted alkylene; substituted or unsubstituted alkenylene; substituted or unsubstituted alkynylene; substituted or unsubstituted heteroalkylene; substituted or unsubstituted heteroalkenylene; substituted or unsubstituted heteroalkynylene; substituted or unsubstituted heterocyclylene; substituted or unsubstituted carbocyclylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; and combinations thereof; O in each instance is the oxygen atom of either the 5' phosphate or the 3' hydroxyl group of the adjacent oligonucleotide strand.

[0103] In some embodiments, the present disclosure provides brightly labeled reactants with star-shaped nucleic acid linkers. As shown in FIG. 6A, star-shaped nucleic acid linkers can provide reactants symmetrically arranged in the outer regions of the linker and one or more luminescent labels closer to the core region of the configuration. In this way, in some embodiments, the reactants may be more amenable to reaction with, for example, a polymerase. In some embodiments, the nucleotide content surrounding the luminescent label attachment site near the core region is selected according to the specifications described herein (e.g., low G / C content to avoid quenching effects between the label and the linker). In some embodiments, the star-shaped nucleic acid linker 600 comprises a Y-shaped oligonucleotide component having three oligonucleotide strands attached via branched linkers. In some embodiments, the three oligonucleotide strands of the Y-shaped oligonucleotide component are each attached to a reactant (e.g., at the end). In some embodiments, the three oligonucleotide strands of the Y-shaped oligonucleotide component are each hybridized to an oligonucleotide strand. In some embodiments, one of the hybridized oligonucleotide strands is attached to a luminescent label. In some embodiments, however, two or three of the hybridized oligonucleotide strands are each attached to a luminescent light-emitting label (601).

[0104] In some embodiments, the Y-shaped oligonucleotide component of the star-shaped nucleic acid linker is attached to more than three reactants. For example, in some embodiments, a brightly labeled reactant having a star-shaped nucleic acid linker 602 includes two reactants on each of the two oligonucleotide strands of the Y-shaped oligonucleotide component. In some embodiments, the two reactants are attached to the strand of the Y-shaped oligonucleotide component that hybridizes with the unlabeled strand as shown. In some embodiments, the two reactants are attached to the strand of the Y-shaped oligonucleotide component that hybridizes with the labeled strand (603).

[0105] In some embodiments, the star-shaped nucleic acid linker includes a labeled oligonucleotide component and a reactant oligonucleotide component, each of which includes a Y-shaped oligonucleotide. For example, in some embodiments, the star-shaped nucleic acid linker 604 includes a first Y-shaped oligonucleotide component attached to three luminescent light-emitting labels. In some embodiments, each luminescent light-emitting label is attached to each oligonucleotide strand of the first Y-shaped component. In some embodiments, as shown, each luminescent light-emitting label is attached near the core region of the first Y-shaped oligonucleotide component. In some embodiments, the star-shaped nucleic acid linker 604 hybridizes to a second Y-shaped oligonucleotide component attached to three reactants. In some embodiments, each reactant is attached to each oligonucleotide strand of the second Y-shaped component. In some embodiments, as shown, each reactant is attached to an outer region of the first Y-shaped oligonucleotide component (e.g., via a terminal attachment site). Star-shaped nucleic acid 606 illustrates an example of non-limiting distance specifications used in the design of brightly labeled reactants with star-shaped nucleic acid linkers. An example of a brightly labeled reactant structure with a star-shaped nucleic acid linker generated according to the present disclosure is shown in FIG. 6B.

[0106] In some embodiments, the star-shaped nucleic acid linker can be described in terms of the covalent coupling compound used for the Y-shaped oligonucleotide component of the linker. For example, in some embodiments, a brightly labeled reactant having a star-shaped nucleic acid linker includes a first oligonucleotide component that includes three or more oligonucleotide strands extending from a covalent coupling compound. In some embodiments, at least one of the oligonucleotide strands is attached to a reactant (e.g., a nucleoside polyphosphate). In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) of the oligonucleotide strands of the first oligonucleotide component are each attached to one or more reactants. In some embodiments, the first oligonucleotide component hybridizes to a second oligonucleotide component. In some embodiments, the second oligonucleotide component includes at least one oligonucleotide strand attached to a luminescent light-emitting label. In some embodiments, the second oligonucleotide component includes three or more oligonucleotide strands extending from a covalent coupling compound. In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) of the oligonucleotide strands of the second oligonucleotide component are each attached to a luminescent light-emitting label.

[0107] In some embodiments, the brightly labeled reactants of the present disclosure include nucleic acid linkers with a tetrahedral core. As shown by the exemplary structure in FIG. 6C, the tetrahedral core represents a four-way covalent coupling compound that facilitates symmetrically constrained spatial arrangement of one or more luminescent labels and one or more nucleoside polyphosphates. FIG. 6D shows an example of a reaction scheme that can be used to synthesize the exemplary structure shown in FIG. 6C. In some embodiments, the tetrahedral core is contemplated for use in combination with any one or more of the brightly labeled reactant design strategies described herein. As an example, the symmetrically constrained advantages provided by the tetrahedral core are implemented at a three-way junction as described herein to generate the brightly labeled reactant shown in FIG. 6E. As shown, in some embodiments, attachment of the luminescent label at or near the three-way junction can advantageously provide steric effects as described elsewhere herein.

[0108] In some embodiments, the present disclosure further contemplates symmetrically constrained configurations using core chemical couplers that allow symmetric attachment. In some embodiments, for example, the brightly labeled reactant comprises a cyclodextrin-based core. An example of a synthetic scheme for generating a cyclodextrin-based nucleic acid linker with a three-way junction is shown in Figure 7. An example of the structure of a cyclodextrin coupling compound is provided in Table 1, along with examples of other coupling compounds that can be used to covalently link three or more oligonucleotide strands according to embodiments described herein.

[0109] [Table 1] Spacer As described herein, in some embodiments, the luminescent label and / or reactant may be attached to the nucleic acid linker via a spacer. In some embodiments, the spacer is attached to the oligonucleotide chain of the nucleic acid linker at an attachment site. In some embodiments, the attachment site occurs at a terminal site on the oligonucleotide chain (e.g., at the 5' or 3' end). Examples of terminal attachment sites are provided in the application, for example, as shown in Figures 3C, 3E, 4C, 6B, 6C, 8, 9, and 10 (terminal attachment of a nucleotide), and Figures 5B, 9, and 10 (terminal attachment of a luminescent label). In some embodiments, the attachment site occurs at an abasic site within the oligonucleotide chain (e.g., at a site that lacks a nucleotide but is adjacent). Examples of abasic attachment sites are provided in the application, for example, as shown in Figures 3C, 3E, 6B, 6C, and 8 (abasic attachment of a luminescent label). In some embodiments, the attachment site occurs at a base site on the oligonucleotide chain (e.g., attached to a nucleotide, such as a nucleobase on the chain, a sugar or a phosphate of a nucleotide). For example, as shown in Figures 4C and 8 (base attachment of a luminescent label), examples of base attachment sites are provided within the application.

[0110] In some embodiments, the spacer comprises a plurality of thymidine nucleotides. In some embodiments, the spacer comprises a branched spacer, e.g., a branched thymidine spacer. In some embodiments, the branched spacer comprises a branched thymidine spacer. For example, in some embodiments, each nucleoside polyphosphate has the formula Nu-T(T) nThe branched thymidine spacer includes a thymidine spacer TR, Nu represents a nucleoside polyphosphate, T represents a thymidine nucleotide, n is an integer having a value of 1 to 30, and R represents a convergence point linking one or more additional nucleoside polyphosphates. In some embodiments, the convergence point is further directly attached to the oligonucleotide chain of the nucleic acid. In some embodiments, the convergence point is further indirectly attached to the oligonucleotide chain, for example, via an additional thymidine linker and / or additional convergence point. An example of a branched thymidine spacer is shown in FIG. 5B, which shows two nucleoside polyphosphates with a thymidine spacer and a convergence point further directly attached to the oligonucleotide chain.

[0111] In some embodiments, the spacer contains one or more branch points, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleoside polyphosphates are linked to each luminescent light-emitting label, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) luminescent labels are linked to each nucleoside polyphosphate, or two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleoside polyphosphates are linked to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) luminescent light-emitting labels.

[0112] In some embodiments, the spacer is attached to the terminal portion of the oligonucleotide strand of the nucleic acid linker. In some embodiments, the luminescent label and / or reactant is attached to either the 5' or 3' end of the oligonucleotide strand via a universal spacer as shown below:

[0113] [ka] In some embodiments, the spacer is attached to an internal abasic site within the oligonucleotide strand of the nucleic acid linker. In some embodiments, the luminescent label and / or reactant is attached to an internal abasic site on the oligonucleotide strand via a universal spacer as shown below:

[0114] [ka] Examples of spacers for attachment of luminescent labels (e.g., via terminal, internal abasic, or base attachment sites) are provided in Table 2 and shown below. Although the spacer structures are shown with luminescent labels, it should be understood that in some embodiments the reactants can be substituted such that any of the spacer structures provided herein can be used to attach a reactant (e.g., a nucleoside polyphosphate) to a nucleic acid linker.

[0115] In some embodiments, the luminescent label and / or reactant is attached to an internal abasic site on the oligonucleotide strand via a glycolamine spacer as shown below:

[0116] [ka] In some embodiments, the luminescent label and / or reactant is attached to an internal abasic site on the oligonucleotide strand via a serinolamine spacer as shown below:

[0117] [ka] In some embodiments, the luminescent label and / or reactant is attached to an internal base site on the oligonucleotide strand via a C6-amino-T spacer, as shown below:

[0118] [ka] In some embodiments, the luminescent label and / or reactant is attached to an internal base site on the oligonucleotide strand via a C2-amino-T spacer, as shown below:

[0119] [ka] In some embodiments, the luminescent label and / or reactant is attached to an internal base site on the oligonucleotide strand via a C8-alkyne-dT spacer, as shown below:

[0120] [ka] In some embodiments, one or more luminescent labels and / or one or more reactants (e.g., one or more nucleoside polyphosphates) can be attached to the nucleic acid linker using chemical coupling techniques known in the art. Any covalent bond forming reaction can be used to conjugate the luminescent labels (e.g., donor chromophores and / or acceptor chromophores) to the nucleotide. Exemplary reactions include, but are not limited to, alkylation reactions, metathesis reactions, addition reactions, substitution reactions, cycloaddition reactions, and the like.

[0121] For example, in some embodiments, "click chemistry" techniques (e.g., copper-catalyzed, strain-promoted, copper-free click chemistry, etc.) can be used to attach one or more luminescent labels and one or more nucleoside polyphosphates to nucleic acids. In some embodiments, the reaction used to conjugate luminescent labels (e.g., donor chromophores and / or acceptor chromophores) to nucleotides is a "click chemistry" reaction. Any "click chemistry" reaction known in the art can be used for this purpose. Click chemistry is a chemical approach introduced by Sharpless in 2001, which describes a chemistry adapted to rapidly and reliably generate substances by linking discreet reactive units. See, for example, Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001), 40:2004-2021; Evans, Australian Journal of Chemistry (2007), 60:384-395. Exemplary coupling reactions (some of which may be classified as "click chemistry") include, but are not limited to, formation of esters, thioesters, amides from activated acids or acyl halides (e.g., peptide coupling, amine-NHS chemistry); nucleophilic substitution reactions (e.g., nucleophilic displacement of halides or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition); and Diels-Alder reactions (e.g., 、 tetrazine [4+2] cycloaddition).

[0122] In some embodiments, the reaction used to conjugate a nucleotidic label to a luminescent light-emitting label is a cycloaddition reaction. In some embodiments, the cycloaddition is a [4+2] cycloaddition. In some embodiments, the cycloaddition is a 1,3-dipolar cycloaddition. In some embodiments, the reaction is an azide-alkyne cycloaddition (i.e., a Huisgen cycloaddition). In some embodiments, the cycloaddition is performed in the presence of a catalyst. In some embodiments, the catalyst is a copper catalyst. In some embodiments, the cycloaddition reaction is a copper-free reaction. In some embodiments, the azide-alkyne cycloaddition involves a cyclic alkyne (e.g., cyclooctyne) and is strain-promoted. In some embodiments, these reactions conjugate a luminescent light-emitting label to a nucleotidic label to form a "click spacer" as described herein.

[0123] In one embodiment, the nucleotide is functionalized with an azide, the luminescent label is functionalized with an alkyne, and the nucleotide is conjugated to the luminescent label via azide-alkyne cycloaddition (i.e., Huisgen cycloaddition) as described herein. In another embodiment, the nucleotide is functionalized with an alkyne, the luminescent label is functionalized with an azide, and the nucleotide is conjugated to the luminescent label via azide-alkyne cycloaddition (i.e., Huisgen cycloaddition) as described herein. An example of this reaction scheme is shown below, where "Nuc" is a nucleotide (e.g., linked to the 3' or 5' position of R1);

[0124] [ka] For example, in one embodiment, a luminescent label is conjugated by reacting an azide-functionalized luminescent label with a C1-alkynyl-deoxyribose on the nucleotide backbone (see scheme below).

[0125] [ka] In some embodiments, the nucleoside polyphosphates are attached to the nucleic acid linker via an azide-conjugate dN6P (e.g., dN6P-N3), according to the general structure shown below:

[0126] [ka] where the base is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, and derivatives thereof; -YZ-=-CH2CH2-, -CONH-, or -NHCO-; and X=NH or O. For example, in some embodiments, an azide-conjugated dN6P (e.g., dN6P-N3) and / or an azide-conjugated luminescent light-emitting label (e.g., dye-N3) is attached to a nucleic acid linker in a copper-catalyzed reaction by contacting the azide of dN6P-N3 or dye-N3 with the terminal alkyne of the alkyne-conjugated nucleic acid linker, so as to form a triazole bond between the nucleic acid linker and the dN6P or dye under suitable reaction conditions. In some embodiments, azide-conjugated dN6P (e.g., dN6P-N3) and / or azide-conjugated luminescent label (e.g., dye-N3) are attached to the nucleic acid linker in a copper-free reaction by contacting the azide of dN6P-N3 or dye-N3 with the internal alkyne of the cyclooctyne-conjugated nucleic acid linker, so as to form a polycyclic bond between the nucleic acid linker and the dN6P or dye under suitable reaction conditions. Cyclooctyne modification of the nucleic acid linker can be achieved using a suitable cyclooctyne reagent to generate copper-free click chemistry moieties known in the art. For example, cyclooctyne-modified nucleic acid linkers are prepared by contacting a suitable cyclooctyne reagent (e.g., (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate) with the terminal amine of the nucleic acid linker.

[0127] In some embodiments, the reaction used to conjugate the luminescent label (e.g., donor chromophore and / or acceptor chromophore) to the nucleotide is an ester bond or amide bond forming reaction. In some embodiments, amine-N-hydroxysuccinimide (amine-NHS) chemistry is used. In some embodiments, the nucleotide is functionalized with an NHS ester, the luminescent label is functionalized with an amine, and the nucleotide is conjugated to the luminescent label via an amine-NHS coupling reaction. In another embodiment, the nucleotide is functionalized with an amine, the luminescent label is functionalized with an NHS ester, and the nucleotide is conjugated to the luminescent label via an amine-NHS coupling reaction. An example of this reaction scheme is shown below. In some embodiments, these reactions conjugate the luminescent label to the nucleotide to form the "click spacer" described herein.

[0128] [ka] In the case of the FRET system described herein, the donor chromophore and the acceptor chromophore can be conjugated to the nucleotide via different click chemistry reactions (e.g., to allow for their controlled / selective incorporation). In some embodiments, one or more donors are conjugated via the azide-alkyne cycloaddition (i.e., Huisgen cycloaddition) described herein, and one or more acceptors are conjugated via a different reaction. In some embodiments, one or more donor chromophores are conjugated via the azide-alkyne cycloaddition (i.e., Huisgen cycloaddition) described herein, and one or more acceptors are conjugated via the amine-NHS reaction described herein. In some embodiments, the reactions used are reversed. For example, in some embodiments, one or more donors are conjugated via the amine-NHS described herein, and one or more acceptors are conjugated via the azide-alkyne cycloaddition (i.e., Huisgen cycloaddition) described herein.

[0129] The conjugation reactions described herein form a "spacer" that links the nucleotide to the luminescent light-emitting label (or, in other embodiments, the reactant). Thus, in some embodiments, the spacer comprises a linking group (e.g., BCN, tetrazine, tetrazole, triazole, amide, or other product generated through the coupling of reactive moieties suitable for click reactions and similar coupling techniques). In some embodiments, the spacer is of the formula:

[0130] [ka] In the formula, R1 is a first linking group that is attached to an attachment site on the first oligonucleotide strand; R2 is a second linking group that includes a coupling moiety (e.g., triazole, amide) that is formed in a coupling reaction to covalently link R1 and R3; and R3 is a third linking group that is attached to a luminescent light-emitting label.

[0131] In some embodiments, the spacer is a formula selected from the following formulas:

[0132] [ka] In the formula, R1 and R3 are each independently a bond or linking group selected from the group consisting of substituted or unsubstituted alkylene; substituted or unsubstituted alkenylene; substituted or unsubstituted alkynylene; substituted or unsubstituted heteroalkylene; substituted or unsubstituted heteroalkenylene; substituted or unsubstituted heteroalkynylene; substituted or unsubstituted heterocyclylene; substituted or unsubstituted carbocyclylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; and combinations thereof.

[0133] In some embodiments, the spacer is a formula selected from the following formulas:

[0134] [ka] In some embodiments, examples of spacers generated using click chemistry are provided in Table 2.

[0135] [Table 2-1] [Table 2-2] As used herein, a "nucleic acid linker" generally refers to a nucleic acid that links one or more luminescent labels to one or more nucleoside polyphosphates. In some embodiments, a nucleic acid linker may generally refer to a construct having any number of oligonucleotide strands linked via covalent attachment or via base pairing (e.g., hybridization). In some embodiments, a linker may alternatively be referred to as a "core" or "base" construct to which different functional components (e.g., one or more luminescent labels, one or more nucleoside polyphosphates) may be attached. The term "construct" is used in some embodiments throughout various contexts to generally refer to a linker, which may or may not include various other components (e.g., spacers, labels, nucleoside polyphosphates, etc.) described herein.

[0136] In some embodiments, the nucleic acid linkers described herein are not attached to particles of material (e.g., not attached to metal particles, magnetic particles, polymeric particles, or particles of other materials). In some embodiments, the nucleic acid linkers are linear molecules (e.g., "rod-shaped" nucleic acids). In some embodiments, the nucleic acid linkers are circular molecules. In some embodiments, the nucleic acid linkers are single-stranded (e.g., with or without a stem-loop structure). In some embodiments, the nucleic acid linkers are double-stranded (e.g., with or without a stem-loop structure). In some embodiments, the two strands of the double-stranded nucleic acid linker hybridize (due to complementary sequences) and are not covalently attached. However, in some embodiments, one or more covalent bonds can be introduced to covalently attach the two strands of the double-stranded linker (e.g., using one or more chemical linkers). In some embodiments, the nucleic acid linkers can include one or more additional moieties as described herein. In some embodiments, the nucleic acid linker comprises i) one or more additional moieties within or at the sugar phosphate backbone, ii) one or more modifications (e.g., one or more modified bases or sugars), or a combination of i) and ii). However, in some embodiments, the nucleic acid linker does not comprise i), ii), or either i) or ii).

[0137] In the context of a nucleic acid linker, it is understood that a "nucleotide" or "nucleoside polyphosphate" attached to the nucleic acid linker refers to one or more nucleotides (e.g., nucleoside polyphosphates) configured to be incorporated into a growing nucleic acid chain (e.g., during a sequencing reaction). In some embodiments, the one or more nucleotides include one or more nucleoside monophosphates or nucleoside polyphosphates. Examples of nucleoside polyphosphates include, in some embodiments, nucleosides having more than three 5' phosphates, such as nucleoside diphosphates or nucleoside triphosphates, or nucleoside hexaphosphates. Thus, in some embodiments, a "labeled nucleotide" refers to a nucleoside polyphosphate linked to one or more luminescent light-emitting labels by a linker of the present application, which nucleoside polyphosphate serves as a substrate for a polymerase enzyme under nucleic acid synthesis reaction conditions. In some embodiments, the nucleoside polyphosphate comprises at least two or three phosphate groups that can be acted upon by a suitable polymerase enzyme in a phosphoryl transfer reaction (e.g., transfer of the α-phosphate from the β-phosphate of the nucleoside polyphosphate to the 3′ hydroxyl group of a growing nucleic acid chain).

[0138] In some embodiments, one or more nucleoside phosphates (e.g., nucleoside polyphosphates) may be attached to an oligonucleotide (e.g., a labeled or unlabeled oligonucleotide strand) forming part of a linker of the present application via a terminal phosphate, and function as a protective molecule to protect the polymerase from label-induced photodamage (e.g., as described elsewhere in this application). In some embodiments of any of the compositions or methods described in this application, the phosphate moiety (e.g., polyphosphate moiety) of a nucleoside phosphate (e.g., of a nucleoside polyphosphate) comprises one or more phosphates (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate groups) or variants thereof. For example, in some embodiments, the phosphate moiety (e.g., of a nucleoside polyphosphate) may comprise a phosphate ester, a thioester, a phosphoramidate, a phosphonate alkyl linkage, other suitable linkages, or more than one such modification, or a combination of two or more thereof. In some embodiments, the labeled and unlabeled strands of the nucleic acid linker are substantially complementary to one another (e.g., over the length of the dimerization domain, where the strands within the dimerization domain can be, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to one another).

[0139] Nucleoside polyphosphates may have n phosphate groups, where n is equal to or greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of nucleoside polyphosphates include nucleoside diphosphates and nucleoside triphosphates. The labeled nucleotide may be a terminal phosphate-labeled nucleoside polyphosphate, where the terminal phosphate of the nucleoside polyphosphate is attached to a linker (e.g., a nucleic acid linker) that contains one or more luminescent labels, thereby forming a labeled nucleoside polyphosphate. Such labels may be luminescent (e.g., fluorescent or chemiluminescent) labels, fluorogenic labels, colored labels, chromogenic labels, mass tags, electrostatic labels, or electrochemical labels. The labels (or markers) may be attached to the terminal phosphates via linkers, such as spacers, as described herein. The linker (e.g., spacer) may include, for example, at least one or more hydroxyl groups, sulfhydryl groups, amino groups, or haloalkyl groups, which may be suitable for forming, for example, a phosphate ester, a thioester, a phosphoramidite, or a phosphonate alkyl bond at the terminal phosphate of a natural or modified nucleotide. The linker (e.g., spacer) may be cleavable, such as with the aid of a polymerase, to separate the label from the terminal phosphate. Examples of nucleotides and linkers (e.g., spacers) are provided in U.S. Patent No. 7,041,812, which is incorporated by reference in its entirety.

[0140] Nucleotides (e.g., nucleoside polyphosphates) can include adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or any of their variants. Nucleotides (e.g., nucleoside polyphosphates) can include methylated nucleobases. For example, methylated nucleotides can be nucleotides that include one or more methyl groups attached to the nucleobase (e.g., directly attached to the ring of the nucleobase, attached to a substituent of the ring of the nucleobase). Exemplary methylated nucleobases include 1-methylthymine, 1-methyluracil, 3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N6-methyladenine, N6,N6-dimethyladenine, 1-methylguanine, 7-methylguanine, N2-methylguanine, and N2,N2-dimethylguanine.

[0141] As used herein, the term "nucleic acid" generally refers to a molecule that includes one or more nucleic acid subunits. A nucleic acid may include one or more subunits selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. In some examples, a nucleic acid is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a derivative thereof. In some embodiments, a nucleic acid is a modified nucleic acid, including, without limitation, locked nucleic acid (LNA), peptide nucleic acid (PNA), triazole-linked nucleic acid, 2'-F-modified nucleic acid, and derivatives and analogs thereof. A nucleic acid may be single-stranded or double-stranded. In some embodiments, a nucleic acid generally refers to any polymer of nucleotides.

[0142] In some embodiments, the present disclosure provides a new composition for identifying single molecules based on one or more luminescence properties of those molecules. In some embodiments, a molecule (e.g., a luminescence-labeled nucleoside polyphosphate) is identified based on its brightness, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, luminescence intensity, or a combination of two or more thereof. Identifying may mean assigning the exact molecular identity of a molecule, or may distinguish a particular molecule from a set of possible molecules. In some embodiments, multiple single molecules may be distinguished from each other based on different brightness, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, luminescence intensity, or a combination of two or more thereof. In some embodiments, a single molecule is identified (e.g., distinguished from other molecules) by exposing the molecule to a series of separate light pulses and evaluating the timing or other properties of each photon emitted from the molecule. In some embodiments, the information of multiple photons emitted sequentially from a single molecule is aggregated and evaluated to identify the molecule. In some embodiments, the luminescence emission lifetime of a molecule is determined from a plurality of photons emitted sequentially from the molecule, and the luminescence emission lifetime can be used to identify the molecule. In some embodiments, the luminescence emission intensity of a molecule is determined from a plurality of photons emitted sequentially from the molecule, and the luminescence emission intensity can be used to identify the molecule. In some embodiments, the luminescence emission lifetime and luminescence emission intensity of a molecule are determined from a plurality of photons emitted sequentially from the molecule, and the luminescence emission lifetime and luminescence emission intensity can be used to identify the molecule.

[0143] Thus, in some aspects of the present application, a reaction sample is exposed to multiple separate light pulses and a series of emitted photons is detected and analyzed. In some embodiments, the series of emitted photons provides information about a single molecule that is present and does not change in the reaction sample over the time of the experiment. However, in some embodiments, the series of emitted photons provides information about a series of different molecules that are present at different times in the reaction sample (e.g., as a reaction or process progresses).

[0144] Luminescent markers As used herein, a "luminescent label" is a molecule that may absorb one or more photons and then emit one or more photons after one or more time durations. In some embodiments, the term may be used to generally refer to the non-reactant portion of a labeled reactant (e.g., a luminescent label may include at least a portion of a fluorophore and a spacer). In some embodiments, the term specifically refers to a molecule that absorbs and / or emits a photon (e.g., a fluorophore). In some embodiments, the term is used interchangeably with "luminescent molecule" depending on the context. In some embodiments, the luminescent label or luminescent molecule is a fluorophore (e.g., a "dye" or a "fluorophore dye" as used interchangeably herein). In some embodiments, the luminescent label or luminescent molecule is a rhodamine-based molecule. In some embodiments, the luminescent label or luminescent molecule is a cyanine-based molecule. In some embodiments, the luminescent label or luminescent molecule is a BODIPY-based molecule.

[0145] Typically, the luminescent label or molecule comprises an aromatic or heteroaromatic compound and may be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluoroscein, rhodamine, or other similar compounds. Examples of dyes include xanthene dyes such as fluorescein dyes or rhodamine dyes, including 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX). Examples of dyes also include naphthylamine dyes with amino groups at the alpha or beta positions. For example, naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonic acid, 1-anilino-8-naphthalenesulfonic acid, and 2-p-toluidinyl-6-naphthalenesulfonic acid, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Other examples of dyes include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; indodicarbocyanine 3 (Cy®3), (2Z)-2-[(E)-3-[3-(5-carboxypentyl)-1,1-dimethyl-6,8- Disulfobenzo[e]indol-3-ium-2-yl]prop-2-enylidene]-3-ethyl-1,1-dimethyl-8-(trioxidanylsulfanyl)benzo[e]indole-6-sulfonate (Cy® 3.5), 2-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18- Octahydrobenzo[2'',3'']indolizino[8'',7'':5',6']pyrano[3',2':3,4]pyrido[1,2-a]indol-5-ium-14-sulfonate (Cy® 3B), Indodicarbocyanine 5 (Cy® 5), Indodicarbocyanine 5.5 (Cy® 5.5), 3-(-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine Cyanines such as anthraquinone (CyA); 1H,5H,11H,15H-xanthen[2,3,4-ij:5,6,7-i'j']diquinolizin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or Texas Red®); BODIPY® dyes; benzoxazoles; stilbenes; pyrenes; and the like. In some embodiments, the luminescent label or luminescent molecule is a dye selected from Table 3. The dyes listed in Table 3 are not limiting, and the luminescent label or luminescent molecule of the present application may include dyes not listed in Table 3.In some embodiments, the luminescent label or molecule of one or more luminescently labeled nucleotides is selected from Table 3. In some embodiments, the luminescent label or molecule of four or more luminescently labeled nucleotides is selected from Table 3. In some embodiments, the luminescent label or molecule of the present application comprises a dye derived from a dye listed in Table 3. In some embodiments, the luminescent label or molecule is derived from ATTO Rho6G. In some embodiments, the tertiary amide side chain (carboxybutyl) of ATRho6G may be modified to an azidopropyl moiety. Without wishing to be bound by theory, this modification results in a shorter, more rigid dye. In some embodiments, the luminescent label or molecule has the following structure:

[0146] [ka]

[0147] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Dyes may be classified based on the wavelength of maximum absorbance or emitted luminescence. Table 4 provides exemplary fluorophores grouped into columns according to the approximate wavelength of maximum absorbance. The dyes listed in Table 4 are non-limiting, and the luminescent label or luminescent molecule of the present application may include dyes not listed in Table 4. The exact maximum absorbance or emission wavelength may not correspond to the indicated spectral range. In an embodiment, the luminescent label or luminescent molecule of one or more luminescent labeled nucleotides is selected from the "red" group listed in Table 4. In an embodiment, the luminescent label or luminescent molecule of one or more luminescent labeled nucleotides is selected from the "green" group listed in Table 4. In an embodiment, the luminescent label or luminescent molecule of one or more luminescent labeled nucleotides is selected from the "yellow / orange" group listed in Table 4. In one embodiment, the four nucleoside luminescent labels or molecules are selected such that they are all selected from the "red", "yellow / orange", or "green" groups listed in Table 4. In one embodiment, the four nucleoside luminescent labels or molecules are selected such that three are selected from the first group of the "red", "yellow / orange", and "green" groups listed in Table 4, and the fourth is selected from the second group of the "red", "yellow / orange", and "green" groups listed in Table 4. In one embodiment, the four nucleoside luminescent labels or molecules are selected such that two are selected from the first group of the "red", "yellow / orange", and "green" groups listed in Table 4, and the third and fourth are selected from the second group of the "red", "yellow / orange", and "green" groups listed in Table 4. In one embodiment, four nucleotide luminescent labels or molecules are selected such that two are selected from a first group of "red", "yellow / orange", and "green" listed in Table 4, a third is selected from a second group of "red", "yellow / orange", and "green" listed in Table 4, and a fourth is selected from a third group of "red", "yellow / orange", and "green" listed in Table 4.

[0148] [Table 4-1] [Table 4-2] In some embodiments, the luminescent label may include a first chromophore and a second chromophore. In some embodiments, the excited state of the first chromophore (e.g., also referred to as a donor luminescent molecule, donor label, or donor molecule) can be relieved via energy transfer to a second chromophore (e.g., also referred to as an acceptor luminescent molecule, acceptor label, or acceptor molecule). In some embodiments, the energy transfer is Foerster resonance energy transfer (FRET). Such a FRET pair may be useful for providing a luminescent label with properties that make it easier to distinguish among multiple luminescent labels. In some embodiments, the FRET pair may absorb excitation energy in a first spectral range and emit luminescence in a second spectral range.

[0149] In some embodiments, the luminescent label or luminescent molecule is attached within a linker molecule described herein (e.g., incorporated into an oligomeric or polymeric linker). Table 5 provides some examples of fluorophores that fit such conjugation strategies in the context of an oligonucleotide chain. As shown, the cyanine-based fluorophores in Table 5 are conjugated to the 3' end of the first strand portion and the 5' end of the second strand portion to form covalently linked portions within the oligonucleotide chain (e.g., the luminescent label or luminescent label is attached without the use of a spacer). It should be understood that in some embodiments, these or similar types of fluorophores can be attached within any class of oligomeric or polymeric structures. For example, in some embodiments, the fluorophore is attached within the peptide via conjugation to the C-terminus of the first peptide portion and the N-terminus of the second peptide portion. In some embodiments, a single linker molecule includes two or more fluorophores attached within the linker (e.g., 2, 3, 4, 5, 6, or more than 6 dyes attached within a single linker). In some embodiments, the linker comprises one or more fluorophores attached within the linker and one or more fluorophores attached via a spacer. For example, in some embodiments, the brightly labeled reactants described herein comprise one or more (e.g., 1, 2, 3, 4, 5, 6, or more than 6) fluorophores attached within the linker without a spacer and one or more (e.g., 1, 2, 3, 4, 5, 6, or more than 6) fluorophores attached to the linker via a spacer.

[0150] [Table 5] In the case of a set of luminescent labeled molecules (e.g., luminescent labeled nucleotides), the properties of the luminescent labeled FRET pair may allow the selection of a plurality of distinguishable molecules (e.g., nucleotides). In some embodiments, the second chromophore of the FRET pair (e.g., the acceptor molecule of the FRET label) has a luminescent emission lifetime that is different from the plurality of other luminescent labeled molecules. In some embodiments, the second chromophore of the FRET pair (e.g., the acceptor molecule of the FRET label) has a luminescent emission intensity that is different from the plurality of other luminescent labeled molecules. In some embodiments, the second chromophore of the FRET pair (e.g., the acceptor molecule of the FRET label) has a luminescent emission lifetime and a luminescent emission intensity that are different from the plurality of other luminescent labeled molecules. In some embodiments, the second chromophore of the FRET pair (e.g., the acceptor molecule of the FRET label) emits photons in a different spectral range from the plurality of other luminescent labeled molecules. In some embodiments, the first chromophore of the FRET pair (e.g., the donor molecule of a FRET label) has a different luminescence emission lifetime than the plurality of luminescence labeled molecules. In certain embodiments, the FRET pair (e.g., the donor and acceptor molecules of a FRET label) may absorb excitation energy in a different spectral range than the plurality of other luminescence labeled molecules. In certain embodiments, the FRET pair (e.g., the donor and acceptor molecules of a FRET label) may absorb excitation energy in the same spectral range as one or more of the plurality of other luminescence labeled molecules.

[0151] For sequencing reactions, certain combinations of brightly labeled reactants may be preferred. In some embodiments, at least one of the brightly labeled reactants comprises a cyanine dye, or an analog thereof. In some embodiments, at least one of the brightly labeled reactants comprises a rhodamine dye, or an analog thereof. In some embodiments, at least two of the brightly labeled reactants each comprise a cyanine dye, or an analog thereof. In some embodiments, at least two of the brightly labeled reactants each comprise a rhodamine dye, or an analog thereof. In some embodiments, at least three of the brightly labeled reactants each comprise a cyanine dye, or an analog thereof. In some embodiments, at least three of the brightly labeled reactants each comprise a rhodamine dye, or an analog thereof. In some embodiments, at least four of the brightly labeled reactants each comprise a cyanine dye, or an analog thereof. In some embodiments, at least four of the brightly labeled reactants each comprise a rhodamine dye, or an analog thereof. In some embodiments, three of the brightly labeled reactants each comprise a cyanine dye, or an analog thereof, and the fourth brightly labeled reactant comprises a rhodamine dye, or an analog thereof. In some embodiments, two of the brightly labeled reactants each comprise a cyanine dye, or an analog thereof, and the third and, optionally, the fourth brightly labeled reactant comprises a rhodamine dye, or an analog thereof, In some embodiments, three of the brightly labeled reactants each comprise a rhodamine dye, or an analog thereof, and the third and, optionally, the fourth brightly labeled reactant comprises a cyanine dye, or an analog thereof.

[0152] As described herein, a luminescent label or luminescent molecule is a molecule that absorbs one or more photons and then emits one or more photons after one or more time durations. The luminescence of a molecule is described by several parameters, including but not limited to luminescence emission lifetime, absorption spectrum, emission spectrum, luminescence emission quantum yield, and luminescence emission intensity. The terms absorption and excitation are used interchangeably throughout this application. In some embodiments, the terms luminescence and emission are used interchangeably. A typical luminescent molecule may absorb or be excited by light at multiple wavelengths. Excitation at one wavelength or within a certain spectral range may be relieved by a luminescence emission event, while excitation at some other wavelengths or spectral range may not be relieved by a luminescence emission event. In some embodiments, a luminescent molecule is only adequately excited for luminescence at a single wavelength or within a single spectral range. In some embodiments, a luminescent molecule is suitably excited for luminescence at two or more wavelengths or within two or more spectral ranges, hi some embodiments, a molecule is identified by measuring the wavelength of its excitation photon or its absorption spectrum.

[0153] Photons emitted from an luminescence emission event radiate at wavelengths within a spectral range of possible wavelengths. Typically, the emitted photons have a longer wavelength (e.g., less energy or red-shifted) compared to the wavelength of the excitation photon. In some embodiments, a molecule is identified by measuring the wavelength of the emitted photon. In some embodiments, a molecule is identified by measuring the wavelength of a plurality of emitted photons. In some embodiments, a molecule is identified by measuring an emission spectrum.

[0154] The luminescence emission lifetime represents the duration between an excitation event and an emission event. In some embodiments, the luminescence emission lifetime is expressed as a constant in an exponential decay equation. In some embodiments where there is one or more pulse events that deliver excitation energy, the duration is the time between the pulse and the subsequent emission event.

[0155] Determining the luminescence lifetime of a molecule may be performed using any suitable method (e.g., by measuring the lifetime using a suitable technique or by determining a time-dependent characteristic of the emission). In some embodiments, determining the luminescence lifetime of a molecule includes determining the lifetime relative to one or more molecules (e.g., different luminescence-labeled nucleoside polyphosphates in a sequencing reaction). In some embodiments, determining the luminescence lifetime of a molecule includes determining the lifetime relative to a reference. In some embodiments, determining the luminescence lifetime of a molecule includes measuring its lifetime (e.g., fluorescence lifetime). In some embodiments, determining the luminescence lifetime of a molecule includes determining one or more temporal characteristics indicative of the lifetime. In some embodiments, the luminescence emission lifetime of a molecule can be determined based on the distribution of multiple emission events occurring over one or more time-gated windows relative to an excitation pulse (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more emission events). For example, the luminescence emission lifetime of a single molecule can be distinguished from multiple molecules having different luminescence emission lifetimes based on the distribution of photon arrival times measured relative to an excitation pulse.

[0156] It should be understood that the luminescence emission lifetime of a single molecule indicates the timing of photons emitted after the single molecule reaches an excited state, and a single molecule can be distinguished by information indicating the timing of the photons. Some embodiments may include distinguishing a molecule from a plurality of molecules based on its luminescence emission lifetime by measuring the time associated with the photons emitted by the molecule. The distribution of times may provide an indication of the luminescence emission lifetime that may be determined from the distribution. In some embodiments, a single molecule can be distinguished from a plurality of molecules based on the distribution of times, such as by comparing the distribution of times to a reference distribution corresponding to known molecules. In some embodiments, a value of the luminescence emission lifetime is determined from the distribution of times.

[0157] Luminescence emission quantum yield represents the fraction of excitation events that lead to emission events at a given wavelength or within a given spectral range, and is typically less than 1. In some embodiments, the luminescence emission quantum yield of a molecule described herein is 0 to about 0.001, about 0.001 to about 0.01, about 0.01 to about 0.1, about 0.1 to about 0.5, about 0.5 to 0.9, or about 0.9 to 1. In some embodiments, a molecule is identified by determining or estimating the luminescence emission quantum yield.

[0158] As used herein for a single molecule, luminescence emission intensity represents the number of emitted photons per unit time emitted by a molecule that has been excited by the delivery of pulsed excitation energy. In some embodiments, luminescence emission intensity represents the detected number of emitted photons per unit time emitted by a molecule that has been excited by the delivery of pulsed excitation energy and detected by a particular sensor or set of sensors.

[0159] The luminescence emission lifetime, luminescence emission quantum yield, and luminescence emission intensity may be different for a given molecule under different conditions. In some embodiments, a single molecule will have an observed luminescence emission lifetime, luminescence emission quantum yield, or luminescence emission intensity that is different from a collection of molecules. In some embodiments, a molecule confined to a sample well will have an observed luminescence emission lifetime, luminescence emission quantum yield, or luminescence emission intensity that is different from a molecule that is not confined to a sample well. In some embodiments, a luminescence emission label or luminescence emission molecule that is attached to another molecule will have a luminescence emission lifetime, luminescence emission quantum yield, or luminescence emission intensity that is different from a luminescence emission label or luminescence emission molecule that is not attached to another molecule. In some embodiments, a molecule that interacts with a macromolecular complex will have a luminescence emission lifetime, luminescence emission quantum yield, or luminescence emission intensity that is different from a molecule that does not interact with a macromolecular complex.

[0160] In some embodiments, the luminescent molecules described herein absorb one photon and emit one photon after a duration. In some embodiments, the luminescent emission lifetime of a molecule can be determined or estimated by measuring the duration. In some embodiments, the luminescent emission lifetime of a molecule can be determined or estimated by measuring the duration of multiple pulse and radiation events. In some embodiments, the luminescent emission lifetime of a molecule can be distinguished between the luminescent emission lifetimes of multiple types of molecules by measuring the duration. In some embodiments, the luminescent emission lifetime of a molecule can be distinguished between the luminescent emission lifetimes of multiple types of molecules by measuring the duration of multiple pulse and radiation events. In some embodiments, a molecule is identified or distinguished between multiple types of molecules by determining or estimating the luminescent emission lifetime of the molecule. In some embodiments, a molecule is identified or distinguished between multiple types of molecules by distinguishing the luminescent emission lifetime of the molecule between the luminescent emission lifetimes of multiple types of molecules.

[0161] In some embodiments, the luminescent emission event is fluorescence. In some embodiments, the luminescent emission event is phosphorescence. As used herein, the term luminescent emission encompasses all luminescent emission events, including both fluorescence and phosphorescence.

[0162] Sequencing Some aspects of the present application are useful for sequencing biological polymers such as nucleic acids and proteins. In some embodiments, the compositions and techniques described in the present application can be used to identify a series of nucleotide or amino acid monomers incorporated into a nucleic acid or protein (e.g., by detecting the time course of incorporation of a series of labeled nucleotide or amino acid monomers). In some embodiments, the compositions and techniques described in the present application can be used to identify a series of nucleotides incorporated into a template-dependent nucleic acid sequencing reaction product synthesized by a polymerase enzyme. In some embodiments, one or more of the multiple types of luminescent labeled nucleotides (e.g., one, two, three, four, or more types of labeled nucleotides) comprise a nucleotide (e.g., a nucleoside polyphosphate) linked to a FRET label that comprises at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule. The FRET label of each type of nucleotide is distinguishable among multiple labeled nucleotides (e.g., one type of FRET-labeled nucleotide is distinguishable from all other types of FRET-labeled nucleotides and / or labels that do not undergo FRET).

[0163] Upon base pairing between the nucleobase of the target nucleic acid and the complementary nucleoside polyphosphate (e.g., dNTP), the polymerase incorporates the dNTP into the newly synthesized nucleic acid strand by forming a phosphodiester bond between the 3' hydroxyl end of the newly synthesized strand and the alpha phosphate of the dNTP. In the example where the luminescent label conjugated to the dNTP (e.g., via the linker of the present application) is a fluorophore, its presence is signaled by excitation and a pulse of emission is detected during and / or after the incorporation step. In the case of a detection label (e.g., a luminescent label) conjugated to the terminal (gamma) phosphate of the dNTP via the linker of the present application, incorporation of the dNTP into the newly synthesized strand dissociates the beta and gamma phosphates, and the linker contains the detection label which is free to nucleate in the sample well, resulting in a decrease in the emission detected from the fluorophore.

[0164] In some embodiments, the template-dependent nucleic acid sequencing reaction product is synthesized in a sequencing reaction carried out by a naturally occurring nucleic acid polymerase. In some embodiments, the polymerase is a mutant or modified variant of a naturally occurring polymerase. In some embodiments, the template-dependent nucleic acid sequencing product comprises one or more nucleotide segments that are complementary to a complementary template nucleic acid strand. In one aspect, the present application provides a method for determining the sequence of a template (or target) nucleic acid strand by determining the sequence of its complementary nucleic acid strand.

[0165] As used herein, the term "polymerase" generally refers to any enzyme (or polymerizing enzyme) that can catalyze a polymerization reaction. Examples of polymerases include, without limitation, nucleic acid polymerases, transcriptases, or ligases. A polymerase can be a polymerizing enzyme. An embodiment directed to single molecule nucleic acid extension (e.g., for nucleic acid sequencing) may use any polymerase that can synthesize a nucleic acid complementary to a target nucleic acid molecule. In some embodiments, the polymerase can be a DNA polymerase, an RNA polymerase, a reverse transcriptase, and / or one or more mutants or modified versions thereof.

[0166] Examples of polymerases include, but are not limited to, DNA polymerase, RNA polymerase, thermostable polymerase, wild-type polymerase, modified polymerase, E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase, phi29 DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum The polymerase includes Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfuturbo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerase with 3'-5' exonuclease activity, and their variants, modified products, and derivatives. In some embodiments, the polymerase is a single subunit polymerase. Non-limiting examples of DNA polymerases and their properties are described in detail in, among others, DNA Replication, 2nd Edition, Kornberg and Baker, WH Freeman, New York, NY (1991).

[0167] In some embodiments, the polymerase is a polymerase with high processivity. However, in some embodiments, the polymerase is a polymerase with reduced processivity. The processivity of a polymerase generally refers to the ability of a polymerase to continuously incorporate dNTPs into a nucleic acid template without releasing the nucleic acid template. In some embodiments, the polymerase is a polymerase with low 5'-3' exonuclease activity and / or 3'-5' exonuclease activity. In some embodiments, the polymerase is modified (e.g., by amino acid substitution) to have reduced 5'-3' exonuclease activity and / or 3'-5' activity compared to the corresponding wild-type polymerase. Further non-limiting examples of DNA polymerases include 9°Nm™ DNA polymerase (New England Biolabs) and the P680G mutant of Klenow exo-polymerase (Tuske et al., (2000), JBC, 275(31):23759-23768). In some embodiments, polymerases with reduced processivity provide improved accuracy for sequencing templates that contain one or more stretches of nucleotide repeats (e.g., two or more consecutive bases of the same type). In some embodiments, the polymerase is one that has a higher affinity for labeled nucleotides than for unlabeled nucleic acids.

[0168] In another aspect, the present application provides a method of sequencing target nucleic acid by sequencing multiple nucleic acid fragments, and the target nucleic acid comprises the fragments.In some embodiments, the method comprises combining multiple fragment sequences to provide the sequence or partial sequence of parent target nucleic acid.In some embodiments, the combining step is performed by computer hardware and software.The method described herein can allow for a set of related target nucleic acids, such as an entire chromosome or genome to be sequenced.

[0169] During sequencing, the polymerase can be coupled (e.g., attached) to a priming position of the target nucleic acid molecule. The priming position can be a primer that is complementary to a portion of the target nucleic acid molecule. Alternatively, the priming position is a gap or nick provided in the double-stranded segment of the target nucleic acid molecule. The gap or nick can be 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. The nick can provide a break in one strand of the double-stranded sequence, which can provide a priming position for the polymerase, such as, for example, a strand-displacing polymerase enzyme.

[0170] In some cases, the sequencing primer may anneal to a target nucleic acid molecule that may or may not be immobilized to a solid support. The solid support may include, for example, a sample well (e.g., nano-aperture, reaction chamber) on a chip used for nucleic acid sequencing. In some embodiments, the sequencing primer may be immobilized to a solid support, and target nucleic acid molecule hybridization also immobilizes the target nucleic acid molecule to the solid support. In some embodiments, the polymerase is immobilized to a solid support, and the soluble primer and target nucleic acid contact the polymerase. However, in some embodiments, a complex including the polymerase, the target nucleic acid, and the primer is formed in solution, and the complex is immobilized to a solid support (e.g., via immobilization of the polymerase, the primer, and / or the target nucleic acid). In some embodiments, none of the components in the sample well (e.g., nano-aperture, reaction chamber) are immobilized to a solid support. For example, in some embodiments, a complex including the polymerase, the target nucleic acid, and the primer is formed in solution, and the complex is not immobilized to a solid support.

[0171] Under suitable conditions, the polymerase enzyme contacting the annealed primer / target nucleic acid can add or incorporate one or more nucleotides onto the primer, and nucleotides can be added to the primer 5' to 3' in a template-dependent manner. The incorporation of such nucleotides onto the primer (e.g., via the action of a polymerase) can be generally referred to as a primer extension reaction. Each nucleotide can be associated with a detectable label that can be detected and identified during the nucleic acid extension reaction (e.g., based on its luminescence emission lifetime and / or other characteristics) and used to determine each nucleotide incorporated into the sequence of the extended primer and, therefore, the newly synthesized nucleic acid molecule. Through the sequence complementarity of the newly synthesized nucleic acid molecule, the sequence of the target nucleic acid molecule can also be determined. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule and the incorporation of nucleotides into the sequencing primer can occur under similar reaction conditions (e.g., the same or similar reaction temperature) or under different reaction conditions (e.g., different reaction temperatures). In some embodiments, the sequencing by synthesis method may include a step of amplifying the target nucleic acid to achieve the presence of a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and / or a population of target nucleic acids. However, in some embodiments, sequencing by synthesis is used to determine the sequence of a single molecule in each reaction being evaluated (no nucleic acid amplification is required to prepare the target template for sequencing). In some embodiments, multiple single molecule sequencing reactions are performed in parallel (e.g., on a single chip) according to multiple aspects of the present application. For example, in some embodiments, multiple single molecule sequencing reactions are each performed in separate reaction chambers (e.g., nano-apertures, sample wells) on a single chip.

[0172] Embodiments are capable of sequencing a single nucleic acid molecule species with high accuracy and long read lengths, such as at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% accuracy and / or read lengths of about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or more. In some embodiments, the target nucleic acid molecule used in single molecule sequencing is a single-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid (RNA), RNA derivatives) template that is added or immobilized to a sample well (e.g., nano-aperture) that contains at least one additional component of the sequencing reaction (e.g., a polymerase such as a DNA polymerase, a sequencing primer) immobilized or attached to a solid support such as the bottom or sidewall of the sample well. The target nucleic acid molecule or the polymerase can be attached directly or via a linker to a wall of the sample, such as the bottom or sidewall of the sample well. The sample well (e.g., nanoaperture) can also contain any other reagents necessary for nucleic acid synthesis via a primer extension reaction, such as appropriate buffers, cofactors, enzymes (e.g., polymerases), and deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates, including deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including luminescent labels, such as fluorophores, which can be linked to dNTPs via linkers of the present application.In some embodiments, each class of dNTP (e.g., adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs (e.g., dUTP), and thymine-containing dNTPs (e.g., dTTP)) is conjugated (e.g., via a linker of the present application) to a separate luminescent light-emitting label, such that detection of light emitted from the label indicates the identity of the dNTP incorporated into the newly synthesized nucleic acid. A "separate luminescent light-emitting label" may, in some embodiments, refer to a dNTP that contains a different luminescent light-emitting label (e.g., a different fluorophore) than another dNTP. In some embodiments, a separate luminescent light-emitting label refers to a dNTP that contains a different number of the same or similar luminescent light-emitting labels than another dNTP. In some embodiments, a separate luminescent light-emitting label refers to a dNTP that contains one or more light-emitting properties that are detectably different from another dNTP. The light emitted from the luminescent label is detected through any suitable device and / or method and attributed to the appropriate luminescent label (and thus to the associated dNTP).The luminescent label can be conjugated to the dNTP at any position (e.g., via the linker of the present application) so that the presence of the luminescent label does not inhibit the incorporation of the dNTP into newly synthesized nucleic acid strand or the activity of polymerase.In some embodiments, the luminescent label is conjugated to the terminal phosphate (e.g., gamma phosphate) of the dNTP (e.g., via the linker of the present application).

[0173] In some embodiments, the single-stranded target nucleic acid template can be contacted with sequencing primers, dNTPs, polymerase and other reagents required for nucleic acid synthesis. In some embodiments, all suitable dNTPs can be contacted with the single-stranded target nucleic acid template simultaneously (e.g., all dNTPs are present at the same time) so that the incorporation of dNTPs can occur continuously. In other embodiments, dNTPs can be contacted with the single-stranded target nucleic acid template sequentially, and the single-stranded target nucleic acid template is contacted with each suitable dNTP individually, with a washing step between contacting the single-stranded target nucleic acid template with different dNTPs. The cycle of contacting the single-stranded target nucleic acid template with each dNTP individually, followed by washing, can be repeated for each successive base position of the single-stranded target nucleic acid template to be identified.

[0174] In some embodiments, the sequencing primer anneals to the single-stranded target nucleic acid template, and the polymerase sequentially incorporates dNTPs (or other nucleoside polyphosphates) into the primer based on the single-stranded target nucleic acid template. The unique luminescence label associated with each incorporated dNTP can be excited with a suitable excitation light during or after incorporation of the dNTP into the primer, and the emission can then be detected using any suitable device and / or method. The detection of a specific emission of light (e.g., having a specific emission lifetime, intensity, spectrum, and / or combination thereof) is attributed to the specific dNTP incorporated. The sequence obtained from the collection of detected luminescence labels can then be used to determine the sequence of the single-stranded target nucleic acid template via sequence complementarity.

[0175] In some embodiments, the present disclosure relates to any of the following co-pending U.S. patent applications: 14 / 543,865, 14 / 543,867, 14 / 543,888, 14 / 821,656, 14 / 821,686, 14 / 821,688, 15 / 161,067, 15 / 161,088, 15 / 161,125, 15 / 255,245, 15 / 255,303, 15 / 255,624, 15 / 255,624, 15 / 255,725, 15 / 255,825, 15 / 255,926, 15 / 255,102, 15 / 255,125, 15 / 255,245, 15 / 255,303, 15 / 255,102 ...25, 15 / 255,125, 15 / 255 Nos. 15 / 261,697, 15 / 261,724, 15 / 600,979, 15 / 846,967, 15 / 847,001, 15 / 971,493, 62 / 289,019, 62 / 296,546, 62 / 310,398, 62 / 339,790, 62 / 343,997, 62 / 344,123, and 62 / 426,144.

[0176] kit In yet another aspect, the present application provides a kit for sequencing a template nucleic acid. In some embodiments, the kit comprises a plurality of types of luminescently labeled nucleotides as described herein. In some embodiments, each type of labeled nucleotide comprises two or more luminescent labels attached to one or more nucleoside polyphosphates via linkers according to the present application. In some embodiments, each type of labeled nucleotide comprises a nucleoside polyphosphate linked to a FRET label comprising at least three luminescently emitting molecules, including at least one donor molecule and at least one acceptor molecule, according to the present application. In some embodiments, the plurality of nucleotides is selected from the labeled nucleotides shown in Figures 3A-3C, 3E-3G, 4A-4C, 5A-5B, 6A-6C, and 8-10. For example, in some embodiments, the plurality of nucleotides is designed according to the structure shown in Figure 3G. In some embodiments, the plurality of nucleotides is designed according to the structure shown in Figure 3B (306), Figure 3B (308), and Figure 5A (502). In some embodiments, the plurality of nucleotides are designed according to the structures shown in Figures 11A, 11E, 12, 13A, 13B, 13C, 14, 15, and 16. In some embodiments, the kit further comprises a polymerizing enzyme (e.g., a DNA polymerase, as described elsewhere herein). In some embodiments, the kit further comprises a primer complementary to the template nucleic acid to be sequenced.

[0177] In some embodiments, the application provides a reaction mixture comprising one or more brightly labeled reactants described herein. In some embodiments, the reaction mixture comprises a mixture to be added to a sequencing reaction. In some embodiments, the reaction mixture comprises a polymerizing enzyme. In some embodiments, the polymerizing enzyme is configured to be immobilized on a solid support (e.g., at the bottom of a sample well, as described elsewhere herein). In some embodiments, the reaction mixture comprises a template nucleic acid to be sequenced. In some embodiments, the reaction mixture comprises a primer complementary to a portion of the template nucleic acid. In some embodiments, the reaction mixture comprises one or more components necessary to initiate a sequencing reaction (e.g., a divalent metal ion such as magnesium or iron). In some embodiments, the reaction mixture comprises one or more components necessary to stabilize a sequencing reaction (e.g., one or more buffering agents, one or more reducing agents, etc.).

[0178] FRET Nucleic Acid Linker In some embodiments, the present application provides a labeled nucleotide comprising a nucleotide (e.g., a nucleoside polyphosphate) linked to at least two luminescent labels or molecules comprising a FRET pair via a nucleic acid linker, as described herein. In some embodiments, the at least two luminescent labels or molecules comprise at least one donor label (also referred to herein as a donor molecule) and at least one acceptor label (also referred to herein as an acceptor molecule), and the emission spectrum of the donor label overlaps with the absorption spectrum of the acceptor label to form a FRET pair. The term "FRET label" refers to a luminescent label comprising at least one donor molecule and at least one acceptor molecule that can undergo FRET. In some embodiments, a nucleic acid linker comprising a FRET pair or a FRET label can be referred to herein as a FRET nucleic acid linker.

[0179] In some embodiments, the FRET nucleic acid linker comprises one donor label and one acceptor label. In some embodiments, the FRET nucleic acid linker comprises one donor label and more than one acceptor label (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more acceptor labels). In some embodiments, the FRET nucleic acid linker comprises more than one donor label (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more donor labels) and one acceptor label. In some embodiments, the FRET nucleic acid linker comprises at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule. In some embodiments, the donor molecule to acceptor molecule ratio is 1:1. In some embodiments, the donor molecule to acceptor molecule ratio is greater than 1:1 (e.g., 1.5:1, 2:1, 3:1, 3.5:1, 4:1, or more). In some embodiments, a donor molecule to acceptor molecule ratio of greater than 1:1 leads to increased brightness of the FRET label compared to that observed with a 1:1 ratio pair. In some embodiments, the improvement is greater than 5%, greater than 10%, greater than 15%, greater than 20%, or more. In some embodiments, the improvement is about 5-10%, 10-15%, 15-20%, 20%-25%, or more. In some embodiments, the acceptor molecule to donor molecule ratio is greater than 1:1 (e.g., 1.5:1, 2:1, 3:1, 3.5:1, 4:1, or more). In some embodiments, an acceptor molecule to donor molecule ratio of greater than 1:1 leads to improved FRET efficiency compared to that observed with a 1:1 ratio pair. In some embodiments, the improvement is greater than 5%, greater than 10%, greater than 15%, greater than 20%, or more. In some embodiments, the improvement is about 5-10%, 10-15%, 15-20%, 20%-25%, or more.

[0180] In some embodiments, the FRET nucleic acid linker comprises at least two donor molecules and at least one acceptor molecule. In some embodiments, the FRET nucleic acid linker comprises two donor molecules and one acceptor molecule. In some embodiments, the FRET nucleic acid linker comprises three donor molecules and one acceptor molecule. In some embodiments, the FRET nucleic acid linker comprises four donor molecules and one acceptor molecule. In some embodiments, the FRET nucleic acid linker comprises two donor molecules and two acceptor molecules. In some embodiments, increasing the number of donor molecules relative to the number of acceptor molecules leads to improved brightness of the FRET label compared to the brightness observed for a 1:1 ratio pair. In some embodiments, the improvement is greater than 5%, greater than 10%, greater than 15%, greater than 20%, or more. In some embodiments, the improvement is about 5-10%, 10-15%, 15-20%, 20%-25%, or more. In some embodiments, the FRET nucleic acid linker comprises at least two acceptor molecules and at least one donor molecule. In some embodiments, the FRET nucleic acid linker comprises two acceptor molecules and one donor molecule. In some embodiments, the FRET nucleic acid linker comprises three acceptor molecules and one donor molecule. In some embodiments, the FRET nucleic acid linker comprises four acceptor molecules and one donor molecule. In some embodiments, increasing the number of acceptor molecules relative to the number of donor molecules leads to improved FRET efficiency compared to the FRET efficiency observed with a 1:1 ratio pair. In some embodiments, the improvement is greater than 5%, greater than 10%, greater than 15%, greater than 20%, or more. In some embodiments, the improvement is about 5-10%, 10-15%, 15-20%, 20%-25%, or more.

[0181] In some embodiments, the FRET nucleic acid linker comprises one or more stem loops (e.g., 1, 2, 3, 4, 5, or more stem loops). In some embodiments, the FRET nucleic acid linker comprises one or more stem loops, and at least one of the donor and / or acceptor labels or molecules is / are located within the loop of the stem loop structure. In some embodiments, each of the donor molecules is located in the loop of the stem loop structure. In some embodiments, each of the donor molecules and the acceptor molecules is located in the loop of the stem loop structure. In some embodiments, the one or more acceptor molecules are located at the junction where two or more stem loops meet. In some embodiments, the FRET nucleic acid linker comprises two or more stem loops. In some embodiments, the FRET nucleic acid linker comprises three or more stem loops. In some embodiments, the FRET nucleic acid linker comprises four or more stem loops.

[0182] In some embodiments, the FRET nucleic acid linker is a linear linker. In some embodiments, the FRET nucleic acid linker is a branched linker. In some embodiments, the FRET nucleic acid linker has a three-way DNA junction.

[0183] In some embodiments, the nucleic acid comprises a first oligonucleotide strand and a second oligonucleotide that hybridizes to the first oligonucleotide strand. In some embodiments, the donor molecule and the acceptor molecule are arranged on the same strand. In some embodiments, the acceptor molecule is arranged on a different strand than the donor molecule. In some embodiments, one or more donor molecules and one or more acceptor molecules are arranged on the same strand or on different strands.

[0184] In some embodiments, one or more acceptor molecules are 5' to one or more donor molecules. In some embodiments, one or more acceptor molecules are 3' to one or more donor molecules. In some embodiments, an acceptor molecule may be between two donor molecules.

[0185] In some embodiments, one or more donor molecules (eg, 1, 2, 3, 4, 5 or more) and / or one or more acceptor molecules (eg, 1, 2, 3, 4, 5 or more) are incorporated into a nucleic acid linker.

[0186] In some embodiments, the donor and acceptor molecules are separated by 3 bases (or base pairs or nucleotides). In some embodiments, the donor and acceptor molecules are separated by 4 bases. In some embodiments, the donor and acceptor molecules are separated by 5 bases. In some embodiments, the donor and acceptor molecules are separated by 6 bases. In some embodiments, the donor and acceptor molecules are separated by 7 bases. In some embodiments, the donor and acceptor molecules are separated by 8 bases. In some embodiments, the donor and acceptor molecules are separated by 9 bases. In some embodiments, the donor and acceptor molecules are separated by 10 bases. In some embodiments, the donor and acceptor molecules are separated by 11 bases. In some embodiments, the donor and acceptor molecules are separated by 12 bases. In some embodiments, the donor and acceptor molecules are separated by 13 bases. In some embodiments, the donor and acceptor molecules are separated by more than 3 bases. In some embodiments, the donor and acceptor molecules are separated by 4 or more bases. In some embodiments, the donor and acceptor molecules are separated by 5 or more bases. In some embodiments, the donor and acceptor molecules are separated by 6 or more bases. In some embodiments, the donor and acceptor molecules are separated by 3-4, 4-5, 5-6, 3-6, 3-10, or 5-10 bases. In some embodiments, each of the donor-acceptor molecule pairs is separated by the same number of bases. In some embodiments, different donor-acceptor pairs are separated by the same or different numbers of bases. In general, a longer distance may work for donor and acceptor molecule pairs with greater overlap between the emission spectrum of the donor molecule and the absorption spectrum of the acceptor label.

[0187] In some embodiments, the distance between the donor molecule and the acceptor molecule is 1-3 nm. In some embodiments, the distance between the donor molecule and the acceptor molecule is 1-4 nm, 1-5 nm, 1-6 nm, 1-7 nm, 1-8 nm, 1-9 nm, or 1-10 nm. In some embodiments, the distance between the donor molecule and the acceptor molecule is 2-4 nm, 2-5 nm, 2-8 nm, or 2-10 nm. In some embodiments, the distance between the donor molecule and the acceptor molecule is 3-5 nm, 3-8 nm, or 3-10 nm. In general, a longer distance can work with donor and acceptor molecule pairs with greater overlap between the emission spectrum of the donor molecule and the absorption spectrum of the acceptor label.

[0188] In some embodiments, the FRET efficiency between the donor-acceptor molecule pair ranges from about 65% to about 100%. In some embodiments, the FRET efficiency between the donor-acceptor molecule pair is about 75% to about 95%. In some embodiments, the FRET efficiency between the donor-acceptor molecule pair is about 80% to about 90%. In some embodiments, the FRET efficiency between the donor-acceptor molecule pair is about 85%. In some embodiments, the FRET efficiency between the donor-acceptor molecule pair is greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, or greater than about 90%.

[0189] In some embodiments, FRET nucleic acid linker comprises DNA, RNA, PNA, LNA or their derivatives.In some embodiments, FRET nucleic acid linker comprises LNA.Without wishing to be bound by theory, GC pair quenches signal, so replacing GC pair with LNA leads to improved brightness and / or efficiency.

[0190] Exemplary configurations of nucleic acid linkers and results of sequencing experiments using labeled nucleotides containing FRET nucleic acid linkers are shown in Figures 11A-11F and 12-17. In some embodiments, the FRET nucleic acid linkers of the present application exhibit a large Stokes shift. For example, in some embodiments, an excitation wavelength of 532 nm for the donor label and an emission wavelength of 590 nm for the acceptor label provide a large Stokes shift that enhances detection with a spectral filter of approximately 570 nm. In some embodiments, the FRET nucleic acid linkers of the present application aid in the exclusion of lasers from redder emission and / or redder spectral filters. In some embodiments, the FRET nucleic acid linkers of the present application allow the use of bluer (e.g., 457, 488, 515 nm) excitation wavelengths.

[0191] Thus, in some embodiments, a FRET nucleic acid linker may be combined with one or more other linkers or labeled nucleotides described in this application. Working Example Example 1: Strategies for attaching dyes to nucleic acid linkers Various next generation sequencing technologies implement labeled reaction components. For example, dye-labeled nucleotides can be used to make specific base calls during incorporation events based on the detection or observation of unique emission properties corresponding to each base type. These properties, such as lifetime and intensity, therefore need to be easily identifiable for each base in the set. Initial efforts at enhancing the fluorescence intensity of labeled nucleotides revealed that the brightness of dye-labeled nucleotides increased when a second dye molecule was added to the construct. However, the fluorescence lifetime was significantly reduced compared to the single-labeled variant. In developing improved labeled nucleotides, nucleic acids were investigated as a core structure for linking fluorescent dyes to nucleotides.

[0192] One potential source of change in the fluorescence lifetime of multiply labeled nucleotides is the degree of interaction between dye molecules of the same construct, which results in a quenching effect. This possibility was further explored by generating dye-labeled nucleotides shown in Figure 8. Two dye molecules (DyLight530R2) were linked to a nucleoside polyphosphate via a nucleic acid linker. An unlabeled oligonucleotide strand was hybridized to a labeled oligonucleotide strand, providing rigidity to the nucleic acid linker. A first construct 800 was created using a C6-amino-T spacer for dye attachment to the nucleic acid, and a second construct 802 was created using a glycolamine spacer for dye attachment.

[0193] Analysis of the first construct revealed a fluorescence lifetime of approximately 1.4 ns, while the second construct exhibited a lifetime of approximately 3.5 ns. The increased lifetime measured in the second construct was attributed to the use of a shorter spacer for dye attachment compared to the first construct. As shown in FIG. 8, the C6-amino-T spacer in the first construct is longer than the glycolamine spacer in the second construct. One possible explanation for the improved lifetime of the second construct is that the shorter spacer length reduces dye-dye interactions by limiting the extent to which the motional ranges of the attached dyes overlap.

[0194] Example 2: Increased linker rigidity extends fluorescence lifetime Following the observation that spacer length can affect fluorescence lifetime, potentially due to spatial overlap of the dyes, it was thought that symmetrical arrangement of the dyes within the multi-labeled molecule could have a similar effect. A Y-shaped nucleic acid linker was generated and is shown in FIG. 9. The initial construct has three oligonucleotide strands covalently linked via the branched linker shown. Two strands are each end-attached to a dye molecule (Chromis530N) and the third strand is end-attached to a nucleoside polyphosphate. The third strand is further hybridized to an unlabeled strand to provide rigidity between the nucleoside polyphosphate and the labeled region. A second version of this construct is generated by hybridization with oligonucleotide component 902, which hybridizes to the first and second strands.

[0195] Analysis of the original construct revealed a fluorescence lifetime of approximately 2.3 ns, while the construct with the additional oligonucleotide moiety 902 exhibited a lifetime of approximately 4.2 ns. One possible explanation for the measured increase in lifetime in the latter construct is that oligonucleotide moiety 902 imparts rigidity to the labeled region. The increased rigidity would likely facilitate dye separation by constraining each dye to a more limited range of motion.

[0196] Example 3: Effect of dye spacer length in constrained linker configurations The geometrically constrained linker configuration was further developed by generating the Tris-dye labeled construct shown in FIG. 10. As shown, the nucleic acid linker moiety includes three main oligonucleotide components. The first component includes four oligonucleotide strands that are covalently attached via a four-way branched linker as shown. Three of these strands are each terminally attached to a dye molecule (AttoRho6G), and the fourth strand hybridizes to a second oligonucleotide component. The second oligonucleotide component is terminally attached to two nucleoside polyphosphates via a branched linker as shown. A third oligonucleotide component, including three oligonucleotide strands covalently linked via a branched linker as shown, hybridizes to the three dye-labeled strands of the first component to provide rigidity to the labeled region. Two separate Tris-dye constructs with different spacer lengths were generated according to the boxed regions shown in FIG. 10.

[0197] The first Tris-dye labeled nucleotide construct with a longer spacer (see FIG. 10, boxed area, top) showed a three-fold increase in fluorescence intensity compared to the one dye labeled nucleotide construct. In addition, it showed a slight decrease in lifetime when compared to the two dye one nucleotide construct with the same dye molecule (not shown). Measurements obtained with the second Tris-dye labeled nucleotide construct with a shorter spacer (see FIG. 10, boxed area, bottom) showed a slight increase in fluorescence lifetime compared to the first Tris-dye labeled construct.

[0198] Previously, Tris-dye labeled nucleotides produced multiple lifetimes during sequencing reactions, which were believed to be the result of two dyes interacting to produce the first lifetime and a non-interacting dye producing the second lifetime. Importantly, only a single lifetime was observed for any of the Tris-dye labeled molecules shown in Figure 10 during sequencing reactions.

[0199] Example 4: Characterization of FRET Nucleic Acid Linkers Figure 12 shows an example of a FRET nucleic acid linker structure with a linear nucleic acid having a central acceptor molecule (A) and two donor molecules (D) on either side. In this example, each donor molecule is 9 bases away from the acceptor molecule. The donor molecule-acceptor molecule distances for the two are similar, but not necessarily identical. A FRET efficiency of 83% was observed.

[0200] We developed FRET nucleic acid linkers with various numbers of AT and GC pairs between and around the donor and acceptor molecules. These FRET nucleic acid linkers are shown in Figures 13A-13C. ATRho6G was modified to generate a shorter, more rigid molecule, ATRho6G-C3. Specifically, the tertiary amide side chain (carboxybutyl) of ATRho6G was changed to azidopropyl. ATRho6G-C3 allowed the donor-acceptor distance to be reduced by one base pair (Figure 13A). The use of an internal cyanine dye and / or replacement of the abasic amino conjugation site with a BODIPY dye conjugated to C2-amino-T, a modifier that does not disrupt the double-stranded structure, also allowed the donor-acceptor distance to be reduced (Figure 13B). As shown in Figure 13B, a FRET efficiency of 90% was observed with the cyanine-BODIPY FRET pair. An additional FRET nucleic acid linker with a 6-base pair spacer is shown in Figure 13C.

[0201] A systematic study of how the relative positions of the donor and acceptor molecules affect FRET efficiency and brightness was performed for the ATRho6GC3-BODIPY pair, showing that the optimal position of the acceptor molecule is 5 bases away from the donor molecule, with a FRET efficiency of 90% (Figure 14).

[0202] Next, we tested the effect of the distance between the luminescent molecule and the labeled nucleotide (or polymerase-template complex) on the sequencing signal intensity. Luminescent molecules located more than 15-16 nm away from the labeled nucleotide (or polymerase-template complex) were not efficiently irradiated. The distance between the luminescent molecule and the labeled nucleotide in a linear structure compared to a three-way DNA junction structure is shown in Figure 15.

[0203] The effect of the number of acceptor molecules on FRET efficiency was also tested. The FRET efficiency of Cy3-Cy5 pairs in either a 1-donor-1-acceptor or 1-donor-2-acceptor configuration was compared. When additional acceptor molecules were added, the FRET efficiency improved from about 60-70% to about 90% (Figure 16). Thus, using multiple copies of the acceptor to emit donor emission results in more efficient energy transfer.

[0204] FIG. 17 provides a summary of the FRET efficiencies obtained for different donor and acceptor molecule pairs, ratios, and orientations. Equivalence and Scope Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such change and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific inventive embodiments described herein. Thus, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0205] All definitions defined and used herein should be understood to be controlling of dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0206] 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 include the entire document.

[0207] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0208] As used herein and in the claims, the term "and / or" should be understood to mean "either or both" of the elements so connected, i.e., conjunctively present in some cases and non-conjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., as "one or more" of the elements so connected. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used with open-ended language such as "comprising", may in one embodiment refer to only A (optionally including elements other than B); in another embodiment, it may refer to only B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so forth.

[0209] When used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of a number or list of elements, but including two or more of a number or list of elements, and optionally additional unlisted items. "Only one of" or "exactly one of," or "consisting of," and the like, as used in the claims, refers to the inclusion of exactly one element of a number or list of elements only if clearly indicated to the contrary. In general, when used herein, the term "or" when preceded by a term of exclusivity, such as "either," "one of," or "exactly one of," shall be interpreted only as indicating exclusive alternative terms (i.e., "one or the other, but not both"). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0210] As used in this specification and the claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each of every element specifically listed in the list of elements, and not excluding any combinations within the list of elements. This definition also allows for the optional presence of elements other than those specifically identified elements in the list of elements that the phrase "at least one" refers to, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can represent, in one embodiment, at least one A, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment, at least one B, optionally more than one B, and no A (and optionally including elements other than A); in yet another embodiment, at least one A, optionally more than one A, and at least one B, optionally more than one B (and optionally including other elements), etc.

[0211] It is also to be understood that, unless specifically stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0212] In the claims and the above specification, 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 intended to be closed or semi-closed, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. It should be understood that embodiments described in this document using open-ended transitional phrases (e.g., "comprising") also contemplate, in alternative embodiments, "consisting of" and "consisting essentially of" the feature described by that open-ended transitional phrase. For example, if the present disclosure describes "a composition comprising A and B," the present disclosure also contemplates alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. A labeled nucleotide comprising a nucleoside polyphosphate linked to a nucleic acid comprising a FRET label comprising at least three luminescent molecules, the FRET label comprising at least one donor molecule and at least one acceptor molecule, The FRET labels are labeled nucleotides having a donor molecule to acceptor molecule ratio of greater than 1:1 or an acceptor molecule to donor molecule ratio of greater than 1:1, and each luminescent molecule is attached to the nucleic acid at an attachment site of the nucleic acid that is at least two bases away from a guanine or cytosine on the nucleic acid.

2. The labeled nucleotide of claim 1, wherein the FRET label has a donor molecule to acceptor molecule ratio of greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, or 4:1, or an acceptor molecule to donor molecule ratio of greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, or 4:

1.

3. A labeled nucleotide described in claim 1 or 2, wherein the FRET label comprises at least two donor molecules and at least one acceptor molecule, or at least one donor molecule and at least two acceptor molecules.

4. The nucleic acid comprises one or more stem-loop structures, Optionally, at least one donor molecule or at least one acceptor molecule is located in a loop region of a stem-loop structure.

5. The labeled nucleotide of claim 4, wherein each stem-loop structure comprises a loop region including at least four unpaired bases, and / or wherein the loop region of each stem-loop structure comprises a sequence having a G / C content of less than 33%.

6. A labeled nucleotide described in any one of claims 1 to 5, wherein each donor molecule and acceptor molecule are separated by about 1 to 3 nm, or each donor molecule and acceptor molecule are separated by 3 to 13 bases.

7. A labeled nucleotide described in any one of claims 1 to 6, wherein each donor molecule and acceptor molecule has a FRET efficiency of at least 85%.

8. The nucleic acid comprises a first oligonucleotide strand and a second oligonucleotide strand hybridized to the first oligonucleotide strand, and optionally, at least one luminescent molecule is on a different strand from other luminescent molecules. A labeled nucleotide according to any one of claims 1 to 7.

9. A labeled nucleotide according to any one of claims 1 to 8, wherein one or more luminescent molecules are incorporated within the nucleic acid.

10. The labeled nucleotide of any one of claims 1 to 9, wherein the nucleic acid comprises fewer than 150, fewer than 100, or fewer than 50 bases.

11. A labeled nucleotide according to any one of claims 1 to 10, wherein one or more luminescent molecules are attached to the nucleic acid at an attachment site that is at least 5 bases away from any other attachment site.

12. The labeled nucleotide of claim 1, wherein at least one luminescent molecule is attached to an abasic site on the nucleic acid.

13. 13. The labeled nucleotide of any one of claims 1 to 12, wherein the luminescent molecule is a fluorescent dye, optionally wherein the fluorescent dye is a rhodamine dye, a BODIPY dye, or a cyanine dye.

14. A labeled nucleotide according to any one of claims 1 to 13, wherein the nucleotide is approximately 1 to 10 nm away from any of the luminescent molecules.

15. A method for determining a sequence of a template nucleic acid, the method comprising: (i) exposing a complex in a target area to one or more different types of luminescently labeled nucleotides, the complex comprising the template nucleic acid, a primer, and a polymerizing enzyme, each type of luminescently labeled nucleotide comprising a labeled nucleotide according to any one of claims 1 to 14; (ii) directing a series of one or more pulses of excitation energy proximate the target area; (iii) detecting a plurality of emitted photons from the luminescently labeled nucleotides during their successive incorporation into the nucleic acid containing the primer; and (iv) identifying the sequence of the incorporated nucleotides by determining at least one of a luminescence emission intensity and a luminescence emission lifetime based on the emitted photons.

16. A kit for sequencing a template nucleic acid, the kit comprising: A kit comprising two or more different types of luminescently labeled nucleotides, each type of luminescently labeled nucleotide comprising a labeled nucleotide according to any one of claims 1 to 14.

17. A nucleic acid sequencing reaction composition comprising two or more different types of luminescently labeled nucleotides in a reaction mixture, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide described in any one of claims 1 to 14.

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