Methods and Compositions for Nucleic Acid Sequencing Using Photo-Switchable Tags

Abstract

Embodiments of the present disclosure relate to nucleotides labeled with a photo-switchable compound. Also provided herein are methods and kits for using these labeled nucleotides for sequencing applications.

Description

Background Technical Field

[0001] The present disclosure generally relates to nucleotides labeled with photoswitchable markers and their use in polynucleotide sequencing methods and applications.

[0002] The detection of analytes (such as nucleic acid sequences) present in biological samples has been used as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying the genetic susceptibility to diseases, and measuring the response to various types of treatments. A common technique for detecting analytes such as nucleic acid sequences in biological samples is nucleic acid sequencing.

[0003] Clearly, the need for high-throughput, smaller, and cheaper DNA sequencing technologies will facilitate the benefits of genomic sequencing. Personalized healthcare is moving towards the forefront and will benefit from such technologies; sequencing an individual's genome to identify potential mutations and abnormalities is crucial for determining whether a person has a specific disease and subsequent treatment methods tailored to that person. To accommodate this strong effort, sequencing should move forward and conform to high-throughput technologies, not only having high-throughput capabilities, but also taking into account ease of use, time and cost efficiency, and clinicians' access to instruments and reagents.

[0004] Photoswitchable fluorescent probes are an important new development in super-resolution fluorescence microscopy technology, in which dyes that can be switched from a dark state to a luminescent state are required to obtain higher spatial resolution. See Vaughan et al., FEBS Letters 588 (2014) 3603–3612. Photoswitchable fluorescent probes utilize photochromic compounds that can be reversibly switched between states with different absorption spectra when irradiated with light of appropriate wavelength and intensity. The photogenerated species is usually restored to the starting species by thermodynamic methods or further illumination. For example, Anderson et al. recently reported the synthesis of a small molecule dyad composed of a red-emitting fluorophore covalently linked to a photoswitchable quencher and used this dyad for bioimaging. See Anderson et al., Org. Lett. 2016, 18, 3666-3669. In similar work, Bossi et al. reported the synthesis of a dyad with a coumarin fluorophore and an oxazine photochromic agent, which was conjugated to an antibody for super-resolution imaging. See Bossi et al., J. Phys. Chem. C 2016, 120, 12860-12870.

[0005] There is a need to develop new photoswitchable fluorescent labels that can be used in bioimaging applications, including next-generation sequencing applications. SUMMARY OF THE INVENTION

[0006] Some embodiments of the present disclosure relate to nucleotide conjugates comprising a photoswitchable label, the photoswitchable label comprising a fluorescent moiety optionally covalently bonded to a photochromic moiety via a linking group. Upon irradiation with a light source such as laser or LED light, the photoswitchable label can undergo a reversible change from an "on" state to an "off" state and vice versa. In some embodiments, the fluorescent moiety of the nucleotide conjugate can be a red-emitting fluorophore, such as silicon rhodamine. In some embodiments, the photochromic moiety of the photoswitchable label comprises a spiropyran or spirothiopyrano moiety having the structure of formula (I):

[0007]

[0008] wherein X is O (oxygen) or S (sulfur);

[0009] Each R 1a and R 2a are independently selected from: H, halogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy;

[0010] R 3 is selected from H, C 1-6 alkyl and –(CH2) n -R 4 ;

[0011] R 4 is selected from C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic group and 3- to 7-membered heterocyclic group, each of which is optionally substituted;

[0012] Ring A is C 6-10 aryl or 5- to 10-membered heteroaryl, each of which is substituted with at least one electron-withdrawing group; and

[0013] n is an integer from 1 to 6.

[0014] In some other embodiments, the photochromic moiety of the photoswitchable label comprises a structure of formula (II):

[0015]

[0016] wherein Y is O (oxygen) or S (sulfur);

[0017] Each R 1b and R 2b are independently selected from: H, halogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy; and

[0018] Ring B is C 6-10 aryl or a 5- to 10-membered heteroaryl; each of which is substituted with at least one electron-withdrawing group; and wherein * represents the point of attachment to the fluorophore moiety.

[0019] Some embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the nucleotide conjugates described herein.

[0020] Some further embodiments of the present disclosure relate to methods for determining the nucleotide sequence of a target polynucleotide, which include performing a sequencing reaction comprising one or more of the following cycles:

[0021] (i) Incorporating different types of nucleotide conjugates into a plurality of polynucleotides complementary to the target polynucleotide to produce extended polynucleotides, wherein each first type of nucleotide conjugate comprises a first photoswitchable label, each second type of nucleotide conjugate comprises a second photoswitchable label, and each third type of nucleotide conjugate comprises a third label;

[0022] (ii) Detecting a first set of signals from the extended polynucleotides in each cycle via a first imaging event;

[0023] (iii) Irradiating the extended polynucleotides with a light source after the first imaging event to cause a change in the emission signals of the first photoswitchable label and the second photoswitchable label;

[0024] (iv) Detecting a second set of signals from the extended polynucleotides via a second imaging event; and

[0025] Determining the sequence of a target polynucleotide based on sequentially incorporated nucleotide conjugates. In some embodiments, four different types of nucleotide conjugates are present simultaneously and compete for incorporation into a polynucleotide complementary to the target polynucleotide during each cycle of incorporation. In some such embodiments, incorporation of the first type of nucleotide conjugate is determined from the signal state in the first imaging event and the dark state in the second imaging event. In some such embodiments, incorporation of the second type of nucleotide conjugate is determined from the dark state in the first imaging event and the signal state in the second imaging event. In some such embodiments, incorporation of the third type of nucleotide conjugate is determined from the signal states in the first and second imaging events. In some such embodiments, incorporation of the fourth type of nucleotide conjugate is determined from the dark states in the first and second imaging events. In some further embodiments, the first type of nucleotide conjugate comprises a photochromic moiety of formula (I), and the second type of nucleotide conjugate comprises a photochromic moiety of formula (II).

[0026] Some further embodiments of the present disclosure relate to kits comprising one or more nucleotide conjugates with photo-switchable labels as described herein. The kits can be used in applications such as sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cell assays (e.g., cell binding or cell function analysis) or protein assays (e.g., protein binding assay or protein activity assay). It can be used on an automated instrument for performing a particular technique, such as an automated sequencing instrument. The sequencing instrument may only contain one laser to distinguish different detectable labels. The sequencing instrument can comprise additional lasers or light sources operating at different wavelengths for activating the photo-switchable label from an "on" state to an "off" state and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A Shows a flow chart of a standard Illumina single-channel sequencing-by-synthesis (SBS) chemistry.

[0028] Figure 1B Shows how images 1 and 2 from standard single-channel SBS are processed by image analysis software to identify which bases have been incorporated.

[0029] Figure 2 Shows a flow chart of a single-channel sequencing process using the photo-switchable dyes disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0031] Illumina's next-generation sequencing system, the iSeq 100TM Use CMOS-based technology to provide a simplified and accessible benchtop sequencing solution. The standard sequencing workflow is shown in Figure 1A and 1B and is also referred to as 1-channel sequencing. Each sequencing cycle includes two chemical steps and two imaging steps. In Figure 1A , the first chemical step exposes the flow cell to a mixture of nucleotides with fluorescently labeled adenine and thymine. In the first imaging step, the luminescence of each cluster is recorded by a CMOS sensor. The second chemical step removes the fluorescent label from adenine and adds a fluorescent label to cytosine. Guanine is dark (unlabeled) in both chemical steps. The second imaging is recorded. In Figure 1B , the combination of Image 1 and Image 2 is processed by image analysis software to identify which bases are incorporated at each cluster position. The sequencing cycle is repeated "n" times to produce a read length of "n" bases. Different from the four-channel SBS chemistry where the sequencer uses different dyes for each nucleotide, the iSeq 100 TM system uses one dye in each sequencing cycle. In single-channel chemistry, adenine has a removable label and is labeled only in the first imaging. Cytosine has a linking group that can bind a label and is labeled only in the second imaging. Thymine has a permanent fluorescent label and is therefore labeled in both imagings, and guanine is permanently dark. The nucleotides are identified by analyzing the different emission patterns of each base in the two images.

[0032] Embodiments of the present disclosure relate to new methods for determining the nucleotide sequence of a target polynucleotide. In particular, the method involves using nucleotides labeled with a photo-switchable dye that is capable of undergoing a photo-induced color change when irradiated with a light source.

[0033] There are several advantages to using photo-switchable fluorophores. The photo-switchable dyes do not need to exhibit switching stability since they are only needed for a single imaging event and are cleaved immediately thereafter. These dyes are known to be able to switch multiple times without showing any fatigue resistance. The method also eliminates the use of dedicated cleavage mixtures or exchange reagents since the conversion of the emission state of the dye is induced "remotely" by photon energy. When the switching occurs in the time range of sub-microseconds, the total cycle time should also be minimized, which will greatly reduce the time required to pump reagents in and out of the flow cell. The irradiation-based conversion typically has a high yield, which is an essential feature to avoid mixed signals or suppressed emission.

[0034] In addition, the ability to control the luminescence state of fluorescent labels is very useful in nucleic acid sequencing applications. For example, a commonly observed problem with many similar tags is that after they are successfully used in processing steps, residual emission can lead to harmful background signals. Remotely inducing changes in the emission curve based on isomerization, conformational changes, or reversible ring-opening processes offers the possibility of improving the signal-to-noise ratio.

[0035] In some embodiments, the method may include the following steps:

[0036] (a) Performing a sequencing reaction comprising the following repeating cycles:

[0037] (i) Incorporating four different types of nucleotide conjugates into a plurality of polynucleotides complementary to a target polynucleotide to produce extended polynucleotides, wherein each nucleotide conjugate of the first type comprises a first photo-switchable label, each nucleotide conjugate of the second type comprises a second photo-switchable label, and each nucleotide conjugate of the third type comprises a third label;

[0038] (ii) Detecting a first set of signals from the extended polynucleotides in each cycle via a first imaging event;

[0039] (iii) Irradiating the extended polynucleotides with a light source after the first imaging event to cause a change in the emission signals of the first photo-switchable label and the second photo-switchable label;

[0040] (iv) Detecting a second set of signals from the extended polynucleotides via a second imaging event;

[0041] (b) Determining the sequence of the target polynucleotide based on the sequentially incorporated nucleotide conjugates.

[0042] In some embodiments, the incorporation of the nucleotide conjugate of the first type is determined based on the signal state in the first imaging event and the dark state in the second imaging event; the incorporation of the nucleotide conjugate of the second type is determined based on the dark state in the first imaging event and the signal state in the second imaging event; the incorporation of the nucleotide conjugate of the third type is determined based on the signal states in the first imaging event and the second imaging event; and the incorporation of the nucleotide conjugate of the fourth type is determined based on the dark states in the first imaging event and the second imaging event.

[0043] When using "signal state" in connection with a detection event, it refers to the condition in which a specific signal is generated in the detection event. For example, a nucleotide subunit can be in a signal state and detectable when linked to a fluorescent label, and in a sequencing method, the fluorescent label is detected in a fluorescence detection step by excitation and emission of the fluorescent label. When using the term "dark state" in connection with a detection event, it refers to the condition in which no specific signal is generated in the detection event. For example, when a nucleotide lacks a fluorescent label and / or does not emit fluorescence specific for detection in a fluorescence detection step of a sequencing method, the nucleotide subunit can be in a dark state. Dark state detection can also include any background fluorescence that may be present in the absence of a fluorescent label. For example, some reaction components may exhibit minimal fluorescence when excited at some wavelengths. Thus, even in the absence of a fluorescent moiety, there may be background fluorescence from such components. Additionally, background fluorescence may be attributable to light scattering, such as light scattering from an adjacent sequencing reaction, which can be detected by a detector. Thus, a "dark state" can include such background fluorescence, in cases where a fluorescent moiety is not specifically included, such as when using nucleotides lacking a fluorescent label in the methods described herein. However, such background fluorescence is expected to be distinguishable from the signal state and thus the nucleotide incorporation of an unlabeled nucleotide (or "dark" nucleotide) can still be discerned.

[0044] In some embodiments of the methods described herein, step (a) is repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 times. In some embodiments, the four different types of nucleotide conjugates are present simultaneously and compete for incorporation in each cycle. In some further embodiments, the incorporation of the nucleotide conjugates is effected by a polymerase.

[0045] In some embodiments, the different types of nucleotide conjugates comprise reversible terminator moieties. In some further embodiments, step (a) further comprises cleaving the reversible terminator moiety from the incorporated nucleotide conjugate prior to the next incorporation cycle. In some further embodiments, the nucleotide conjugates comprise nucleotide types selected from: dATP, dTTP, dUTP, dCTP, dGTP, and their unnatural nucleotide analogs.

[0046] In some embodiments of the methods described herein, the labels of the first type of nucleotide conjugate and the third type of nucleotide conjugate can comprise the same fluorescent moiety. In another embodiment, the labels of the second type of nucleotide conjugate and the third type of nucleotide conjugate can comprise the same fluorescent moiety. In other embodiments, the labels of the first type of nucleotide conjugate, the second type of nucleotide conjugate, and the third type of nucleotide conjugate can comprise different fluorescent moieties. The same emission filter or different emission filters can be used to detect the different fluorescent moieties. In some embodiments, the fourth type of nucleotide conjugate is not labeled with a fluorescent moiety. In some embodiments of the methods described herein, the irradiation in step (a)(iii) does not change or substantially does not change the signal detected from the third label of the third nucleotide conjugate.

[0047] In some embodiments, the irradiation source in step (a)(iii) can comprise a laser, a light emitting diode (LED), or a combination thereof. In some embodiments, the irradiation source in step (a)(iii) has a wavelength different from the excitation wavelength used in the first imaging event. In some such embodiments, the irradiation source in step (a)(iii) can have a wavelength of about 350 nm to about 450 nm. In one embodiment, the irradiation source in step (a)(iii) has a wavelength of about 405 nm.

[0048] In some embodiments of the methods described herein, the first imaging event and the second imaging event have the same or substantially the same excitation wavelength. In such embodiments, one detection channel can be used to detect the first, second, and third labels. In some such embodiments, the first imaging event and the second imaging event can have an excitation wavelength of about 550 nm to about 650 nm. In one embodiment, the first imaging event and the second imaging event have an excitation wavelength of about 633 nm.

[0049] First optically switchable label

[0050] In some embodiments of the methods described herein, when irradiated with the light source of step (a)(iii) described herein, the emission signal of the first photoswitchable label changes, for example, from a signal state to a dark state. In other words, the observed emission signal can switch from "on" to "off".

[0051] In some embodiments, the first photo-switchable tag comprises a first fluorescent moiety covalently bonded, optionally via a first linking group, to a first photochromic moiety. In some embodiments, the first linking group can be part of the π-conjugated system of the first fluorescent moiety. In some such embodiments, the first fluorescent moiety comprises a fluorophore that emits red light (e.g., red light having a wavelength between about 600 nm and about 700 nm). In some such embodiments, the first fluorescent moiety comprises a silicon rhodamine fluorophore. In one embodiment, the rhodamine fluorophore comprises the following structure:

[0052] In some embodiments, the first photochromic moiety can comprise a spiropyran or a spirothianine moiety. In some such embodiments, the first photochromic moiety comprises a structure of formula (I):

[0053]

[0054] wherein X is O (oxygen) or S (sulfur);

[0055] Each R 1a and R 2a are independently selected from: H, halogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy;

[0056] R 3 is selected from H, C 1-6 alkyl and –(CH2) n -R 4 ;

[0057] R 4 is selected from C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic group and 3- to 7-membered heterocyclic group, each of which is optionally substituted;

[0058] Ring A is C 6-10 aryl or 5- to 10-membered heteroaryl, each of which is substituted with at least one electron-withdrawing group; and n is an integer from 1 to 6.

[0059] In some such embodiments, the irradiation step (a)(iii) causes the cleavage of the spiro-carbon*―X bond in formula (I), resulting in a ring-opening reaction that converts the first photochromic moiety into a quencher capable of inhibiting the emission of the first fluorophore moiety. In certain cases, the cleavage of the spiro-carbon*―X bond is reversible.

[0060] In some embodiments of the first photochromic moiety of formula (I), the phenyl moiety fused to the pyrrolidine may be optionally substituted. In some embodiments, X is S. In some further embodiments, each R 1a and R 2a is C 1-6 alkyl, for example, each R 1a and R 2a is methyl. In some embodiments, ring A is a phenyl or naphthyl group substituted with at least one electron-withdrawing group. Non-limiting examples of electron-withdrawing groups may be selected from: nitro, cyano, fluoro, bromo, -S(O)2OH, trifluoromethanesulfonyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluorine or bromine atoms, and sulfonyl groups substituted with one or more fluorine or bromine atoms.

[0061] In one embodiment, the first photochromic moiety comprises a structure of formula (Ia):

[0062]

[0063] In one embodiment, the first photoswitchable label comprises the following structure:

[0064] wherein L 1 is a first linking group. In some such embodiments, the first linking group L 1 may comprise an azide moiety. In some further embodiments, the first photoswitchable label is covalently linked to a nucleotide via the first linking group or via a different position linked to the first photochromic moiety or the first fluorescent moiety.

[0065] Alternatively, the first photoswitchable label may only comprise a photochromic moiety and an optional linking group, wherein the photochromic moiety itself can act as a fluorophore and is capable of switching from a signal state to a dark state when irradiated with the light source described in step (a)(iii).

[0066] Second optically switchable label

[0067] In some embodiments of the methods described herein, when irradiated with the light source described in step (a)(iii), the emission signal of the second photoswitchable label changes, for example, from a non-emissive dark state to a signal state. In other words, the observed emission signal can be switched from "off" to "on".

[0068] In some embodiments, the second photo-switchable label comprises a second fluorescent moiety optionally covalently bonded via a second linking group to a second photochromic moiety. In some embodiments, the second linking group can be part of the π-conjugated system of the second fluorescent moiety. In some embodiments, the second fluorescent moiety comprises a fluorophore that emits red light (e.g., red light having a wavelength between about 600 nm and about 700 nm). In some such embodiments, in some such embodiments, the second fluorescent moiety comprises a coumarin fluorophore. In one embodiment, the coumarin fluorophore comprises the following structure:

[0069] In some embodiments, the second photochromic moiety can comprise an oxazine moiety or a thiazine moiety. In some such embodiments, the second photochromic moiety comprises a structure of formula (II):

[0070]

[0071] wherein Y is O (oxygen) or S (sulfur);

[0072] each R 1b and R 2b is independently selected from: H, halogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy;

[0073] Ring B is C 6-10 aryl or a 5- to 10-membered heteroaryl; each of which is substituted with at least one electron-withdrawing group; and wherein * represents the point of attachment to the fluorescent moiety.

[0074] In some such embodiments, the irradiation step (a)(iii) causes the cleavage of the carbon *-Y bond in formula (II), resulting in a ring-opening reaction that activates the second fluorescent moiety to the emissive state. In certain cases, the cleavage of the carbon *-Y bond is reversible.

[0075] In some embodiments of the second photochromic moiety of formula (II), the phenyl moiety fused to the five-membered pyrrolidine can be optionally substituted. In some embodiments, Y is O. In some further embodiments, each R 1b and R 2b is C 1-6 alkyl, for example, each R 1b and R 2bis methyl. In some embodiments, Ring B is a phenyl or naphthyl group substituted with at least one electron-withdrawing group. In other embodiments, Ring B can be selected from six-membered heteroaryl groups, such as pyridyl or pyrimidinyl. Non-limiting examples of electron-withdrawing groups can be selected from: nitro, cyano, fluorine, bromine, -S(O)2OH, trifluoromethanesulfonyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl groups, and sulfonyl groups substituted with one or more fluorine or bromine atoms.

[0076] In one embodiment, the second photochromic moiety comprises a structure of formula (IIa):

[0077]

[0078] In one embodiment, the second photoswitchable label comprises the following structure:

[0079] wherein L 2 is a second linking group. In some such embodiments, the second linking group L 2 can comprise a π-conjugated structure (e.g., one or more double bonds). In one instance, L 2 is a double bond connecting the coumarin and the second photochromic moiety. In some embodiments, the second photoswitchable label is covalently linked to the nucleotide via the second linking group or via the point indicated by the wavy line or.

[0080] Alternatively, the second photoswitchable label can comprise only a photochromic moiety and an optional linking group, wherein the photochromic moiety itself can act as a fluorophore and is capable of switching from a non-emissive dark state to an emissive state when irradiated with the light source described in step (a)(iii).

[0081] Non-limiting examples of photoswitchable labels or photochromic moieties that can be used in the present application also include diarylethene (e.g., bisthienylethene derivatives shown below), azine (e.g., azobenzene shown below), photochromic quinone (e.g., phenoxytetracenequinone), spirooxazine, spirothiazine, meso aldehyde 1-allyl-1-phenyl-2-phenylosazone, tetrachloro-1,2-ketonaphthalenone, thioindigoides, dinitrobenzylpyridine, and chromene, etc.

[0082]

[0083] In any embodiment of the methods described herein, the plurality of extended polynucleotides are attached to a substrate, such as the surface of a flow cell. In a further embodiment, the polynucleotides are attached to nanopores on the surface of the flow cell.

[0084] In any embodiment of the methods described herein, detecting the first set of signals and the second set of signals includes obtaining an image of the substrate.

[0085] Other illustrative embodiments are described below.

[0086] In some embodiments, methods of nucleic acid sequencing include directly or indirectly detecting three different nucleotide types using a single fluorescent label and a nucleotide type that is detected not by the presence of a fluorescent signal but by the absence or lack of a fluorescent signal. In some embodiments, methods for nucleic acid sequencing include using two or more different fluorescent labels that have the same or similar excitation / emission spectra for directly or indirectly detecting three different nucleotide types and a nucleotide type that is detected not by the presence of a fluorescent signal but by the absence or lack of a fluorescent signal. The same or similar excitation and emission spectra allow a laser to excite two or more different fluorescent labels, and a filter captures the fluorescent signals emitted therefrom. Fluorescent detection to determine the sequence of a nucleic acid sample is performed in a temporal-spatial context, e.g., at different times during a sequencing reaction (i.e., before and after a change in reaction conditions such as enzymatic cleavage, a change in ambient pH, addition of additional reagents) to provide fluorescent patterns (e.g., fluorescent transition patterns), the cumulative pattern of which determines the sequence of the nucleic acid target. Thus, the methods described herein are time- and cost-effective and allow for the simplification of associated sequencing instruments.

[0087] Exemplary applications of using temporal-spatial fluorescent pattern differences to determine a target nucleic acid sequence are the sequencing-by-synthesis (SBS) methods and techniques. Thus, the embodiments described herein find particular use in sequencing through synthetic fluorescence applications. Even though the embodiments described herein are examples of innovative methods of fluorescent sequencing, the disclosed embodiments can be used in a variety of other applications that require the detection of more than one analyte (i.e., nucleotide, protein, or fragment thereof) in a sample.

[0088] In developing embodiments of sequencing using a minimal set of dyes, experiments have revealed alternative strategies for differentiating nucleotide incorporations using only one or two fluorophore moieties. These strategies provide for all four nucleotide types to be present simultaneously during a sequencing cycle, and use a minimal number of dyes and optical filter sets. In some embodiments, one or two excitation and emission filters are used, and at most three fluorescent labels are utilized to determine incorporations of all four nucleotide types present during the reaction. In a preferred embodiment, one light excitation range and one detection emission filter are used, and no more than two fluorescent labels (or two or three identical or similar excitation / emission spectra) are utilized to determine incorporations of all four nucleotide types present during the reaction.

[0089] In some embodiments, sequencing using a minimal set of dyes is performed on a substrate (e.g., glass, plastic, semiconductor chip, or composite-derived substrate). In some embodiments, a nucleic acid species is provided on the substrate, e.g., for single target sequencing. In other embodiments, sequencing can also be in multiplex form, where multiple nucleic acid targets are detected and sequenced in parallel, e.g., in the form of a flow cell or an array type. The embodiments described herein are particularly advantageous when performing parallel sequencing or massively parallel sequencing. Platforms for performing fluorescent parallel sequencing include, but are not limited to, platforms provided by Illumina, Inc. (e.g., HiSeq, Genome Analyzer, MiSeq, iSeq, iScan platforms), Life Technologies (e.g., SOLiD), Helicos Biosciences (e.g., Heliscope), 454 / Roche Life Sciences (Branford, Conn.), and Pacific Biosciences (e.g., SMART). Examples of substrates for parallel sequencing are flow cells, chips, and other types of surfaces that can accommodate multiple nucleic acid species. In multiplex forms in which multiple nucleic acid species are sequenced in parallel, the clonally amplified target sequences (e.g., by emulsion PCR (emPCR) or bridge amplification) are typically covalently immobilized on the substrate. For example, when performing emulsion PCR, the target targets are immobilized on beads, while the clonally amplified targets are immobilized at specific locations on the channels or arrays or chips of a flow cell.

[0090] Flow cells used in conjunction with the compositions and methods described herein can be used for sequencing in a variety of ways. For example, a DNA sample (e.g., a DNA library) can be applied to a flow cell or fluidic device that includes one or more etched flow channels, where the flow cell can also include a population of probe molecules covalently linked to its surface. Advantageously, the probes in the flow cell channels are located at different addressable positions in the channels, and DNA library molecules can be added to the flow cell channels where complementary sequences can bind (as described herein and further as described in WO2012 / 096703, which is incorporated herein by reference in its entirety). Another example of a flow cell used in the present application includes the CMOS flow cell as described in U.S. Patents 8,906,320 and 9,990,381, which are incorporated herein by reference in their entirety. Bridge amplification can be performed as described herein and then sequencing can be performed by the methods and compositions of sequencing-by-synthesis as described herein. Methods for creating and utilizing flow cells for sequencing are known in the art; references are provided herein and are incorporated herein by reference in their entirety. It is contemplated that the methods and compositions described herein are not limited to any particular fabrication or method of flow cell-directed sequencing methodologies.

[0091] Sequencing using the methods and compositions described herein can also be performed in microtiter plates, e.g., in high-density reaction plates or slides (Margulies et al., 2005, Nature 437(7057):376 - 380, which is incorporated herein by reference in its entirety). For example, genomic targets can be prepared by emPCR technology. The reaction plate or slide can be made of a fiber optic material capable of capturing and recording light generated by a reaction, e.g., by a fluorescence or luminescence reaction. The core material can be etched to provide discrete reaction wells capable of accommodating at least one emPCR reaction bead. Such slides / reaction plates can accommodate more than 1.6 million wells. The target sequencing reaction emPCR beads can be loaded onto the created slide / reaction plate and mounted onto an instrument that provides sequencing reagents and performs sequencing.

[0092] When implementing a patterned substrate containing DNA nanoballs on a chip or slide by Complete Genomics (Mountain View, CA), examples of array substrates for target sequencing using the compositions and methods disclosed herein are provided. As described in Drmanac et al., 2010, Science 327(5961):78 - 81, silica and titanium can be laminated on a silicon wafer and then patterned using photolithography and dry etching techniques. The wafer can be treated with HMDS and a photoresist layer can be coated thereon to define discrete regions for silanization and for subsequent covalent attachment of DNA nanoballs for sequencing. Those skilled in the art will understand that there are many methods for creating slides / chips with discrete positions for nucleic acid immobilization for sequencing methodologies, and the present method is not limited to the substrate preparation methods for sequencing.

[0093] For purposes of illustration and not meant to limit the embodiments described herein, a sequencing cycle of a general strategy can be described by a sequencing step. The following example is based on sequencing by a sequencing - by - synthesis reaction; however, the methods described herein are not limited to any particular sequencing reaction methodology.

[0094] Four nucleotides A, C, T, and G (usually modified nucleotides designed for sequencing reactions, e.g., reversibly blocked (rb) nucleotides (e.g., rbA, rbT, rbC, rbG), where three of the four types are fluorescently labeled) are added, along with other reaction components, to the location (e.g., flow cell, chip, slide, etc.) where the target template sequence is located and where the sequencing reaction occurs. After incorporation of the nucleotides into the growing sequence nucleic acid strand based on the target sequence, the reaction is exposed to light and the fluorescence is observed and recorded; this constitutes the first imaging event and the first fluorescence detection pattern. After the first imaging event, the sample is irradiated with a light source to cause a recognizable and measurable change in the emission signals of the first and second fluorescent labels. The reaction location is irradiated again, and any change in fluorescence is captured and recorded; this constitutes the second imaging event (i.e., the second fluorescence detection pattern). The protecting groups present on the incorporated nucleotides are removed and washed away along with other reagents present after the second imaging event to prepare for the next sequencing cycle. In some embodiments, the methods of the present disclosure do not involve the use of any chemical reagents that may directly or indirectly cause a recognizable and measurable change in fluorescence from the first imaging event to the second imaging event. The fluorescence patterns and nucleotide incorporations from the two imaging events are compared to determine the target nucleic acid sequence for that particular cycle.

[0095] Definition

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "comprising" and other forms thereof (e.g., "include", "includes", and "included") is not limiting. The use of the term "having" and other forms thereof (e.g., "have", "has", and "had") is not limiting. As used in this specification, whether in a transitional phrase or in the body of a claim, the term "comprise(s)" and "comprising" shall be interpreted to have an open-ended meaning. That is, the above terms shall be interpreted synonymously with the phrase "having at least" or "including at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include other steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include other features or components.

[0097] As used herein, common organic abbreviations are defined as follows:

[0098] ℃ temperature (degrees Celsius)

[0099] dATP deoxyadenosine triphosphate

[0100] dCTP deoxycytidine triphosphate

[0101] dGTP deoxyguanosine triphosphate

[0102] dTTP deoxythymidine triphosphate

[0103] ddNTP dideoxynucleotide triphosphate

[0104] ffN fully functionalized nucleotide

[0105] LED light emitting diode

[0106] As used herein, the term "array" refers to a population of different probe molecules attached to one or more substrates such that the different probe molecules can be distinguished from one another based on their relative positions. An array can include different probe molecules, each located at a different addressable position on the substrate. Optionally or additionally, an array can include separate substrates each bearing a different probe molecule, where the different probe molecules can be identified based on the position of the substrate on the surface to which it is attached or based on the position of the substrate in a liquid. Exemplary arrays in which the separate substrates are located on a surface include, but are not limited to, those arrays that include beads in wells, such as those described in U.S. Patent No. 6,355,431 B1, US 2002 / 0102578, and PCT Publication WO 00 / 63437. Exemplary forms that can be used in the present invention for distinguishing beads in a liquid array using a microfluidic device such as a fluorescence-activated cell sorter (FACS) are described, for example, in U.S. Patent No. 6,524,793. Other examples of arrays that can be used in the present invention include, but are not limited to, those arrays described in U.S. Patent Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751, and 6,610,482; and International Patent WO 93 / 17126; WO 95 / 11995; WO 95 / 35505; European Patent EP 742 287, and EP 799 897.

[0107] As used herein, the term "covalently linked" or "covalently bonded" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently linked polymer coating refers to a polymer coating that is attached to a functionalized surface by forming a chemical bond as opposed to other means such as adhesion or electrostatic interaction. It should be understood that a polymer covalently linked to a surface can also be bonded by other means in addition to covalent linkage.

[0108] As used herein, any "R" group represents a substituent that can be attached to the indicated atom. The R group can be substituted or unsubstituted. If two "R" groups are described as "together with the atoms to which they are attached" forming a ring or ring system, it means that the collective unit of the inserted bond and the atoms of the two R groups is the recited ring. For example, when the following substructure is present:

[0109]

[0110] and R 1 and R 2 are defined as selected from hydrogen and alkyl or R 1 and R 2 together with the atom to which they are attached form an aryl or carbocyclic group, which means that R 1 and R 2 can be selected from hydrogen or alkyl or have a substructure with the following structure:

[0111]

[0112] wherein A is an aryl ring or carbocyclic group containing the indicated double bond.

[0113] It should be understood that, depending on the context, the naming conventions for some groups may include mono-radical or di-radical. For example, when a substituent requires two points of attachment to the rest of the molecule, it should be understood that the substituent is a di-radical. For example, substituents recognized as alkyl groups that require two points of attachment include di-radicals such as –CH2–, –CH2CH2–, –CH2CH(CH3)CH2–, etc. Other group naming conventions clearly indicate that the group is a di-radical, such as "alkylene" or "alkenylene".

[0114] As used herein, the term "halogen" or "halo" refers to any radiostable atom in column 7 of the periodic table, such as fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0115] As used herein, "alkyl" refers to a straight-chain or branched-chain hydrocarbon chain that is completely saturated (i.e., does not contain double or triple bonds). The alkyl can have 1 to 20 carbon atoms (whenever a numerical range such as "1 to 20" appears, it refers to each integer within the given range; for example, "1 to 20 carbon atoms" means that the alkyl can include 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although this definition also covers the occurrence of the term "alkyl" where no numerical range is specified). The alkyl can also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl can be designated as "C 1-4 alkyl" or a similar name. By way of example only, "C 1-6 alkyl" indicates the presence of one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyls include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc.

[0116] As used herein, "alkoxy" refers to the formula -OR, where R is an alkyl group as defined above. For example, "C1-C9 alkoxy" includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy, etc.

[0117] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing one or more double bonds. The alkenyl group can have 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkenyl" where no numerical range is specified. The alkenyl can also be a medium-sized alkenyl having 2 to 9 carbon atoms. The alkenyl group can also be a lower alkenyl having 2 to 6 carbon atoms. The alkenyl can be designated as "C 2-6 alkenyl" or a similar name. By way of example only, "C 2-6 alkenyl" means that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from vinyl, prop-1-enyl, prop-2-enyl, prop-3-enyl, but-1-enyl, but-2-enyl, but-3-enyl, but-4-enyl, 1-methylprop-1-enyl, 2-methylprop-1-enyl, 1-ethyl-vinyl-1-enyl, 2-methyl-prop-3-enyl, buta-1,3-dienyl, buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyls include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl, etc.

[0118] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more triple bonds. The alkynyl can have 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkynyl" where no numerical range is specified. The alkynyl can also be a medium-sized alkynyl having 2 to 9 carbon atoms. The alkynyl can also be a lower alkynyl having 2 to 6 carbon atoms. The alkynyl can be designated as "C 2-6 alkynyl" or a similar name. By way of example only, "C 2-6 alkynyl" means that there are 2 to 6 carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-3-ynyl, but-4-ynyl, and 2-butynyl. Typical alkynyls include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, etc.

[0119] As used herein, "aryl" refers to an aromatic ring or ring system containing only carbon in the ring skeleton (i.e., two or more fused rings sharing two adjacent carbon atoms). When the aryl is a ring system, each ring in the system is aromatic. The aryl can have 6 to 18 carbon atoms, although this definition also encompasses occurrences of the term "aryl" where no numerical range is specified. In some embodiments, the aryl has 6 to 10 carbon atoms. The aryl can be referred to as "C6-10 "aryl", "C6 or C 10 "aryl" or similar names. Examples of aryl include, but are not limited to, phenyl, naphthyl, azulyl, and anthryl.

[0120] "Aralkyl" or "arylalkyl" is an aryl as a substituent linked via an alkylene group, such as "C 7-14 aralkyl", etc., including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 alkylene).

[0121] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur) in the ring skeleton. When the heteroaryl is a ring system, each ring in the system is aromatic. The heteroaryl may have 5 - 18 ring members (i.e., the number of atoms constituting the ring skeleton, including carbon atoms and heteroatoms), although this definition also encompasses occurrences of the term "heteroaryl" where no numerical range is specified. In some embodiments, the heteroaryl has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl may be designated as "5 - 7 membered heteroaryl", "5 - 10 membered heteroaryl", or similar names. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0122] "Heteroaralkyl" or "heteroarylalkyl" is a heteroaryl as a substituent linked via an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolidinyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 alkylene).

[0123] As used herein, "carbocyclic group" refers to a non-aromatic ring or ring system that contains only carbon atoms in the backbone of the ring system. When the carbocyclic group is a ring system, two or more rings can be joined together in a fused, bridged, or spiro fashion. The carbocyclic group can have any degree of saturation, provided that at least one of the rings in the ring system is not aromatic. Thus, carbocyclic groups include cycloalkyl, cycloalkenyl, and cycloalkynyl groups. The carbocyclic group can have from 3 to 20 carbon atoms, although this definition also encompasses occurrences of the term "carbocyclic group" where no numerical range is specified. The carbocyclic group can also be a medium-sized carbocyclic group having from 3 to 10 carbon atoms. The carbocyclic group can also be a carbocyclic group having from 3 to 6 carbon atoms. The carbocyclic group can be designated as "C 3-6 carbocyclic group" or a similar name. Examples of carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydroindene, bicyclo[2.2.2]octyl, adamantyl, and spiro[4.4]nonyl.

[0124] As used herein, "cycloalkyl" refers to a fully saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0125] As used herein, "heterocyclic group" refers to a non-aromatic ring or ring system that contains at least one heteroatom in the ring skeleton. Heterocyclic groups can be joined together in a fused, bridged, or spiro-linked manner. Heterocyclic groups can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom can be present in a non-aromatic or aromatic ring of the ring system. The heterocyclic group can have from 3 to 20 ring members (i.e., the number of atoms making up the ring skeleton, including carbon atoms and heteroatoms), although this definition also encompasses occurrences of the term "heterocyclic group" where no numerical range is specified. The heterocyclic group can also be a medium-sized heterocyclic group having from 3 to 10 ring members. The heterocyclic group can also be a heterocyclic group having from 3 to 6 ring members. The heterocyclic group can be designated as a "3-6 membered heterocyclic group" or a similar name. In a preferred six-membered monocyclic heterocyclic group, the heteroatoms are selected from at most three of O, N, or S, and in a preferred five-membered monocyclic heterocyclic group, the heteroatoms are one or two heteroatoms selected from O, N, or S. Examples of heterocyclic groups include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxolanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidinone, pyrrolidinedione, 4-piperidinone, pyrazolinyl, pyrazolidinyl, 1,3-dioxolenyl, 1,3-dioxolanyl, 1,4-dioxolenyl, 1,4-dioxolanyl, 1,3-oxathiolanyl, 1,4-oxathioladienyl, 1,4-oxathiolanyl, 2H-1,2-oxazinyl, trioxolanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolenedienyl, 1,3-dioxolanyl, 1,3-dithiolenedienyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, dihydroindolyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolinyl, and tetrahydroquinoline.

[0126] "O-carboxyl" means "-OC(=O)R" where R is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclic group.

[0127] "C-carboxyl" means the group "-C(=O)OR", where R is selected from hydrogen, C1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group. Non-limiting examples include carboxymethyl (i.e., -C(=O)OH).

[0128] "sulfonyl" means "-SO2R", where R is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group as defined herein.

[0129] "S-sulfonamido" means "-SO2NR A R B ", where R A and R B are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group as defined herein.

[0130] The "N-sulfonamido" group means the "-N(R A )SO2R B " group, where R A and R B are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group as defined herein.

[0131] The "C-amide" group means the "-C(=O)NR A R B " group, where R A and R B are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group as defined herein.

[0132] The "N - amide" group refers to the "-N(R A )C(=O)R B " group, where R A and R B are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5 - 10 - membered heteroaryl, and 3 - 10 - membered heterocyclic group as defined herein.

[0133] The "amino" group refers to the "-NR A R B " group, where R A and R B are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic group, C 6-10 aryl, 5 - 10 - membered heteroaryl, and 3 - 10 - membered heterocyclic group as defined herein. Non - limiting examples include a free amino group (i.e., -NH2).

[0134] The "aminoalkyl" group refers to an amino group linked via an alkylene group.

[0135] The "alkoxyalkyl" group refers to an alkoxy group linked via an alkylene group, such as "C 2- C8 alkoxyalkyl", etc.

[0136] As used herein, a substituent is derived from an unsubstituted parent group in which one or more hydrogen atoms have been exchanged for another atom or group. Unless otherwise specified, when a group is considered to be "substituted", it means that the group is substituted with one or more substituents independently selected from the following: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclic group (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclic-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered heterocyclic group (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered heterocyclic-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogen, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl and mercapto, halogen(C1-C6)alkyl (e.g. –CF3), halogen(C1-C6)alkoxy (e.g. –OCF3), (C1-C6)alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfonyl, -SO3H, -OSO2C 1-4 alkyl and oxo(=O). Wherever a group is described as "optionally substituted", the group can be substituted with the above substituents.

[0137] As used herein, the term "azido" refers to the –N3 group.

[0138] As used herein, "nucleotide" includes a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, while in DNA it is deoxyribose, i.e., a sugar lacking a hydroxyl group present in ribose. The nitrogenous heterocyclic base can be a purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of the deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine.

[0139] As used herein, "nucleoside" is structurally similar to a nucleotide but lacks the phosphate moiety. An example of a nucleoside analog is one in which a label is attached to the base and no phosphate is attached to the sugar molecule. As will be understood by those skilled in the art, the term "nucleoside" is used herein in its general sense. Examples include, but are not limited to, ribonucleosides containing a ribose moiety and deoxyribonucleosides containing a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced by carbon and / or a carbon has been replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that can have substituted bases and / or sugar moieties. Additionally, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0140] As will be understood by those skilled in the art, the term "purine base" is used herein in its ordinary meaning and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in the ordinary meaning understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, isoxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0141] As used herein, "derivative" or "analog" refers to a synthetic nucleotide or nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, by Scheit in Nucleotide Analogs (John Wiley&Son, 1980) and by Uhlman et al. in Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also include modified phosphodiester linkages, including phosphorothioate, dithiophosphate, alkyl-phosphate, anilino-phosphate, and amino-phosphate linkages. The terms "derivative", "analog", and "modified" as used herein are used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein.

[0142] As used herein, the term "phosphate" is used in its ordinary meaning as understood by one of ordinary skill in the art and includes its protonated form (e.g., ). As used herein, the terms "monophosphate", "diphosphate", and "triphosphate" are used in their ordinary meaning as understood by one of ordinary skill in the art and include protonated forms.

[0143] As used herein, the term "protecting group" refers to any atom or group of atoms added to a molecule to prevent undesired chemical reactions of existing groups in the molecule. Sometimes, "protecting group" and "blocking group" can be used interchangeably.

[0144] As used herein, the prefix "photo" or "photo-" means related to light or electromagnetic radiation. The term can encompass all or part of the electromagnetic spectrum, including but not limited to one or more ranges of the radio, microwave, infrared, visible light, ultraviolet, X-ray, or gamma-ray portions, which are commonly referred to as the spectrum. The portion of the spectrum can be the portion blocked by the surface of the metal region of the metals such as those described herein. Optionally or additionally, the portion of the spectrum can be the portion passing through the interstitial region of the surface of a region made of materials such as glass, plastic, silica, or other materials described herein. In certain embodiments, radiation capable of passing through the metal can be used. Optionally or additionally, radiation masked by glass, plastic, silica, or other materials described herein can be used.

[0145] Labeled nucleotide

[0146] According to one aspect of the present disclosure, the nucleotides described for incorporation contain a detectable label (or marker), and such nucleotides are referred to as labeled nucleotides. The label (e.g., a fluorescent dye) can be attached by an optional linking group, by a variety of methods, including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspects, the dye is attached to the substrate by covalent attachment. More particularly, the covalent attachment is by means of a linking group. In some cases, such labeled nucleotides are also referred to as "modified nucleotides". The dye is covalently attached to the nucleotide by a cleavable linking group. In some such embodiments, the cleavable linking group can comprise one or more moieties, including an azide moiety, an azidomethyl moiety, a disulfide moiety, –(CH2CH2O)-, or any other covalent linking group described herein.

[0147] The labeled nucleotides can be used to label polynucleotides formed by enzymatic synthesis, for example, by PCR amplification, isothermal amplification, solid-phase amplification, polynucleotide sequencing (e.g., solid-phase sequencing), nick translation reactions, etc., as non-limiting examples.

[0148] In some embodiments, the dye can be covalently attached to the oligonucleotide or nucleotide via the nucleobase. For example, the labeled nucleotide or oligonucleotide can have a label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linking group moiety.

[0149] Unless otherwise indicated, references to nucleotides are also intended to apply to nucleosides. Unless otherwise indicated, the present invention will further refer to DNA for further description, although this description also applies to RNA, PNA, and other nucleic acids.

[0150] Some embodiments of the present disclosure relate to nucleotide conjugates comprising a photoconvertible label, wherein the photoconvertible label comprises a fluorescent moiety optionally covalently bonded to a photochromic moiety via a linking group. In some embodiments, the photoswitchable label is linked to the nucleobase by a cleavable linking group. In some further embodiments, the photoswitchable label is linked to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. In some embodiments, the nucleotide is labeled with a photoswitchable label. Upon irradiation with a light source, the emission signal of the photoswitchable label will, for example, change from a signal state to a dark state. In other words, the observed emission signal can be switched from "on" to "off". Optionally, the photoswitchable label can change a non-emissive dark state to a signal state. In other words, the observed emission signal can be switched from "off" to "on".

[0151] In some embodiments, the photo-switchable label comprises a fluorescent moiety optionally covalently bonded to a photochromic moiety via a linking group. In some such embodiments, the fluorescent moiety comprises a fluorophore that emits red light (e.g., red light having a wavelength between about 600 nm and about 700 nm).

[0152] In some embodiments, the fluorescent moiety of the photo-switchable label comprises a silicon rhodamine fluorophore. In one embodiment, the rhodamine fluorophore comprises the following structure: In some such embodiments, the photochromic moiety of the photo-switchable label may comprise a spiropyran or a spirothiopyran moiety. For example, the photochromic moiety comprises a structure of formula (I):

[0153]

[0154] wherein X, R 1a 、R 2a 、R 3 、R 4 and ring A are as defined herein, and wherein the carbon marked with an asterisk * is a spiro carbon that will undergo a chemical transformation upon irradiation with an appropriate light source.

[0155] In some embodiments of the photochromic moiety of formula (I), the phenyl moiety fused to the five-membered pyrrolidine may be optionally substituted. In some embodiments, X is S. In some further embodiments, each R 1a and R 2a is a C 1-6 alkyl group, e.g., each R 1a and R 2a is a methyl group. In some embodiments, ring A is a phenyl or naphthyl group substituted with at least one electron-withdrawing group. Non-limiting examples of electron-withdrawing groups may be selected from: nitro, cyano, fluoro, bromo, -S(O)2OH, trifluoromethanesulfonyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluorine or bromine atoms, and sulfonyl groups substituted with one or more fluorine or bromine atoms. In one embodiment, the photochromic moiety comprises a structure of formula (Ia): In one embodiment, the photo-switchable label comprises the following structure: wherein L 1 is a first linking group. In some such embodiments, the first linking group L 1 may comprise an azide moiety. In some further embodiments, the first photo-switchable label is covalently linked to a nucleotide via the first linking group or via a different position linked to the first photochromic moiety or the first fluorescent moiety.

[0156] In some other embodiments, the photo-switchable fluorescent moiety comprises a coumarin fluorophore. In one embodiment, the coumarin fluorophore comprises the following structure: In some such embodiments, the photochromic moiety of the photo-switchable label may comprise an oxazine or thiazine moiety. For example, the photochromic moiety comprises the structure of formula (II):

[0157]

[0158] wherein Y, R 1b , R 2b and ring B are defined as described herein, and wherein the carbon marked with an asterisk * indicates the point of attachment directly or via a second linking group to the fluorescent moiety. The carbon*-Y bond in formula (II) will undergo a chemical transformation upon irradiation with an appropriate light source.

[0159] In some embodiments of the photochromic moiety of formula (II), the phenyl moiety fused to the five-membered pyrrolidine may be optionally substituted. In some embodiments, Y is O. In some further embodiments, each R 1b and R 2b is C 1-6 alkyl, for example, each R 1b and R 2b is methyl. In some embodiments, ring B is a phenyl or naphthyl group substituted with at least one electron-withdrawing group. In other embodiments, ring B may be selected from six-membered heteroaryl groups, such as pyridyl or pyrimidinyl. Non-limiting examples of electron-withdrawing groups may be selected from: nitro, cyano, fluorine, bromine, -S(O)2OH, trifluoromethanesulfonyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluorine or bromine, and sulfonyl groups substituted with one or more fluorine or bromine. In one embodiment, the photochromic moiety comprises the structure of formula (IIa): In one embodiment, the photo-switchable label comprises the following structure:

[0160] wherein L 2 is a second linking group. In some such embodiments, the second linking group L 2 may comprise a π-conjugated structure (e.g., one or more double bonds). In one example, L 2 is a double bond connecting the coumarin and the photochromic moiety. In some embodiments, the photo-switchable label is covalently linked to the nucleotide via the second linking group or via the point indicated by the wavy line .

[0161] Alternatively, the photo-switchable labels described herein may contain only a photochromic moiety and an optional linking group, where the photochromic moiety itself can act as a fluorophore and is capable of switching from a non-emissive dark state to an emissive state, or from an emissive state to a dark state, when irradiated with an appropriate light source.

[0162] Some further embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the nucleotide conjugates described herein.

[0163] Linker group

[0164] In some embodiments described herein, the purine or pyrimidine base of the nucleotide molecule may be linked to a detectable label as described above. In some such embodiments, the linking group used is cleavable. The use of a cleavable linker ensures that, if desired, the label can be removed after detection, thus avoiding any interference signals with any labeled nucleotides incorporated.

[0165] In some other embodiments, the linking group used is non-cleavable. Since labeled nucleotides as described herein are incorporated in each case, there is no need to subsequently incorporate any nucleotides, and thus no need to remove the label from the nucleotides.

[0166] Cleavable linking groups are known in the art, and conventional chemical methods can be applied to link the linking group to the nucleobase and the label. The linking group can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, free radicals, metals, reducing agents or oxidizing agents, light, temperature, enzymes, etc. The linking groups described herein can also be cleaved with the same catalysts used to cleave the bond of the 3'-O-protecting group. Suitable linking groups can be adapted to standard chemical protecting groups as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Other suitable cleavable linking groups for solid-phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100: 2092 - 2157, 2000).

[0167] In the case where the detectable label is linked to the base, the linking group can be linked to any position of the nucleobase as long as Watson-Crick base pairing can still occur. In the case of purine bases, the linking group is preferably linked via the 7-position of the purine or a preferred deazapurine analogue, via an 8-modified purine, via an N-6-modified adenosine or an N-2-modified guanine. For pyrimidines, the linking is preferably via the 5-position of cytosine, thymine or uracil and the N-4 position of cytosine.

[0168] In some embodiments, the linking group may comprise a spacer unit. The length of the linking group is not critical as long as the label is kept at a sufficient distance from the nucleotide so as not to interfere with any interactions between the nucleotide and an enzyme (such as a polymerase).

[0169] In some embodiments, the linking group may be composed of a functional group similar to a 3'-OH protecting group. This would make the deprotection and deprotection process more efficient since only one treatment would be required to remove both the label and the protecting group.

[0170] The use of the term "cleavable linking group" does not imply that the entire linking group needs to be removed. The cleavage site can be at a position on the linking group that ensures that a portion of the linking group remains attached to the dye and / or the substrate moiety after cleavage. As a non-limiting example, a cleavable linking group can be an electrophilic cleavable linking group, a nucleophilic cleavable linking group, a photocleavable linking group, cleavable under reducing conditions, oxidative conditions (e.g., a linking group containing a disulfide bond or an azide group), cleavable via the use of a safety-catch linking group, and cleavable by an elimination mechanism. Using a cleavable linking group to attach a dye compound to a substrate moiety ensures that the label is removed, if desired, after detection, thus avoiding any interfering signals in downstream steps.

[0171] Useful linking groups can be found in PCT Publication WO 2004 / 018493 (incorporated herein by reference), examples of which include linking groups that can be cleaved by a water-soluble phosphine or a water-soluble transition metal catalyst formed from a transition metal and at least a partially water-soluble ligand (e.g., a Pd(II) complex and THP). The latter forms at least a partially water-soluble transition metal complex in aqueous solution. Such cleavable linking groups can be used to attach the base of a nucleotide to a label, such as a dye as described herein.

[0172] Specific linking groups include those disclosed in PCT Publication WO 2004 / 018493 (incorporated herein by reference), such as those including moieties of the following formula:

[0173]

[0174] (wherein X is selected from O, S, NH, and NQ, where Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from O, S, NH, and N(allyl), T is hydrogen or a C1-C 10 substituted or unsubstituted alkyl group, and * indicates the position at which the moiety is attached to the remainder of the nucleotide). In some aspects, the linking group attaches the base of the nucleotide to a label, such as a dye compound as described herein.

[0175] Other examples of linking groups include those disclosed in U.S. Publication 2016 / 0040225, incorporated herein by reference, such as those including moieties of the formula:

[0176]

[0177] (where * indicates the position at which the moiety is attached to the remainder of the nucleotide). The linking group moieties shown herein can comprise all or part of the linking group structure between the nucleotide / nucleoside and the label.

[0178] In certain embodiments, the length of the linking group between the fluorescent dye (fluorophore) and the guanine base can be varied, for example, by inserting a polyethylene glycol spacer, such that the fluorescence intensity is higher compared to the same fluorophore attached to the guanine base via other linking bonds known in the art. Exemplary linking groups and their properties are set forth in PCT Publication WO2007020457, incorporated herein by reference. The design of the linking group, particularly its increased length, can enhance the brightness of the fluorophore attached to the guanine base of a guanosine nucleotide when incorporated into a polynucleotide (such as DNA). Thus, when the dye is used in any assay method that requires detection of a fluorescent dye label attached to a guanine-containing nucleotide, a linking group comprising a spacer group of the formula –((CH2)2O) n –(where n is an integer between 2 and 50) is advantageous, as described in WO 2007 / 020457.

[0179] Nucleosides and nucleotides can be labeled at the site of the sugar or the nucleobase. As is known in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose, and in DNA the sugar is deoxyribose (i.e., a sugar lacking a hydroxyl group where ribose has one). The nitrogenous base is a derivative of purine or pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) or uracil (U) in the case of RNA. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. A nucleotide is also a phosphate ester of a nucleoside, and the esterification occurs at the hydroxyl group attached to the C-3 or C-5 of the sugar. Nucleotides are typically mono-, di-, or tri-phosphate esters.

[0180] Although the bases are commonly referred to as purines or pyrimidines, those skilled in the art will understand that derivatives and analogs that do not alter the ability of nucleotides or nucleosides to perform Watson-Crick base pairing are available. A "derivative" or "analog" refers to a compound or molecule whose core structure is the same as or very similar to the parent compound, but has a chemical or physical modification (e.g., a different or additional side group that enables the derivatized nucleotide or nucleoside to be linked to another molecule). For example, the base can be a deazapurine. In certain embodiments, the derivative should be able to perform Watson-Crick pairing. "Derivatives" and "analogs" also include, for example, synthetic nucleotide or nucleoside derivatives having modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide analogs (John Wiley&Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also contain modified phosphodiester linkages, including phosphorothioate, dithiophosphate, alkyl-phosphate, anilino-phosphate, aminophosphate linkages, and the like.

[0181] The dye can be attached (e.g., via a linking group) to any position of the nucleobase. In certain embodiments, Watson-Crick base pairing can still occur with the resulting analog. Particular nucleobase labeling sites include the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases. As described above, the dye can be covalently attached to the nucleotide using a linking group.

[0182] In certain embodiments, the labeled nucleoside or nucleotide can be enzyme incorporable and enzyme extendable. Thus, the linking group moiety can have a sufficient length to link the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by nucleic acid replicating enzymes. Thus, the linking group can also contain a spacer unit. The distance of this spacer, e.g., the distance between the nucleobase and the cleavage site or the label.

[0183] A nucleoside or nucleotide labeled with a dye as described herein can have the following formula:

[0184]

[0185] Wherein the dye is a dye compound; B is a nucleobase, such as uracil, thymine, cytosine, adenine, guanine, etc.; L is an optional linking group, which may or may not be present; R' can be H, monophosphate, diphosphate, triphosphate, phosphorothioate, phosphate analog, –O– linked to a group containing active phosphorus or –O– protected by a protecting group; R” can be H, OH, phosphoramidite or a 3′-OH protecting group, and R”' is H or OH. When R” is phosphoramidite, R' is an acid-labile hydroxy protecting group that allows subsequent monomers to couple under automated synthesis conditions.

[0186] In certain embodiments, the protecting group is separate and independent of the dye compound, i.e., not linked thereto. Alternatively, the dye may comprise all or part of the 3'-OH protecting group. Thus, R” can be a 3'-OH protecting group that may or may not comprise the dye compound.

[0187] In another alternative embodiment, there is no protecting group on the 3'-carbon of the pentose, and the dye (or dye and linker construct) attached to the base, e.g., is of a size or structure sufficient to act as a hindrance to the incorporation of additional nucleotides. Thus, the hindrance may be due to steric hindrance or may be due to a combination of size, charge, and structure, regardless of whether the dye is attached to the 3'-position of the sugar.

[0188] In another alternative embodiment, the protecting group is present on the 2'- or 4'-carbon of the pentose and is of a size or structure sufficient to act as a hindrance to the incorporation of other nucleotides.

[0189] The use of protecting groups can control polymerization, e.g., by stopping extension when incorporating nucleotides. If the blocking action is reversible (e.g., by changing chemical conditions or removing chemical protection, non-limiting examples), extension can be stopped at certain points and then continued.

[0190] In another specific embodiment, the 3'-OH protecting group will comprise portions disclosed in WO2004 / 018497 and WO2014 / 139596, which are incorporated herein by reference. For example, the protecting group can be azidomethyl (-CH2N3) or a substituted azidomethyl (e.g., -CH(CHF2)N3 or CH(CH2F)N3 or allyl).

[0191] In certain embodiments, both the linking group (between the dye and the nucleotide) and the protecting group are present and are separate moieties. In certain embodiments, both the linking group and the protecting group can be cleaved under substantially similar conditions. Thus, the deprotection process may be more efficient because only one treatment is required to remove both the dye compound and the protecting group. However, in some embodiments, the linking group and the protecting group need not be cleavable under similar conditions, but rather can be cleaved separately under different conditions.

[0192] The present disclosure also encompasses polynucleotides incorporating the dye compounds. Such polynucleotides can be DNA or RNA composed of deoxyribonucleotides or ribonucleotides, respectively, that participate in forming the phosphodiester linkage. The polynucleotide can comprise a combination of naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof, with at least one modified nucleotide (e.g., labeled with a dye compound) as described herein. The polynucleotides according to the present disclosure can also include non-natural backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures composed of a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

[0193] Non-limiting exemplary labeled nucleotides described herein include:

[0194]

[0195] Wherein L represents the linking group and R represents the sugar residue as described above, or the 5'-position is substituted with one, two, or three phosphates.

[0196] In some embodiments, non-limiting exemplary fluorescent dye conjugates are as follows:

[0197]

[0198] Wherein PG represents the 3'-hydroxy protecting group as described herein.

[0199] Kit

[0200] The present disclosure also provides kits that contain the labeled nucleotides described herein. Such kits generally include at least one nucleotide labeled with a dye (e.g., a photoswitchable dye as described herein) and at least one other component. The other component can be one or more components identified in the methods described herein or in the embodiments section below. Some non-limiting examples of components that can be incorporated into the kits of the present disclosure are set forth below.

[0201] In a particular embodiment, the kit may comprise at least one labeled nucleotide or nucleoside and labeled or unlabeled nucleotides or nucleosides. For example, a nucleotide labeled with a dye may be provided in combination with unlabeled or native nucleotides and / or with fluorescently labeled nucleotides or any combination thereof. The combination of nucleotides may be provided as separate individual components (e.g., one nucleotide type per container or tube), or as a nucleotide mixture (e.g., two or more nucleotides are mixed in the same container or tube). In some embodiments, one or more dyes are selected from the photoswitchable dyes disclosed herein.

[0202] When the kit comprises a plurality, particularly two or three, or more particularly four, nucleotides each labeled with a dye compound, the different nucleotides may be labeled with different dye compounds, or one may be a non-luminescent nucleotide without a dye compound. When the different nucleotides are labeled with different dye compounds, the kit is characterized in that the dye compounds are spectrally distinguishable fluorescent dyes by one or more imaging events. When two nucleotides labeled with a fluorescent dye compound are provided in kit form, some embodiments are characterized in that the spectrally distinguishable fluorescent dyes may be excited at the same wavelength, e.g., by the same laser. When four nucleotides labeled with a fluorescent dye compound are provided in kit form, some embodiments are characterized in that two spectrally distinguishable fluorescent dyes can be excited at one wavelength, while the other two spectrally distinguishable dyes are not excited at that wavelength. In some embodiments, one of the four different types of nucleotides is unlabeled.

[0203] Although the kit is illustrated herein for configurations having different nucleotides labeled with different dye compounds, it should be understood that the kit may comprise 2, 3, 4 or more different nucleotides having the same dye compound.

[0204] In a particular embodiment, the kit may comprise a polymerase capable of catalyzing the incorporation of nucleotides into a polynucleotide. Other components included in such a kit may include buffers and the like. The nucleotides of the present disclosure and any other nucleotide components including mixtures of different nucleotides may be provided in the kit in a concentrated form to be diluted prior to use. In such an embodiment, a suitable dilution buffer may also be included. Similarly, one or more components determined in the methods set forth herein may be included in the kits of the present disclosure.

[0205] Some embodiments of the present disclosure relate to kits of nucleotides comprising at least one type of label, wherein the nucleotides are labeled with a photo-switchable dye as described herein. In some further embodiments, the kit comprises two or more different types of nucleotides, wherein the first type of nucleotide is labeled with a first photo-switchable label as described herein, and the second type of nucleotide is labeled with the second photo-switchable label as described herein. In additional embodiments, the kit may comprise a third type of nucleotide that comprises a label that emits at the same or substantially the same wavelength as the first photo-switchable label, or that comprises a label that can be excited using the same or substantially the same excitation wavelength as the first photo-switchable label. In further embodiments, the kit may comprise an unlabeled fourth type of nucleotide. In some embodiments, the first type, second type, and third type of nucleotides can be measured by detection at the same wavelength.

[0206] In any of the embodiments of the kits described herein, it can be used on an automated sequencing instrument, wherein the automated sequencer comprises two lasers operating at different excitation wavelengths, and a detection system having a single detection channel set to a fixed emission wavelength.

[0207] In addition to the fluorescent moieties disclosed in the photo-switchable labels, other exemplary fluorescent moieties or their derivatives used as fluorescent moieties include, but are not limited to, fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamido fluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazide methylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and its derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and its derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and its derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and its derivatives such as indolium-based cyanine dyes, benzindolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and their derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes (e.g., those described in US2010 / 0009353, which is incorporated herein by reference in its entirety), NR dyes, near-infrared dyes, and other information known in the art such as Haugland,Those described in the Molecular Probes Handbook, (Eugene, Oreg.), 6th Edition; the Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Edition, Plenum Press New York (1999), Hermanson, Bioconjugate Techniques, 2nd Edition, US2010 / 0009353 or WO 98 / 59066, each of which is incorporated by reference in its entirety. In some embodiments, the third label described herein may also be selected from any of the exemplary fluorescent moieties or derivatives thereof described herein.,

[0208] Sequencing application

[0209] The labeled nucleotides or nucleosides according to the present disclosure can be used in any analytical method, for example, including methods for detecting fluorescent labels attached to nucleotides or nucleosides, whether the labeled nucleotides or nucleosides are present alone or incorporated or linked to larger molecular structures or conjugates. As used herein, the term "incorporated into a polynucleotide" can mean that the 5'-phosphate participates in forming a phosphodiester bond to the 3'-hydroxyl group of a second (modified or unmodified) nucleotide, and this second nucleotide itself can form part of a longer polynucleotide chain. The 3'-end of the nucleotide described herein may or may not participate in forming a phosphodiester bond to the 5'-phosphate of other (modified or unmodified) nucleotides. Thus, in a non-limiting embodiment, the present disclosure provides a method for detecting nucleotides incorporated into a polynucleotide, the method comprising: (a) incorporating at least one nucleotide of the present disclosure into a polynucleotide; and (b) detecting the nucleotide incorporated into the polynucleotide by detecting the fluorescence signal of a dye compound linked to the nucleotide.

[0210] The method may include: a synthesis step (a) that incorporates one or more nucleotides according to the present invention into a polynucleotide; and a detection step (b) that detects one or more nucleotides incorporated into the polynucleotide by detecting or quantitatively measuring the fluorescence of one or more nucleotides incorporated into the polynucleotide.

[0211] Some embodiments of the present application relate to sequencing methods, including: (a) incorporating at least one labeled nucleotide described herein into a polynucleotide; and (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting the fluorescence signal of a new fluorescent dye linked to the nucleotide.

[0212] In one embodiment, in the synthesis step, at least one nucleotide is incorporated into a polynucleotide by the action of a polymerase. However, other methods of linking nucleotides to polynucleotides can be used, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Thus, when referring to nucleotides and polynucleotides, the term "incorporation" can encompass polynucleotide synthesis by chemical as well as enzymatic methods.

[0213] In a specific embodiment, the synthesis step is carried out and optionally includes incubating a template polynucleotide strand with a reaction mixture comprising the fluorescently labeled nucleotides of the present disclosure. A polymerase can also be provided under conditions that allow for the formation of a phosphodiester linkage between the free 3'-hydroxyl group on a polynucleotide strand annealed to the template polynucleotide strand and the 5'-phosphate group on the nucleotide. Thus, the synthesis step can include forming a polynucleotide strand guided by complementary base pairing of the nucleotides to the template strand.

[0214] In all embodiments of the method, the detection step can be carried out while annealing the polynucleotide strand incorporating the labeled nucleotide to the template strand, or after a denaturation step in which the two strands are separated. Other steps can be included between the synthesis step and the detection step, such as chemical or enzymatic reaction steps or purification steps. In particular, the target strand incorporating the labeled nucleotide can be isolated or purified and then further processed or used in subsequent analyses. For example, a target polynucleotide labeled with the nucleotides described herein in the synthesis step can subsequently be used as a labeled probe or primer. In other embodiments, the products of the synthesis steps described herein can be subjected to further reaction steps, and if desired, the products of these subsequent steps can be purified or isolated.

[0215] Those familiar with standard molecular biology techniques will be well acquainted with the appropriate conditions for the synthesis step. In one embodiment, the synthesis step can be analogous to a standard primer extension reaction using nucleotide precursors (including the nucleotides described herein) to form an extended target strand complementary to the template strand in the presence of a suitable polymerase. In other embodiments, the synthesis step itself can form part of an amplification reaction, thereby producing a labeled double-stranded amplification product consisting of annealed complementary strands derived from the replication of the target and template polynucleotide strands. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random primed DNA labeling, etc. A particularly useful polymerase for the synthesis step is one that can catalyze the incorporation of the nucleotides described herein. A variety of naturally occurring or modified polymerases can be used. For example, a thermostable polymerase can be used for synthesis reactions carried out using thermal cycling conditions, while for isothermal primer extension reactions, a thermostable polymerase may not be required. Suitable thermostable polymerases capable of incorporating the nucleotides according to the present disclosure include those described in WO 2005 / 024010 or WO 06120433, each of which is incorporated herein by reference. In synthesis reactions carried out at lower temperatures (e.g., 37°C), the polymerase does not necessarily need to be a thermostable polymerase, and thus the choice of polymerase depends on many factors such as reaction temperature, pH, strand displacement activity, etc.

[0216] In certain non-limiting embodiments, the present disclosure encompasses methods for nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, and any other applications involving the detection of the labeled nucleotides or nucleosides described herein when incorporated into a polynucleotide. Any of a variety of other applications that facilitate the use of polynucleotides labeled with fluorescent dyes can use the dye-labeled nucleotides or nucleosides described herein.

[0217] In one particular embodiment, the present disclosure provides for the use of the labeled nucleotides of the present disclosure in a polynucleotide synthesis sequencing reaction. Synthesis sequencing generally involves the sequential addition of one or more nucleotides or oligonucleotides in the 5' to 3' direction to a growing polynucleotide chain using a polymerase or ligase to form an extended polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the bases present in the one or more added nucleotides can be determined in a detection or "imaging" step. The identity of the added bases can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using conventional Watson-Crick base-pairing rules. For example, when scoring single nucleotide polymorphisms, it may be useful to use the labeled nucleotides described herein to determine the identity of a single base, and such single base extension reactions are within the scope of the present disclosure.

[0218] In one embodiment of the present disclosure, the sequence of a template polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced, the incorporation of the nucleotides being effected by detecting a fluorescent label attached to the incorporated nucleotide. Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared as a hairpin construct, which will contain the primer as part of the hairpin), and the nascent strand is extended in a stepwise manner by the addition of nucleotides to the 3' end of the primer under a polymerase-catalyzed reaction.

[0219] In a particular embodiment, each of the different nucleoside triphosphates (A, T, G, and C) can be labeled with a distinct fluorophore and also contain a protecting group at the 3' position to prevent uncontrolled polymerization. Optionally, one of the four nucleotides may be unlabeled (non-fluorescent). The polymerase incorporates the nucleotides into the nascent strand complementary to the template polynucleotide, and the protecting group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescent signal from each incorporated nucleotide can be optically "read" by a suitable means (e.g., a charge-coupled device, which uses laser excitation and a suitable emission filter). The 3'-protecting group and the fluorescent dye compound can then be removed (deprotected) either simultaneously or sequentially to expose the nascent strand for further nucleotide incorporation. Generally, the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly necessary. Similarly, U.S. Patent 5,302,509, which is incorporated herein by reference, discloses methods for sequencing polynucleotides immobilized on a solid support.

[0220] As described above, the method involves incorporating fluorescently labeled 3′-protected nucleotides A, G, C, and T into a growing strand complementary to a immobilized polynucleotide in the presence of a DNA polymerase. The polymerase incorporates bases complementary to the target polynucleotide, but is blocked from further addition by the 3'-protecting group. The label of the incorporated nucleotide can then be determined, and the protecting group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction can be any polynucleotide for which sequencing is desired. The nucleic acid template for the sequencing reaction will typically contain a double-stranded region with a free 3'-OH group, which serves as a primer or as a starting point for further addition of other nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3'-hydroxyl group on the complementary strand. The overhanging end region of the template to be sequenced can be single-stranded or double-stranded, provided that there is a "nick" in the strand complementary to the template strand to be sequenced to provide a free 3'-hydroxyl group for initiation of the sequencing reaction. In such an embodiment, sequencing can be carried out by strand displacement. In some embodiments, a primer with a free 3'-hydroxyl group can be added as a separate component (e.g., a short oligonucleotide) that hybridizes to the single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced can each form part of a partially self-complementary nucleic acid strand capable of forming an intramolecular duplex (e.g., a hairpin loop structure). Hairpin polynucleotides and methods for their attachment to solid supports are disclosed in PCT publications WO 01 / 57248 and WO 2005 / 047301, which are incorporated herein by reference in their entireties. Nucleotides can be added sequentially to the growing primer, resulting in the synthesis of a polynucleotide strand in the 5' to 3' direction. The identity of the base that has been added can be determined, particularly but not necessarily, after each addition of a nucleotide, thereby providing sequence information of the nucleic acid template. Thus, the nucleotide is incorporated into the nucleic acid strand (or polynucleotide) by forming a phosphodiester linkage with the 5'-phosphate group of the nucleotide and the free 3'-hydroxyl group of the nucleic acid strand.

[0221] The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule consisting of deoxynucleotides and ribonucleotides. The nucleic acid template can contain naturally occurring and / or non-naturally occurring nucleotides as well as natural or non-natural backbone linkages, provided that they do not prevent replication of the template in the sequencing reaction.

[0222] In some embodiments, the nucleic acid template to be sequenced can be attached to a solid support via any suitable ligation method known in the art, such as by covalent attachment. In some embodiments, the template polynucleotide can be directly attached to the solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support can be modified in some way to allow for the direct covalent attachment of the template polynucleotide, or the template polynucleotide can be immobilized via a hydrogel or a polyelectrolyte multilayer, which itself can be non-covalently attached to the solid support.

[0223] Arrays in which polynucleotides have been directly attached to silica-based supports are, for example, those disclosed in WO 00 / 06770 (incorporated herein by reference), in which polynucleotides are immobilized on a glass support by reaction of side-chain epoxy groups on the glass with internal amino groups on the polynucleotide. Additionally, polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in WO 2005 / 047301 (incorporated herein by reference). Further examples of solid-phase supported template polynucleotides are those in which the template polynucleotide is attached to a hydrogel loaded on a silica or other solid support, for example, as described in WO 00 / 31148, WO 01 / 01143, WO 02 / 12566, WO03 / 014392, U.S. Patent 6,465,178, and WO 00 / 53812, each of which is incorporated herein by reference.

[0224] A particular surface on which the template polynucleotide can be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO 2005 / 065814, which is incorporated herein by reference. Specific hydrogels that can be used are described in WO 2005 / 065814 and U.S. Published Patent 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly-N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide).

[0225] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Patent 6,172,218 (incorporated herein by reference). Attachment to beads or microparticles can be used for sequencing applications. A bead library can be prepared, where each bead contains a different DNA sequence. Exemplary libraries and methods for creating them are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of an array of such beads using the nucleotides set forth herein is within the scope of the present disclosure.

[0226] The template to be sequenced can form part of an "array" on a solid support, in which case the array can take any convenient form. Thus, the methods of the present disclosure are applicable to all types of high-density arrays, including single-molecule arrays, cluster arrays, and bead arrays. The labeled nucleotides of the present disclosure can be used for template sequencing on substantially any type of array, including but not limited to those formed by immobilizing nucleic acid molecules on a solid support.

[0227] However, the labeled nucleotides of the present invention are particularly advantageous in the context of sequencing of cluster arrays. In a clustered array, different regions (commonly referred to as sites or features) on the array contain multiple polynucleotide template molecules. Typically, the multiple polynucleotide molecules cannot be resolved individually by optical means but are detected as a whole. Depending on how the array is formed, each site on the array can contain multiple copies of a single polynucleotide molecule (e.g., the site is homologous for a particular single-stranded or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Cluster arrays of nucleic acid molecules can be produced using techniques commonly known in the art. By way of example, WO 98 / 44151 and WO 00 / 18957 (each incorporated herein by reference) describe methods of nucleic acid amplification in which the template and the amplification products remain immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present on the cluster arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with the dye compounds of the present invention.

[0228] The labeled nucleotides of the present disclosure can also be used for sequencing of templates on single-molecule arrays. As used herein, the term "single-molecule array" or "SMA" refers to a population of polynucleotide molecules distributed (or arrayed) on a solid support, wherein any individual polynucleotide is spaced from all other molecules of the population by a spacing sufficient to resolve the individual polynucleotide molecules separately. Thus, in some embodiments, the target nucleic acid molecules immobilized on the surface of the solid support can thus be resolved by optical means. This means that one or more distinct signals, each representing a polynucleotide, will occur within a resolvable region of the particular imaging device used.

[0229] The spacing between adjacent polynucleotide molecules on an array that enables single-molecule detection is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, and even more particularly at least 350 nm. Thus, each molecule can be resolved and detected individually as a single-molecule fluorescence spot, and the fluorescence of the single-molecule fluorescence spot also exhibits single-step photobleaching.

[0230] The terms "individually resolvable" and "individual resolution" are used herein to specify that, when visualized, a molecule on an array can be distinguished from its neighboring molecules. The separation between individual molecules on the array will be determined in part by the particular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to the published applications WO 00 / 06770 and WO 01 / 57248, which are hereby incorporated by reference in their entirety. Although one use of the nucleotides of the present disclosure is in a sequencing-by-synthesis reaction, the use of the nucleotides is not limited to such methods. Indeed, the nucleotides can be advantageously used in any sequencing method that requires detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0231] In particular, the labeled nucleotides of the present disclosure can be used in automated fluorescence sequencing protocols, particularly fluorescence dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and his colleagues. Such methods typically use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into a primer extension sequencing reaction. The so-called Sanger sequencing method and related protocols (Sanger-type) utilize random chain termination with labeled dideoxynucleotides.

[0232] Accordingly, the present disclosure also encompasses labeled nucleotides that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, such dideoxynucleotides being suitable for Sanger-type sequencing methods and the like.

[0233] It should be appreciated that the labeled nucleotides of the present disclosure incorporating 3'-protecting groups can also be used in the Sanger method and related protocols, since the same effect obtained with dideoxynucleotides can be achieved by using nucleotides having 3'-OH protecting groups: both prevent the incorporation of subsequent nucleotides. When using the nucleotides having 3' protecting groups according to the invention in a Sanger-type sequencing method, it should be understood that the dye compound or detectable label attached to the nucleotide need not be attached by a cleavable linker, since each instance of the labeled nucleotides incorporated in the present disclosure does not require subsequent incorporation of a nucleotide and thus does not require removal of the label from the nucleotide.

[0234] Additional embodiments disclosed herein provide a method for determining multiple nucleic acid sequences, the method comprising: providing a sample comprising multiple different nucleic acids, each nucleic acid comprising a template and a primer; performing cycles of a sequencing reaction, wherein the cycle comprises extending the primers of the nucleic acids in the sample to form multiple extended primers having at least four different labeled nucleotide types as described herein, thereby forming an extended sample, obtaining a first set of signals from the extended sample, wherein at least two different nucleotide types in the extended primers are in a signal state and at least one different nucleotide type in the extended primers is in a dark state; irradiating the polynucleotides with a light source to change the emission signals of certain nucleotide labels and obtaining a second set of signals from the sample, wherein, compared to the second set of signals, two different nucleotide types in the first set of signals are in different states; and determining the sequences of the multiple different nucleic acids by evaluating the first set of signals and the second set of signals from the cycles. In some embodiments, the multiple different nucleic acids are attached to a substrate. In some embodiments, the extension of the primers comprises polymerase-catalyzed addition of different nucleotide types. In some embodiments, the different nucleotide types comprise reversible protecting moieties, such that a single nucleotide type is added to each extended primer in each cycle. In some embodiments, the extension of the primers comprises ligase-catalyzed addition of oligonucleotides comprising different nucleotide types. In some embodiments, during obtaining the first set of signals from the extended sample, two different nucleotide types in the extended primers are in a dark state. In preferred embodiments, the sequencing reaction cycles as described above are repeated one or more times. Example

[0235] Additional embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0236] Example 1

[0237] In this example, a simplified method for the identification of incorporated dyes based on a modified photoswitchable red-emitting fluorophore is described. Figure 2 The workflow of one incorporation cycle is shown.

[0238] Table 1 shows the structures and functions of the fluorescent labels attached to each base required for this method. The "A" and "C" dyes have photoswitching capabilities. The "T" dye can be a standard red-emitting dye, for example, a red dye that is the same as a part of the photoswitchable dimer of the "A" nucleotide. The linking group that attaches the dye to the nucleotide is the LN3 linking group described herein and is only partially shown in Table 1 and the following photochemical reaction scheme. Other linking groups disclosed herein can also be used. All of these dyes are capable of functioning in a biologically relevant medium.

[0239] Table 1.

[0240]

[0241]

[0242] The design of the novel A dye enables its photo-switching using blue laser or LED (405 nm). Photo-chemical activation results in a ring-opening reaction that converts the bottom of the dyad into a quencher that can inhibit the emission of the silicon rhodamine dye (Scheme 1).

[0243]

[0244] Scheme 1. Photo-chemical quenching of a dyad with a silicon rhodamine fluorophore.

[0245] While converting the A dye to the quenched “off” state, the photo-activatable C dye can be switched to the “on” fluorescent state by exposure to blue laser or LED radiation (405 nm). For the C dye, a similar ring-opening reaction reversibly opens the oxazine ring, which reversibly generates a highly fluorescent state with the maximum absorbance concentrated around 550 - 650 nm (Scheme 2). Therefore, all the dyes proposed here can be excited with the same red LED or laser (633 nm).

[0246]

[0247] Scheme 2. Photo-chemical activation of a dyad with a silicon rhodamine fluorophore.

[0248] As Figure 2 shown, four different types of nucleotides according to Table 1 are exposed to the flow cell for incorporation into the polynucleotide. In the first imaging step, the luminescence of each cluster is recorded. In this first imaging event, fluorescence signals emitted from the “A” and “T” nucleotides are detected. Then, the “A” dye is quenched and the “C” dye is activated using a blue laser with a wavelength of 405 nm. Then a second imaging step is performed, and the luminescence of each cluster is recorded again. In this second imaging event, fluorescence signals emitted from the “C” and “T” nucleotides are detected. In both imaging events, the “G” nucleotide is dark (unlabeled). The nucleotides are identified by analyzing the different emission patterns of each base in the two images. The image analysis software processes the combination of Image 1 and Image 2 to identify which bases are incorporated at each cluster position. After the second imaging event, the incorporated nucleotides are deprotected according to the standard procedure to allow the incorporation of another nucleotide. This sequencing cycle is repeated “n” times to generate “n” bases of the read length.

[0249] In this embodiment, a 405 nm blue laser or LED is used, which can be further tuned by introducing additional substituents in the coumarin moiety of the "C" dye or the spironaphthoxazine moiety of the "A" dye. This will allow for full compatibility with the current iSeq TM system that has a 450 nm laser. The proposed modification will also reduce the likelihood of possible DNA damage caused by repeated exposure to higher energy radiation over multiple sequencing cycles.

Claims

1. A method for determining the nucleotide sequence of a target polynucleotide, comprising: (a) performing a sequencing reaction comprising one or more of the following cycles: (i) incorporating four different types of nucleotide conjugates into a plurality of polynucleotides complementary to the target polynucleotide to produce extended polynucleotides, wherein the first type of nucleotide conjugate comprises a first photo-switchable label, the second type of nucleotide conjugate comprises a second photo-switchable label, and the third type of nucleotide conjugate comprises a third label; (ii) detecting a first set of signals from the extended polynucleotides in each cycle via a first imaging event; (iii) irradiating the extended polynucleotides with a light source to cause a change in the emission signals of the first photo-switchable label and the second photo-switchable label; (iv) detecting a second set of signals from the extended polynucleotides via a second imaging event; (b) determining the sequence of the target polynucleotide based on the sequentially incorporated nucleotide conjugates; wherein: the incorporation of the first type of nucleotide conjugate is determined from the signal state in the first imaging event and the dark state in the second imaging event; the incorporation of the second type of nucleotide conjugate is determined from the dark state in the first imaging event and the signal state in the second imaging event; the incorporation of the third type of nucleotide conjugate is determined from the signal states in the first imaging event and the second imaging event; the incorporation of the fourth type of nucleotide conjugate is determined from the dark states in the first imaging event and the second imaging event; and the four different types of nucleotide conjugates are present simultaneously and compete for incorporation in each cycle.

2. The method of claim 1, wherein step (a) is repeated at least 50 times.

3. The method of claim 1, wherein the incorporation of the nucleotide conjugate is carried out by a polymerase.

4. The method of claim 1, wherein the different types of nucleotide conjugates comprise reversible terminator moieties.

5. The method of claim 4, wherein step (a) further comprises cleaving the reversible terminator moiety from the incorporated nucleotide conjugate prior to the next incorporation cycle.

6. The method of claim 1, wherein the nucleotide conjugate comprises a nucleotide type selected from: dATP, dTTP, dUTP, dCTP, dGTP and their unnatural nucleotide analogs.

7. The method of claim 1, wherein the labels of the first type of nucleotide conjugate and the third type of nucleotide conjugate comprise the same fluorescent moiety.

8. The method of claim 1, wherein the labels of the first type of nucleotide conjugate, the second type of nucleotide conjugate and the third type of nucleotide conjugate comprise different fluorescent moieties.

9. The method of claim 8, wherein different fluorescent moieties are detected using the same emission filter.

10. The method of claim 1, wherein the fourth type of nucleotide conjugate is not labeled with a fluorescent moiety.

11. The method according to claim 1, wherein the irradiation in step (a)(iii) does not change the signal detected from the third label of the third type of nucleotide conjugate.

12. The method according to claim 1, wherein the irradiation light source in step (a)(iii) comprises a laser, a light emitting diode (LED), or a combination thereof.

13. The method according to claim 1, wherein the irradiation light source in step (a)(iii) has a wavelength different from the excitation wavelength used in the first imaging event.

14. The method according to claim 1, wherein the irradiation light source in step (a)(iii) has a wavelength of 350 nm to 450 nm.

15. The method according to claim 14, wherein the irradiation light source in step (a)(iii) has a wavelength of 405 nm.

16. The method according to claim 1, wherein the first imaging event and the second imaging event have the same excitation wavelength.

17. The method according to claim 16, wherein the first imaging event and the second imaging event have an excitation wavelength of 550 nm to 650 nm.

18. The method according to claim 17, wherein the first imaging event and the second imaging event have an excitation wavelength of 633 nm.

19. The method according to claim 1, wherein the first photo-switchable label comprises a first fluorescent moiety covalently bonded to a first photochromic moiety; or a first fluorescent moiety covalently bonded to a first photochromic moiety via a first linking group.

20. The method according to claim 19, wherein the first fluorescent moiety comprises a fluorescent moiety that emits red light.

21. The method according to claim 19, wherein the first fluorescent moiety comprises a silicon rhodamine moiety.

22. The method according to claim 19, wherein the first photochromic moiety comprises a spiropyran or a spirothianine moiety.

23. The method according to claim 19, wherein the first photochromic moiety comprises a structure of formula (I): Wherein: X is O (oxygen) or S (sulfur); Each R 1a and R 2a are independently selected from: H, halogen, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted C 2-6 alkenyl, unsubstituted or substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy; R 3 selected from H, C 1-6 alkyl and -(CH2) n -R 4 ; R 4 selected from C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic group, and 3- to 7-membered heterocyclic group, each of which is substituted or unsubstituted; Ring A is C 6-10 aryl or a 5- to 10-membered heteroaryl, each of which is substituted by at least one electron-withdrawing group; n is an integer from 1 to 6; and wherein the irradiation step (a)(iii) causes the cleavage of the spiro C―X bond.

24. The method according to claim 23, wherein, X is S.

25. The method according to claim 23, wherein each R 1a and R 2a is C 1-6 alkyl.

26. The method according to any one of claims 23 to 25, wherein, Ring A is a phenyl or naphthyl group substituted by at least one electron-withdrawing group.

27. The method according to claim 26, wherein the electron-withdrawing group is selected from nitro, cyano, fluorine, bromine, -S(O)2OH, -S(O)2CF3, ammonium, alkylammonium, and a C 1-6 alkyl group substituted with one or more fluorine or bromine atoms.

28. The method according to claim 19, wherein the first photochromic moiety comprises a structure of formula (Ia):

29. The method according to claim 19, wherein the first photo-switchable label comprises the following structure: where L 1 is the first linking group.

30. The method according to claim 1, wherein the second photo-switchable label comprises a second fluorescent moiety covalently bonded to a second photochromic moiety or a second fluorescent moiety covalently bonded to a second photochromic moiety via a second linking group.

31. The method according to claim 30, wherein the second fluorescent moiety comprises a coumarin moiety.

32. The method according to claim 30, wherein the second photochromic moiety comprises an oxazine moiety or a thiazine moiety.

33. The method according to claim 30, wherein the second photochromic moiety comprises a structure of formula (II): Wherein: Y is O (oxygen) or S (sulfur); Each R 1b and R 2b are independently selected from: H, halogen, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted C 2-6 alkenyl, unsubstituted or substituted C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 1-6 haloalkoxy; Ring B is C 6-10 aryl or 5- to 10-membered heteroaryl; each of which is substituted with at least one electron-withdrawing group; and where * represents a point of attachment to the fluorescent moiety, and said irradiation step (a)(iii) causes cleavage of the carbon*―Y bond.

34. The method according to claim 33, wherein Y is O. The method according to claim 33, wherein each R 1b and R 2b is C 1-6 alkyl.

36. The method according to any one of claims 33 to 35, wherein ring B is a phenyl or naphthyl group substituted by at least one electron-withdrawing group.

37. The method according to claim 36, wherein the electron-withdrawing group is selected from nitro, cyano, fluorine, bromine, -S(O)2OH, -S(O)2CF3, ammonium, alkylammonium, and a C 1-6 alkyl group substituted with one or more fluorine or bromine atoms.

38. The method according to claim 30, wherein said second photochromic moiety comprises a structure of formula (IIa):

39. The method according to claim 30, wherein said second photoswitchable label comprises the following structure: where L 2 is a second linking group.

40. The method according to claim 1, wherein said plurality of polynucleotides are attached to a substrate.

41. The method according to claim 40, wherein detecting said first set of signals and said second set of signals comprises obtaining an image of said substrate.

42. A kit comprising four types of nucleotides, wherein the first type of nucleotide is a labeled nucleotide conjugate comprising a photoswitchable label, wherein said photoswitchable label comprises a fluorescent moiety covalently bonded to a photochromic moiety or a fluorescent moiety covalently bonded to a photochromic moiety via a linking group, wherein said fluorescent moiety comprises a silicon rhodamine moiety and said photochromic moiety comprises a structure of formula (I): Wherein: X is S (sulfur); Each R 1a and R 2a is independently C 1-6 alkyl; R 3 selected from H, C 1-6 alkyl and -(CH2) n -R 4 , R 4 selected from C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic group, and 3- to 7-membered heterocyclic group, each of which is substituted or unsubstituted Where n is an integer from 1 to 6; and wherein ring A is a phenyl or naphthyl group substituted by at least one electron-withdrawing group selected from nitro, cyano, fluorine, bromine, -S(O2)OH, -S(O)2CF3, ammonium, alkylammonium, and C 1-6 alkyl substituted by one or more fluorine or bromine atoms; And the second type of nucleotide is a labeled nucleotide conjugate, wherein said photoswitchable label comprises a fluorescent moiety covalently bonded to a photochromic moiety or a fluorescent moiety covalently bonded to a photochromic moiety via a linking group, wherein said fluorescent moiety comprises a coumarin moiety and said photochromic moiety comprises a structure of formula (I); Wherein the third type of nucleotide comprises a label emitting at the same wavelength as the first type of nucleotide, and The fourth type of nucleotide is unlabeled.

43. The kit according to claim 42, wherein the photochromic moiety of said first type of nucleotide conjugate comprises a structure of formula (Ia):

44. The kit according to claim 42, wherein the photoswitchable label of said first type of nucleotide conjugate comprises the following structure: wherein L 1 is a linking group.

45. The kit according to claim 44, wherein the photochromic moiety of said first type of nucleotide conjugate comprises a structure of formula (IIa):

46. The kit according to claim 44, wherein the photoswitchable label of said first type of nucleotide conjugate comprises the following structure: wherein L 2 is a linking group.

47. The kit according to claim 46, wherein the photoswitchable label of said first type of nucleotide conjugate is linked to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base.

48. The kit according to claim 42, wherein the photoswitchable label of said first type of nucleotide conjugate is linked to the nucleobase via a cleavable linking group.

49. The kit according to claim 42, wherein the photochromic moiety of said second type of nucleotide conjugate comprises an oxazine moiety or a thiazine moiety. The kit according to claim 42, wherein the photochromic moiety of the second type of nucleotide conjugate comprises a structure of formula (II): Wherein: Y is O (oxygen); Each R 1b and R 2b is independently C 1-6 alkyl; and Ring B is phenyl or naphthyl substituted by at least one electron-withdrawing group selected from nitro, cyano, fluorine, bromine, -S(O2)OH, -S(O)2CF3, ammonium, alkylammonium, and C 1-6 alkyl substituted by one or more fluorine or bromine atoms; and where * represents the point of attachment to the fluorescent moiety.

51. The kit according to claim 50, wherein the first type of nucleotide, the second type of nucleotide, and the third type of nucleotide are detected at the same emission wavelength.

52. The kit according to claim 42, for use on an automated sequencer, wherein the automated sequencer comprises two lasers operating at different excitation wavelengths, and a detection system having a single detection channel set to a fixed emission wavelength.

Images