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

Abstract

Embodiments of the present disclosure relate to nucleotides labeled with photoswitchable compounds, and provided herein are methods and kits for using these labeled nucleotides for sequencing applications.

Description

Technical Field

[0001] (Background) (Field) The present disclosure generally relates to nucleotides labeled with optically switchable labels and their use in polynucleotide sequencing methods and applications.

Background Art

[0002] The detection of analytes such as nucleic acid sequences present in biological samples is used as a method for research such as the identification and classification of microorganisms, the diagnosis of infectious diseases, the detection and characterization of genetic abnormalities, the identification of gene changes associated with cancer, genetic susceptibility to diseases, and the measurement of responses to various types of treatment. A common technique for detecting analytes such as nucleic acid sequences in biological samples is nucleic acid sequencing.

[0003] The need for high-throughput, small, and inexpensive DNA sequencing technologies has been shown to be beneficial in enjoying the benefits of genomic sequencing. Personalized medicine is on the forefront and will benefit from such technologies. Sequencing an individual's genome to identify potential mutations and abnormalities is important in determining whether a person has a particular disease, followed by treatment tailored to that individual. To accommodate such proactive efforts, sequencing needs to advance and be able to support high-throughput technologies not only in terms of their high-throughput capabilities but also in terms of ease of use, time and cost efficiency, and access to equipment and reagents by clinicians.

[0004] Photo-switchable fluorescent probes are an important new development in super-resolution fluorescence microscopy, and to access improved spatial resolution, dyes that can be switched from a dark state to an emissive state are required. See Vaughan et al., FEBS Letters 588 (2014) 3603-3612. Photo-switchable fluorescent probes utilize photochromic compounds that reversibly switch between states with different absorption spectra upon irradiation with light of an appropriate wavelength and intensity. The species generated in the photo process usually return to the starting species by thermal means or additional illumination. For example, Anderson et al. recently reported the synthesis of small molecule dyads consisting of a red-emitting fluorophore covalently attached to a photo-switchable quencher and the use of the dyads for biological imaging. See Anderson et al., Org. Lett. 2016, 18, 3666~3669. In a similar effort, Bossi et al. reported the synthesis of dyads equipped with an oxazine photochrome conjugated to a coumarin fluorophore and an antibody for super-resolution imaging. See Bossi et al., J. Phys. Chem. C2016, 120, 12860-12870. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] There is a need to develop new photo-switchable fluorescent labels that can be used in biological imaging applications, including next-generation sequencing applications. MEANS FOR SOLVING THE PROBLEMS

[0006] (Overview) Some embodiments of the present disclosure relate to nucleotide conjugates containing a photoswitchable label, the photoswitchable label comprising a fluorescent moiety covalently attached to a photochromic moiety, optionally via a linker. Upon irradiation with a light source such as a laser or LED light, the photoswitchable label may reversibly change from an "on" state to an "off" state, or 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 spirothiopyran moiety having the structure of formula (I): [Chemical Formula] X is O (oxygen) or S (sulfur), R 1a and R 2a are each independently H, halo, 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 C1-6 haloalkoxy R 3 is selected from the group consisting of H, C 1-6 alkyl, and -(CH2)n-R4, R 4 is selected from the group consisting of C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic, and 3- to 7-membered heterocyclic, each of which may be optionally substituted. 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.

[0007] In some other embodiments, the photochromic moiety of the photoswitchable label comprises the structure of formula (II): [Chemical] Y is O (oxygen) or S (sulfur), R 1b and R 2b are each independently H, halo, 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 Ring B is C 6-10 aryl or 5- to 10-membered heteroaryl; each is substituted with at least one electron-withdrawing group. Here, * indicates the point of attachment to the fluorescent moiety.

[0008] Some embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the nucleotide conjugates described herein.

[0009] Some further embodiments of the present disclosure relate to methods for determining the nucleotide sequence of a target polynucleotide, comprising performing a sequencing reaction comprising one or more of the following cycles: (i) Incorporating different types of nucleotide conjugates into a plurality of polynucleotides complementary to the target polynucleotide to generate extended polynucleotides, each of the first type of nucleotide conjugate comprising a first photoswitchable label, each of the second type of nucleotide conjugate comprising a second type of photoswitchable label, and each of the third type of nucleotide conjugate comprising a third label. (ii) Detecting a first collection of signals from the extended polynucleotide in each cycle via a first imaging event. (iii) Irradiating the extended polynucleotide 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; (iv) detecting a second set of signals from the extended polynucleotide via a second imaging event; and determining the sequence of the target polynucleotide based on the continuously 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 incorporation in each cycle. In some such embodiments, 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. In some such embodiments, 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. In some such embodiments, 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. In some such embodiments, 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. 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).

[0010] Some further embodiments of the present disclosure relate to kits comprising one or more types of nucleotide conjugates and photo-switchable labels, as described herein. The kits can be used in applications such as sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, cell assays (e.g., cell binding or cell function analysis), or protein assays (e.g., protein binding assays or protein activity assays). The use can be performed with an automated instrument for carrying out certain techniques such as an automated sequencing instrument. A sequencing device may include only one laser to distinguish different detectable labels. The sequencing device may include additional lasers or light sources operating at different wavelengths to activate a photo-switchable label from an “on” state to an “off” state, or vice versa.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0012] (Detailed Description) The iSeq100™, Illumina's next-generation sequencing system, uses CMOS-based technology to provide a simplified, accessible benchtop sequencing solution. Standard sequencing workflows are shown in FIGS. 1A and 1B. This is also referred to as single-channel sequencing. Each sequencing cycle includes two chemical steps and two imaging steps. In FIG. 1A, in the first chemical step, the flow cell is exposed to a mixture of nucleotides containing fluorescently labeled adenine and thymine. During the first imaging step, the light emission from each cluster is recorded by a CMOS sensor. In the second chemical step, the fluorescent label is removed from adenine and added to cytosine. In both chemical steps, guanine is dark (unlabeled). A second image is recorded. In FIG. 1B, the combination of Image 1 and Image 2 is processed by image analysis software to identify which base is incorporated at each cluster position. This sequencing cycle is repeated "n" times to create a read length of "n" bases. Unlike 4-channel SBS chemistry where the sequencer uses different dyes for each nucleotide, the iSeq100™ system uses one dye per sequencing cycle. In single-channel chemistry, adenine has a removable label and is labeled only in the first image. Cytosine has a linker group to which a label can be attached and is labeled only in the second image. Thymine has a permanent fluorescent label and is labeled in both images, and guanine is permanently dark. Nucleotides are identified by analyzing the different emission patterns of each base across the two images.

[0013] Embodiments of the present disclosure relate to a new method for determining the nucleotide sequence of a target polynucleotide. In particular, the method includes the use of nucleotides labeled with a photoswitchable dye that can undergo a light-induced color change upon irradiation with a light source.

[0014] There are several advantages associated with the use of photo-switchable fluorophores. Photo-switchable dyes are only required for a single imaging event and are then cleaved immediately afterwards, so there is no need to demonstrate switching robustness. These dyes are known to be able to be switched multiple times without showing fatigue resistance. In this approach, since the conversion of the emission state of the dye is induced "remotely" via photon energy, there is no need to introduce a dedicated cleavage mixture or exchange reagent. Since switching occurs on a time scale of less than a microsecond, the time required for reagent injection and ejection into the flow cell is significantly reduced, so the total cycle time also needs to be minimized. Conversion based on irradiation is usually high-yield and is an essential property to avoid signal mixing or suppression of emission.

[0015] Furthermore, the ability to control the emission state of a fluorescent marker is very useful for nucleic acid sequencing applications. For example, a problem often seen with many similar tags is that residual radiation can contribute to an unwanted background signal after successful use in a process step. Remotely inducing a change in the emission profile based on isomerization, conformational changes, or a reversible ring-opening process may improve the signal-to-noise ratio.

[0016] In some embodiments, the method may include the following steps: (a) Performing a sequencing reaction including the following iterative cycle: (i) Incorporating four different types of nucleotide conjugates into a plurality of polynucleotides complementary to a target polynucleotide to generate an extended polynucleotide, each of the first type of nucleotide conjugate includes a first photo-switchable label, each of the second type of nucleotide conjugate includes a second photo-switchable label, and each of the third type of nucleotide conjugate includes a third label. (ii) Detecting a first collection of signals from the extended polynucleotide in each cycle via a first imaging event. (iii) irradiating the extended polynucleotide with a light source after the first imaging event to cause a change in the emission signals of the first light-switchable label and the second light-switchable label; (iv) detecting a second set of signals from the extended polynucleotide via a second imaging event; (b) determining the sequence of the target polynucleotide based on the continuously incorporated nucleotide conjugates.

[0017] In some embodiments, 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. And 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.

[0018] The "signal state" used with respect to a detection event means a state in which a particular signal is generated in the detection event. For example, a nucleotide subunit is in a signal state and can be detectable when it binds to a fluorescent label detected in a fluorescence detection step by excitation and emission of the fluorescent label in a sequencing method. The term "dark state" used with respect to a detection event means a state in which a particular signal is not generated in the detection event. For example, a nucleotide subunit can be in a dark state when the nucleotide lacks a fluorescent label and / or does not emit fluorescence that is specifically detected in the fluorescence detection step of a sequencing method. Detection of the dark state may include background fluorescence that may be present even without a fluorescent label. For example, some reaction components may exhibit minimal fluorescence when excited at a particular wavelength. Therefore, even in the absence of a fluorescent moiety, there may be background fluorescence from such components. Furthermore, background fluorescence can be due to, for example, light scattering from an adjacent sequencing reaction, which can be detected by a detector. Thus, the "dark state" can include background fluorescence such as when there is no particular inclusion of a fluorescent moiety, such as when a nucleotide lacking a fluorescent label is utilized in the methods described herein. However, such background fluorescence is considered distinguishable from the signal state, and thus nucleotide incorporation of unlabeled nucleotides (or "dark" nucleotides) remains distinguishable.

[0019] 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, four different types of nucleotide conjugates are present simultaneously and compete for incorporation during each cycle. In some further embodiments, incorporation of the nucleotide conjugate is performed by a polymerase.

[0020] In some embodiments, the different types of nucleotide conjugates include reversible terminator moieties. In some further embodiments, step (a) further includes cleaving the reversible terminator moiety from the incorporated nucleotide conjugate prior to the next incorporation cycle. In some further embodiments, the nucleotide conjugate includes a nucleotide type selected from the group consisting of dATP, dTTP, dUTP, dCTP, dGTP, and their unnatural nucleotide analogs.

[0021] 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 include the same fluorescent moiety. In another embodiment, the labels of the second type of nucleotide conjugate and the third type of nucleotide conjugate can include 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 include different fluorescent moieties. The different fluorescent moieties can be detected using the same emission filter or different emission filters. 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 change the signal detected from the third label of the third type of nucleotide conjugate

[0022] In some embodiments, the illumination light source in step (a)(iii) may include a laser, a light emitting diode (LED), or a combination thereof. In some embodiments, the illumination light source in step (a)(iii) has a wavelength different from the excitation wavelength used in the first imaging event. In some such embodiments, the illumination light source in step (a)(iii) may have a wavelength from about 350 nm to about 450 nm. In one embodiment, the illumination light source in step (a)(iii) has a wavelength of about 405 nm.

[0023] 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, the first label, the second label, and the third label can be detected using one detection channel. In some such embodiments, the first imaging event and the second imaging event can have an excitation wavelength from 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.

[0024] (First photoswitchable label) In some embodiments of the methods described herein, when the light source described in step (a)(iii) herein is irradiated, 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 may switch from "on" to "off".

[0025] In some embodiments, the first photoswitchable label comprises a first fluorescent moiety covalently attached to a first photochromic moiety, optionally via a first linker. In some embodiments, the first linker 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, for example, 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: [Chemical Formula] .

[0026] In some embodiments, the first photochromic moiety can comprise a spiropyran or spirothiopyran moiety. In some such embodiments, the first photochromic moiety comprises a structure of formula (I): [Chemical Formula] X is O (oxygen) or S (sulfur), R 1a and R 2a are each independently H, halo, 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 C1-6 haloalkoxy R 3 is selected from the group consisting of H, C 1-6 alkyl, and -(CH2) n -R 4 and is selected from the group consisting of, R 4 is selected from the group consisting of C 6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic, and 3- to 7-membered heterocyclic, each optionally substituted. Ring A is C6-10 Aryl or 5- to 10-membered heteroaryl, each substituted with at least one electron-withdrawing group. n is an integer from 1 to 6.

[0027] In some such embodiments, the irradiation step (a)(iii) causes cleavage of the spiro carbon*-X bond of formula (I), resulting in a ring-opening reaction that converts the first photochromic moiety into a quencher capable of suppressing it. Emission of the first fluorescent moiety. Optionally, the cleavage of the spiro carbon*―X bond is reversible.

[0028] In some embodiments of the first photochromic moiety of formula (I), the phenyl moiety fused to the 5-membered pyrrolidine may be optionally substituted. In some embodiments, X is S. In some further embodiments, each R 1a and R 2a is C1-6 alkyl, for example, each R 1a and R 2a is methyl. In some embodiments, ring A is phenyl or naphthyl substituted with at least one electron-withdrawing group. Non-limiting examples of electron-withdrawing groups are nitro, cyano, fluoro, bromo, -S(O)2OH, triflyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluoros or bromos, and sulfonyl substituted with one or more fluoros or bromos.

[0029] In one embodiment, the first photochromic moiety comprises a structure of formula (Ia):

Chemical formula

[0030] In one embodiment, the first photoswitchable label comprises the following structure:

Chemical formula

[0031] Alternatively, the first photo-switchable label can include only a photochromic moiety and optionally a linker, and the photochromic moiety is in step (a)(iii).

[0032] (The second photo-switchable label) In some embodiments of the methods described herein, upon irradiation with the light source described in step (a)(iii), the emission signal of the second photo-switchable label changes, for example, from a non-emissive dark state to a signal state. In other words, the observed emission signal may switch from "off" to "on".

[0033] In some embodiments, the second photo-switchable label includes a second fluorescent moiety covalently bonded to a second photochromic moiety, optionally via a second linker. In some embodiments, the second linker can be part of the π-conjugated system of the second fluorescent moiety. In some embodiments, the second fluorescent moiety includes a fluorophore that emits red light, for example, red light having a wavelength between about 600 nm and about 700 nm. In some such embodiments, in some of those embodiments, the second fluorescent moiety includes a coumarin fluorophore. In one embodiment, the coumarin fluorophore includes the following structure: [Chemical formula] .

[0034] In some embodiments, the second photochromic moiety may include an oxazine moiety or a thiazine moiety. In some such embodiments, the second photochromic moiety includes a structure of formula (II): [Chemical formula] (II) Y is O (oxygen) or S (sulfur), R 1b and R 2b are each independently H, halo, 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 C1-6 haloalkoxy Ring B is C 6-10 aryl or 5- to 10-membered heteroaryl; each is substituted with at least one electron-withdrawing group. Here, * indicates the bonding point to the fluorescent moiety.

[0035] In some such embodiments, the irradiation step (a)(iii) causes 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. Optionally, the cleavage of the carbon*―Y bond is reversible.

[0036] In some embodiments of the second photochromic moiety of formula (II), the phenyl moiety fused to the 5-membered pyrrolidine moiety may be optionally substituted. In some embodiments, Y is O. In some further embodiments, each R 1b and R 2b is C1-6 alkyl, for example, each R 1b and R 2bis methyl. In some embodiments, ring B is phenyl or naphthyl substituted with at least one electron-withdrawing group. In other embodiments, ring B can be selected from 6-membered heteroaryls such as pyridyl or pyrimidyl. Non-limiting examples of electron-withdrawing groups are nitro, cyano, fluoro, bromo, -S(O)2OH, triflyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluoros or bromos, and sulfonyl substituted with one or more fluoros or bromos.

[0037] In one embodiment, the second photochromic moiety comprises a structure of formula (IIa):

Chemical formula

[0038] In one embodiment, the second photoswitchable label comprises a structure:

Chemical formula

Chemical formula

[0039] Alternatively, the second photoswitchable label can include only the photochromic moiety and optionally a linker, and the photochromic moiety can switch from a non-emissive dark state to an emissive state upon irradiation with the light source described in step (a)(iii) and can itself act as a phosphor.

[0040] Non-limiting examples of light-switchable labels or photochromic moieties include diarylethenes (e.g., bisthienylethene derivatives exemplified below), azines (e.g., azobenzene exemplified below), photochromic quinones (e.g., phthoxynaphthacenequinone), spirooxazines, spirothiazines, mesoaldehyde 1-allyl-1-phenyl-2-phenylsazone, tetrachloro-1,2-ketonaphthalenone, thioindigoid, dinitrobenzylpyridine, chromic, and the like.

Chem.

[0041] 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 polynucleotide is attached to a nanowell on the surface of the flow cell.

[0042] 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.

[0043] Additional exemplary embodiments are described below.

[0044] In some embodiments, a method of sequencing nucleic acids involves the use of one fluorescent label for the direct or indirect detection of three different nucleotide types and one nucleotide type that is not detected by the presence of a fluorescent signal but instead is detected by its absence or omission. The absence of a fluorescent signal. In some embodiments, a method of sequencing nucleic acids involves the use of two or more different fluorescent labels having the same or similar excitation / emission spectra for the direct or indirect detection of three different nucleotide types and one nucleotide type that is not detected by one nucleotide type. The presence of a fluorescent signal is instead detected by the absence or omission of a fluorescent signal. The same or similar excitation and emission spectra are such that a laser excites two or more different fluorescent labels and an optical filter captures their emitted fluorescent signals. Detection of fluorescence for determining the sequence of a nucleic acid sample is performed in time and space, e.g., at different times during a sequencing reaction (i.e., before and after a change in reaction conditions such as enzymatic cleavage, change in environmental pH, addition of additional reagents), providing a fluorescence pattern such as a fluorescence transition pattern, and their cumulative pattern determines the sequence of the nucleic acid target. Thus, the methods described herein are time and cost efficient and enable simplification of the associated sequencing equipment.

[0045] Typical applications that utilize differences in spatio-temporal fluorescence patterns to determine a target nucleic acid sequence are the methods and techniques of sequencing by synthesis (SBS). Thus, the embodiments described herein find particular utility in the sequence by synthetic fluorescence applications. The embodiments described herein are examples of innovative methods of fluorescence sequencing, but the disclosed embodiments are also useful for a variety of other applications where detection of multiple analytes (i.e., nucleotides, proteins, or fragments thereof) in a sample is desired.

[0046] In the development of embodiments of sequencing using a minimal set of dyes, experiments have revealed alternative strategies for distinguishing nucleotide incorporation using only one or two fluorescent moieties. These strategies provide all four nucleotide types that are present simultaneously in a sequencing cycle and use a minimal set of dyes and optical filter sets. In some embodiments, one or two excitation and emission filters are used and three or fewer fluorescent labels are utilized to determine the incorporation of all four nucleotide types that are present during the reaction. In preferred embodiments, two or fewer fluorescent labels (or two or three of the same or similar excitation / emission spectra) are utilized to determine the incorporation of all four nucleotide types that are present during the reaction using one optical excitation range and one excitation range. Detection emission filter.

[0047] In some embodiments, sequencing using a minimal set of dyes is performed on a substrate such as glass, plastic, semiconductor chip, or a composite-derived substrate. In some embodiments, for example, for single target sequencing, one nucleic acid species is provided on the substrate. In other embodiments, the sequencing can also be in a multiplex format where multiple nucleic acid targets are detected and sequenced in parallel, for example, in a flow cell or array type format. The embodiments described herein are particularly advantageous when performing parallel sequencing or massively parallel sequencing. Platforms for performing fluorescence parallel sequencing include, but are not limited to, platforms provided by Illumina, Inc. (HiSeq, Genome Analyzer, MiSeq, iSeq, iScan platforms, etc.), Life Technologies (SOLiD, etc.), Helicos Biosciences (Heliscope, etc.), 454 / Roche Life Sciences (Branford, Connecticut) and Pacific Biosciences (e.g., SMART). Flow cells, chips, and other types of surfaces capable of accommodating multiple nucleic acid species are examples of substrates utilized for parallel sequencing. In a multiplex format where multiple nucleic acid species are sequenced in parallel, clonally amplified target sequences (e.g., via emulsion PCR (emPCR) or bridge amplification) are typically covalently immobilized on the substrate. For example, when performing emulsion PCR, the target of interest is immobilized on beads, while the clonally amplified target is immobilized at specific locations in the channels of a flow cell or on an array or chip.

[0048] The flow cells used with the compositions and methods described herein can be used for sequencing in many ways. For example, a DNA sample, such as a DNA library, can be applied to a flow cell or fluid device that includes one or more etched flow channels, and the flow cell can further include a population of probe molecules covalently attached to its surface. The probes attached to the flow cell channels are advantageously positioned at different addressable locations within the channels, and DNA library molecules can be added to the flow cell channels where complementary sequences can bind (as described herein and as further 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 cells described in U.S. Pat. Nos. 8,906,320 and 9,990,381, which are incorporated herein by reference in their entirety. Bridge amplification can be performed as described herein, followed by sequencing using the synthetic methods and compositions described herein. Methods for creating and utilizing flow cells for sequencing are known in the art. References are provided herein, all of which are incorporated herein by reference. It is contemplated that the methods and compositions described herein are not limited to a particular manufacture or method of a flow cell-directed sequencing methodology.

[0049] Sequencing using the methods and compositions described herein can also be performed on microtiter plates, such as high density reaction plates or slides (see 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. Reaction plates or slides can be made from a fiber optic material that can capture and record light generated from a reaction, such as a fluorescent or luminescent reaction. The core material can be etched to provide individual reaction wells that can hold at least one emPCR reaction bead. Such slides / plates can contain over 1.6 million wells. The fabricated slides / plates can be loaded with target sequencing reaction emPCR beads, provided with sequencing reagents, and attached to an instrument where sequencing is performed.

[0050] Examples of arrayed substrates for sequencing targets using the compositions and methods disclosed herein are provided when performing a patterned substrate containing DNA nanoballs on a chip or slide, as performed by Complete Genomics (Mountain View, CA). As described in Drmanac et al., 2010, Science 327(5961): 78-81, silicon wafers can be formed with layers of silicon dioxide and titanium and then patterned using photolithography and dry etching techniques. The wafers can be treated with HMDS and coated with a photoresist layer to define individual regions for silanization and subsequent covalent attachment of DNA nanoballs for sequencing. One of ordinary skill in the art will understand that there are many ways to create slides / chips with individual positions for immobilization of nucleic acids used in sequencing methodologies, and the present method is not limited to methods for preparing substrates for sequencing.

[0051] Without intending to limit the embodiments described herein, for illustrative purposes, a general strategic sequencing cycle can be described by a series of steps. The following example is based on a sequencing reaction by sequencing, but the methods described herein are not limited to a particular sequencing reaction methodology.

[0052] The four nucleotide types A, C, T, and G are typically modified nucleotides designed for sequencing reactions such as reversibly blocked (rb) nucleotides (e.g., rbA, rbT, rbC, rbG), and three of the four types are fluorescently labeled. The template sequence of interest is placed and added along with other reaction components to the location where the sequencing reaction occurs (flow cell, chip, slide, etc.). After incorporating nucleotides into the nucleic acid strand of the sequence growing 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. Following the first imaging event, the sample is irradiated with a light source, causing a distinguishable and measurable change in the emission signals of the first and second fluorescent labels. The reaction location is illuminated again, and the change in fluorescence is captured and recorded, constituting the second imaging event (i.e., the second fluorescence detection pattern). The blocker present on the incorporated nucleotide is removed and washed away along with other reagents present after the second imaging event as preparation for the next sequencing cycle. In some embodiments, the methods of the present disclosure do not involve the use of chemical reagents that can directly or indirectly cause a distinguishable and measurable change in fluorescence from the first imaging event to the second imaging event. The fluorescence patterns from the two imaging events are compared, nucleotide incorporation occurs, and the sequence of the target nucleic acid for that particular cycle is determined.

[0053] (Definition) 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 terms "including" and other forms such as "include", "includes", "included" is not limiting. The use of the terms "having" and other forms such as "have", "has", "had" is not limiting. As used herein, whether in a transitional phrase of a claim or in the body, the terms "comprise(s)" and "comprising" should be interpreted to have an open-ended meaning. That is, the above terms should 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 also include additional 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 additional features or components.

[0054] The common organic abbreviations used herein are defined as follows. °C Celsius temperature dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP Dideoxynucleotide triphosphate ffN Fully functionalized nucleotide LED Light-emitting diode

[0055] 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 according to their relative positions. An array can comprise different probe molecules disposed at different addressable positions on a substrate. Alternatively or additionally, an array can comprise separate substrates each having a different probe molecule, and the different probe molecules can be identified according to the position of the substrate on the surface to which the substrate is attached or according to the position of the substrate in a liquid. Exemplary formats that can be used in the present invention to distinguish 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. 5,003,003. No. 6,524,793. Further examples of arrays that can be used in the present invention include, but are not limited to, those described in U.S. Patent No. 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 WO93 / 17126; WO95 / 11995; WO95 / 35505; EP742287; and EP799897.

[0056] As used herein, the terms "covalently attached" or "covalently bonded" refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms a chemical bond with a functionalized surface of a substrate as compared to bonding to the surface via other means, such as adhesion or electrostatic interactions. It will be understood that a polymer covalently attached to a surface can also be bonded via means in addition to the covalent bond.

[0057] As used herein, the "R" group represents a substituent that can be attached to the indicated atom. The R group may or may not be substituted. When two "R" groups are described as forming a ring or ring system "together with the atoms to which they are attached", it means that the atoms, intervening bonds, and the set unit of the two R groups are the ring that is listed. For example, the following substructures exist:

Chemical formula

Chemical formula

[0058] It should be understood that certain radical naming rules can include either a monoradical or a diradical, depending on the context. For example, when a substituent requires two points of attachment to the rest of the molecule, the substituent is understood to be a diradical. For example, substituents identified as alkyls that require two points of attachment include diradicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming rules explicitly indicate that the radical is a diradical such as "alkylene" or "alkenylene".

[0059] As used herein, the term "halogen" or "halo" means any one of the radiation-stable atoms in column 7 of the periodic table of the elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0060] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group may have from 1 to 20 carbon atoms (whenever a numerical range such as "1 to 20" appears herein, it refers to each integer within the specified range; e.g., "from 1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, but this definition also covers occurrences of the term "alkyl" where no numerical range is specified). An alkyl group may also be a medium alkyl having from 1 to 9 carbon atoms. An alkyl group may also be a lower alkyl having from 1 to 6 carbon atoms. An alkyl group may be designated as " 1-4 alkyl" or a similar designation. As just one example, " 1-6 alkyl" indicates that there are from 1 to 6 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, etc.

[0061] As used herein, "alkoxy" refers to -OR where R is alkyl as defined above, for example, "C 1-9 alkoxy", and includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

[0062] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have 2 to 20 carbon atoms, but this definition also covers occurrences of the term "alkenyl" where no numerical range is specified. The alkenyl group may also be a medium-sized alkenyl having 2 to 9 carbon atoms. The alkenyl group may also be a lower alkenyl having 2 to 6 carbon atoms. The alkenyl group may be designated as "C 2-6 alkenyl" or a similar designation. By way of example only, "C 2-6 alkenyl" indicates that there are 2 to 6 carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0063] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group may have from 2 to 20 carbon atoms, but this definition also covers occurrences of the term "alkynyl" where no numerical range is specified. The alkynyl group may also be a medium-sized alkynyl having from 2 to 9 carbon atoms. The alkynyl group could also have been a lower alkynyl having from 2 to 6 carbon atoms. The alkynyl group may be designated as "C 2-6 alkynyl" or by a similar designation. By way of example only, "C 2-6 alkynyl" indicates that there are from 2 to 6 carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0064] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) containing only carbon in the ring backbone. When aryl is a ring system, all rings within the system are aromatic. An aryl group may have from 6 to 18 carbon atoms, but this definition also covers occurrences of the term "aryl" where no numerical range is specified. In some embodiments, the aryl group has from 6 to 10 carbon atoms. The aryl group may be designated as "C 6-10 aryl", "C6 or C 10 aryl", or by a similar designation. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0065] "Aralkyl" or "arylalkyl" is "C 7-14An "arylalkyl" is an aryl group bonded via an alkylene group as a substituent, and examples include, but are 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 group).

[0066] 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 heteroaryl is a ring system, each ring within the system is aromatic. A heteroaryl group may have 5 to 18 ring members (i.e., the number of atoms constituting the ring skeleton, including carbon atoms and heteroatoms), but this definition also covers occurrences of the term "heteroaryl" for which a numerical range is not specified. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as "5- to 7-membered heteroaryl", "5- to 10-membered heteroaryl", or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0067] "Heteroalkyl" or "heteroarylalkyl" is a heteroaryl group bonded via an alkylene group as a substituent. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6is an alkylene group).

[0068] As used herein, "carbocyclic" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system skeleton. When the carbocyclic is a ring system, two or more rings may be joined together in a fused, bridged, or spiro-connected manner. The carbocyclic may have any degree of saturation as long as at least one of the rings of the ring system is not aromatic. Thus, examples of carbocyclic include cycloalkyl, cycloalkenyl, and cycloalkynyl. The carbocyclic group may have 3 to 20 carbon atoms, but this definition also covers occurrences of the term "carbocyclic" where no numerical range is specified. The carbocyclic group may also be a medium-sized carbocyclic having 3 to 10 carbon atoms. The carbocyclic group may also be a carbocyclic having 3 to 6 carbon atoms. The carbocyclic group may be designated as "C3-6 carbocyclic" or a similar designation. Examples of carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0069] As used herein, "cycloalkyl" means a completely saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0070] As used herein, "heterocyclyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring skeleton. Heterocyclyls may be joined together in a fused, bridged, or spiro-linked fashion. Heterocyclyls may have any degree of saturation so long as at least one ring of the ring system is not aromatic. The heteroatom may be present in either the non-aromatic or aromatic rings of the ring system. A heterocyclyl group may 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 covers occurrences of the term "heterocyclyl" where no numerical range is specified. A heterocyclyl group may also be a medium-sized heterocyclyl having from 3 to 10 ring members. A heterocyclyl group may also be a heterocyclyl having from 3 to 6 ring members. A heterocyclyl group may be designated as a "3-6 membered heterocyclyl" or a similar designation. In preferred 6-membered monocyclic heterocyclyls, the heteroatoms are selected from one to three of O, N, or S, and in preferred 5-membered monocyclic heterocyclyls, the heteroatoms are selected from one or two heteroatoms of O, N, or S.Examples of heterocyclic rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-dioxinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuran, tetrahydropyranyl, tetrahydroiofephenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0071] The "O-carboxy" group refers to the "-OC(=O)R" group where R is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic as defined herein.

[0072] The "C-carboxy" group refers to the "-C(=O)R" group where R is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic, C 6-10Refers to a "C(=O)OR" group selected from the group consisting of aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl. Non-limiting examples include carboxyl (i.e., -C(=O)OH).

[0073] The "sulfonyl" group means that R is hydrogen, C as defined herein 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C3-7 carbocyclyl, C 6-10 aryl, a " -SO2R" group selected from 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0074] The "S-sulfonamide" group means that R A and R B are each independently hydrogen, C as defined herein 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclyl, C 6-10 aryl, a " -SO2NR A R B " group selected from 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0075] The "N-sulfonamide" group means that R A and R b are each independently hydrogen, C as defined herein 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclyl, C 6-10 aryl, a " -N(R A )SO2R B " group selected from 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0076] The "C-amide" group means that R A and R B are each independently hydrogen, C as defined herein 1-6 alkyl, C 2-6Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic, C 6-10 " -C(=O)NR A R B " group.

[0077] The "N - amide" group means that R A and R B are each independently hydrogen, C as defined herein 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic, C 6-10 aryl, 5 - 10 - membered heteroaryl, and 3 - 10 - membered heterocyclic selected from, "-N(R A )C(=O)R B " group.

[0078] The "amino" group means that R A and R B are each independently hydrogen, C as defined herein 1-6 alkyl, C 2-6 alkenyl, C<o000126>alkynyl, C 3-7 carbocyclic, C 6-10 aryl, 5 - 10 - membered heteroaryl, and 3 - 10 - membered heterocyclic selected from, "-NR A R B " group. Non - limiting examples include free amino (i.e., -NH2).

[0079] The "aminoalkyl" group refers to an amino group connected via an alkylene group.

[0080] The "alkoxyalkyl" group refers to an alkoxy group bonded via an alkylene group, such as "C 2-8 alkoxyalkyl", etc.

[0081] It should be noted that there seems to be a small error in the original text where "<o000126>" should probably be " 2-6 ". This has been maintained in the translation for accuracy.As used herein, a substituent is derived from an unsubstituted parent group in which one or more hydrogen atoms have been replaced with another atom or group. Unless otherwise specified, when a group is considered to be “substituted”, the group is C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclic (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclic-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3- to 10-membered heterocyclic (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3- to 10-membered heterocyclic-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., an ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., -CF3), halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl, cyanato,It means being substituted with one or more substituents independently selected from isocyanato, thiocyanato, isothiocyanato, sulfonyl, -SO3H, -OSO2C1-4 alkyl, and oxo(=O). Whenever a group is described as "optionally substituted", the group can be substituted with the above substituents.

[0082] As used herein, the term "azide" refers to the -N3 group.

[0083] As used herein, "nucleotide" includes a nitrogen-containing 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, and in DNA, it is deoxyribose, i.e., a sugar without the hydroxyl group present in ribose. The nitrogen-containing 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 deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine.

[0084] As used herein, "nucleoside" is structurally similar to nucleotide but lacks the phosphate moiety. Examples of nucleoside analogs are those in which a label is attached to the base and the phosphate group is not attached to the sugar molecule. In this specification, the term "nucleoside" is used in its ordinary meaning as understood by those skilled in the art. 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 is replaced by carbon and / or carbon is replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that can have a substituted base and / or sugar moiety. Furthermore, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0085] As used herein, the term "purine base" is used in its ordinary meaning as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary meaning as understood by those skilled in the art and includes its tautomers. Non-limiting lists of optionally substituted purine bases include purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 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).

[0086] As used herein, "derivative" or "analog" means 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, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Phosphorodithioate, alkylphosphonate, phosphoramidate, and phosphoramidate 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.

[0087] As used herein, the term "phosphate" is used in its ordinary meaning as understood by those skilled in the art and includes its protonated form (e.g.,

Chem.

Chem.

[0088] The terms "protecting group" and "blocking group" as used herein refer to any atom or group of atoms that is added to a molecule to prevent an existing group within the molecule from undergoing an undesired chemical reaction. "Protecting group" and "blocking group" may be used interchangeably.

[0089] The prefix "photo" or "photo-" as used herein means relating to light or electromagnetic radiation. This term can include all or a portion of the electromagnetic spectrum, including, but not limited to, one or more ranges commonly known as radio waves, microwaves, infrared, visible, ultraviolet, X-rays, or the gamma ray portion of the spectrum. A portion of the spectrum may be blocked by a metallic region of a surface, such as the metals described herein. Alternatively or additionally, a portion of the spectrum may pass through an interstitial region of a surface, such as a region made of glass, plastic, silica, or other materials described herein. In certain embodiments, radiation that can pass through metals can be used. Alternatively or additionally, radiation masked by glass, plastic, silica, or other materials described herein can be used.

[0090] (Labeled nucleotide) According to one aspect of the present disclosure, the described incorporated nucleotides contain a detectable label, and such nucleotides are called labeled nucleotides. The label (e.g., a fluorescent dye) can be attached via an optional linker by various means including hydrophobic attraction, ionic attraction, and covalent bonding. In some aspects, the dye is conjugated to the substrate by a covalent bond. More specifically, the covalent bond is by a linker group. In some cases, such labeled nucleotides are also referred to as "modified nucleotides". In some embodiments, the dye is covalently bonded to the nucleotide via a cleavable linker. In some such embodiments, the cleavable linker can include one or more moieties including an azide moiety, an azidomethyl moiety, a disulfide moiety, -(CH2CH2O)-, or other covalent linker described herein.

[0091] Labeled nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, for example, in PCR amplification, isothermal amplification, solid-phase amplification, polynucleotide sequencing (e.g., solid-phase sequencing), nick translation reactions, etc., as non-limiting examples.

[0092] In some embodiments, the dye can be covalently bonded to an oligonucleotide or nucleotide via a nucleotide base. For example, a 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 linker moiety.

[0093] Unless otherwise specified, references to nucleotides shall also apply to nucleosides. This application is further described with reference to DNA, but unless otherwise specified, the description is also applicable to RNA, PNA, and other nucleic acids.

[0094] Some embodiments of the present disclosure relate to nucleotide conjugates comprising a photoswitchable label, the photoswitchable label comprising a fluorescent moiety covalently attached to a photochromic moiety, optionally via a linker. In some embodiments, the photoswitchable label is attached to a nucleobase via a cleavable linker. In some further embodiments, the photoswitchable label is attached 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 changes, for example, from a signal state to a dark state. In other words, the observed emission signal may switch from “on” to “off”. Alternatively, the photoswitchable label may change from a non-emissive dark state to a signal state. In other words, the observed emission signal may switch from “off” to “on”.

[0095] In some embodiments, the photoswitchable label comprises a fluorescent moiety covalently attached to a photochromic moiety, optionally via a linker. In some such embodiments, the fluorescent moiety comprises a fluorophore that emits red light, for example, red light having a wavelength between about 600 nm and about 700 nm.

[0096] In some embodiments, the fluorescent moiety of the photoswitchable label comprises a silicon rhodamine fluorophore. In one embodiment, the rhodamine fluorophore comprises the following structure:

Chemical formula

Chemical formula

[0097] In some embodiments of the photochromic moiety of formula (I), the phenyl moiety fused to the 5-membered pyrrolidine may be optionally substituted. In some embodiments, X is S. In some further embodiments, each R 1a and R 2a is C1-6 alkyl, for example, each R 1a and R 2a is methyl. In some embodiments, Ring A is phenyl or naphthyl substituted with at least one electron-withdrawing group. Non-limiting examples of electron-withdrawing groups are nitro, cyano, fluoro, bromo, -S(O)2OH, triflyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluoro or bromo, and sulfonyl substituted with one or more fluoro or bromo. In one embodiment, the photochromic moiety comprises the structure of formula (Ia):

Chemical formula

Chemical formula

[0098] In some other embodiments, the fluorescent moiety of the photo-switchable one comprises a coumarin fluorophore. In one embodiment, the coumarin fluorophore comprises the following structure: [Chemistry] In some such embodiments, the photochromic moiety of the photoswitchable label may include an oxazine or thiazine moiety. For example, the photochromic moiety includes the structure of formula (II). [Chemistry] Here, the definitions of Y, R1b, R2b, and ring B are described herein, and the carbon labeled with an asterisk * indicates the point of attachment to the fluorescent moiety, either directly or via a second linker. The carbon *-Y bond of formula (II) undergoes a chemical change upon irradiation with an appropriate light source.

[0099] In some embodiments of the photochromic moiety of formula (II), the phenyl moiety fused to the five-membered pyrrolidine moiety may be optionally substituted. In some embodiments, Y is O. In some further embodiments, each R 1b and R 2b is C1-6 alkyl. For example, each R 1b and R 2b is methyl. In some embodiments, ring B is phenyl or naphthyl substituted with at least one electron-withdrawing group. In other embodiments, ring B may be selected from 6-membered heteroaryls such as pyridyl or pyrimidyl. Non-limiting examples of electron-withdrawing groups are nitro, cyano, fluoro, bromo, -S(O)2OH, triflyl (-S(O)2CF3), -OS(O)2CF3, ammonium, alkylammonium, C 1-6 alkyl substituted with one or more fluoros or bromos, and sulfonyl substituted with one or more fluoros or bromos. In one embodiment, the photochromic moiety includes the structure of formula (IIa): [Chemistry] In one embodiment, the photoswitchable label includes the following structure: [Chemistry] Here, L2 is the second linker. In some such embodiments, the second linker L2 may include a π-conjugated structure (e.g., one or more double bonds). In one example, L2 is a double bond that connects coumarin and the photochromic moiety. In some embodiments, the photoswitchable label is covalently attached to the nucleotide via the second linker L2 or via the point indicated by the wavy line

Chemical formula

[0100] Alternatively, the photoswitchable label described herein can include only the photochromic moiety and optionally a linker, and the photochromic moiety itself functions as a fluorophore and becomes non-emissive when irradiated with an appropriate light source that can switch it from a non-emissive dark state to an emissive state or from an emissive state to a dark state.

[0101] Some further embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the nucleotide conjugates described herein.

[0102] (Linker) In some embodiments described herein, the purine or pyrimidine base of the nucleotide molecule can be attached to the detectable label described above. In some such embodiments, the linker used is cleavable. Using a cleavable linker allows the label to be reliably removed after detection if necessary, thereby avoiding interference signals with subsequently incorporated labeled nucleotides.

[0103] In some other embodiments, the linker used is non-cleavable. In each case where the labeled nucleotide described herein is incorporated, there is no need to subsequently incorporate a nucleotide, and thus no need to remove the label from the nucleotide.

[0104] Cleavable linkers are known in the art and conventional chemistry can be applied to attach the linker to the nucleotide base and the label. The linker can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. It is used to cleave the 3'-O-blocking group bond. Suitable linkers can be adapted from standard chemical protecting groups disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Further suitable cleavable linkers used in solid-phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

[0105] When a detectable label is attached to the base, the linker can be attached at any position on the nucleotide base as long as Watson-Crick base pairing can still be carried out. In the context of purine bases, it is preferred that the linker is attached via the 7-position of the purine or a preferred deazapurine analog, via an 8-modified purine, via an N-6-modified adenosine or an N-2-modified guanine. In the case of pyrimidines, the attachment preferably occurs via the 5-position of cytosine, thymidine or uracil and the N-4 position of cytosine.

[0106] In some embodiments, the linker can include a spacer unit. The length of the linker is not critical as long as the label is kept at a sufficient distance from the nucleotide so as not to interfere with the interaction between the nucleotide and an enzyme, such as a polymerase.

[0107] In some embodiments, the linker can consist of a functional group similar to a trivalent OH protecting group. This makes the deprotection and de-labeling processes more efficient as both the label and the protecting group can be removed in a single treatment.

[0108] The use of the term "cleavable linker" does not mean that the entire linker needs to be removed. The cleavage site can be located at a position on the linker that ensures that a portion of the linker remains bound to the dye and / or substrate moiety after cleavage. Cleavable linkers include, by way of non-limiting example, electrophilic cleavable linkers, nucleophilic cleavable linkers, photocleavable linkers, cleavable under reducing conditions (e.g., disulfide or azide-containing linkers), cleavable under oxidizing conditions, cleavable via the use of a safety catch linker, or cleavable by an elimination mechanism. By using a cleavable linker to attach a dye compound to a substrate moiety, it becomes possible to remove the label after detection, if desired, and avoid interfering signals in downstream steps.

[0109] Useful linker groups can be found in PCT Publication No. WO 2004 / 018493 (incorporated herein by reference), examples of which include linkers that can be cleaved using a water-soluble phosphine or a water-soluble transition metal catalyst formed from a transition metal and at least partially water-soluble ligands. In an aqueous solution, the latter forms at least a partially water-soluble transition metal complex, e.g., a Pd(II) complex and THP. Such cleavable linkers can be used to connect the base of a nucleotide to a label such as a dye described herein.

[0110] Special linkers include those disclosed in PCT Publication No. WO 2004 / 018493 (incorporated herein by reference), such as those containing a moiety of the following formula:

Chemical Formula

[0111] Additional examples of linkers include those disclosed in U.S. Publication No. 2016 / 0040225, which is incorporated herein by reference, such as those containing moieties of the following formula: [Chemical formula] (wherein * indicates the location where the moiety is connected to the remainder of the nucleotide). The linker moieties shown herein may include the entire or a portion of the linker structure between the nucleotide / nucleoside and the label.

[0112] In a particular embodiment, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be varied, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore bound to the guanine base via other linkages known in the art. Exemplary linkers and their properties are shown in PCT Publication No. WO 2007 / 020457, which is incorporated herein by reference. The design of the linker, particularly the increase in their length, can improve the brightness of the fluorophore bound to the guanine base of the guanosine nucleotide when incorporated into polynucleotides such as DNA. Thus, when used in an analytical method that requires detection of a fluorescent dye label in which the dye is bound to a guanine-containing nucleotide, it is advantageous for the linker to be a spacer group of the formula -((CH2)2O) n - wherein n is an integer between 2 and 50.

[0113] Nucleosides and nucleotides can be labeled at sites on the sugar or 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, it is deoxyribose, a sugar lacking the hydroxy group present in ribose. The nitrogenous base is a derivative of purine or pyrimidine. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T), or in the context of RNA, uracil (U). 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 esterification occurs at the hydroxy group bonded to C-3 or C-5 of the sugar. Nucleotides are usually mono-, di-, or triphosphates.

[0114] Bases are usually referred to as purines or pyrimidines, but those skilled in the art will understand that derivatives and analogs are available that do not alter the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing. A "derivative" or "analog" means a compound or molecule that has a core structure that is the same as or very similar to the parent compound but has chemical or physical modifications, such as different or additional side chains, that allow the derivative nucleotide or nucleoside to be attached to another molecule. For example, the base may be a deazapurine. In particular embodiments, the derivative should be capable of undergoing Watson-Crick pairing. "Derivatives" and "analogs" also include synthetic nucleotide or nucleoside derivatives having, for example, 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 include modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoranylidate, phosphoramidate linkages, and the like.

[0115] The pigment can be attached at any position on the nucleotide base, for example via a linker. In a particular embodiment, Watson-Crick base pairing can still be carried out with the resulting analog. Specific nucleic acid base labeling sites include the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases. As described above, a linker group can be used to covalently attach the pigment to a nucleoside or nucleotide.

[0116] In a particular embodiment, the labeled nucleotide can be enzymatically incorporated and enzymatically extendable. Thus, the linker moiety can be of sufficient length to connect 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 linker can also include a spacer unit. The spacer, for example, separates the nucleotide base from the cleavage site or label.

[0117] The nucleoside or nucleotide labeled with the pigment described herein may have the following formula:

Chemical formula

[0118] In a particular embodiment, the blocking group is separate and independent from the dye compound, i.e., it is not attached thereto. Alternatively, the dye may comprise all or part of the 3'-OH blocking group. Thus, R'' can be a 3'-OH blocking group that may or may not contain the dye compound.

[0119] In yet another alternative embodiment, there is no blocking group on the 3'-carbon of the pentose sugar, and for example, a dye (or a dye and linker construct) attached to the base can be of a size or structure sufficient to act as a block to the incorporation of further nucleotides. Thus, the block can be due to steric hindrance, regardless of whether the dye is attached to the 3'-position of the sugar, or can be due to a combination of size, charge, and structure.

[0120] In yet another alternative embodiment, the blocking group is present on the 2'- or 4'-carbon of the pentose sugar and can be of a size or structure sufficient to act as a block to the incorporation of further nucleotides.

[0121] The use of a blocking group enables the control of polymerization, such as by stopping elongation when a nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting examples such as a change in chemical conditions or removal of a chemical block, elongation can be stopped at a specific point and then continued.

[0122] In another particular embodiment, the 3'-OH blocking group will include portions disclosed in International Publication No. WO 2004 / 018497 and International Publication No. WO 2014 / 139569, which are incorporated herein by reference. For example, the blocking group can be azidomethyl (-CH2N3) or a substituted azidomethyl (e.g., -CH(CHF2)N3 or CH(CH2F)N3), or allyl.

[0123] In certain embodiments, both a linker (between the dye and the nucleotide) and a blocking group are present and are distinct moieties. In certain embodiments, both the linker and the blocking group are cleavable under substantially the same conditions. Thus, the deprotection and deblocking process can be more efficient since only one treatment is required to remove both the dye compound and the blocking group. However, in some embodiments, the linker and the blocking group need not be cleavable under the same conditions and instead may be cleavable individually under distinct conditions.

[0124] The present disclosure also encompasses polynucleotides incorporating a dye compound. Such polynucleotides may be DNA or RNA each composed of deoxyribonucleotides or ribonucleotides linked by phosphodiester bonds. The polynucleotide may comprise naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof in combination with at least one nucleotide described herein (e.g., labeled with a dye compound). Polynucleotides according to the present disclosure may also include non-natural backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures consisting of a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

[0125] Non-limiting exemplary labeled nucleotides described herein include the following:

Chemical formula

[0126] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

Chemical formula

[0127] (Kit) The present disclosure also provides a kit comprising the labeled nucleotides described herein. Such kits will generally include at least one nucleotide labeled with a dye (e.g., a photoswitchable dye as described herein), along with at least one additional component. The additional components may be one or more components identified by the methods described herein or in the Examples section below. Some non-limiting examples of components that can be combined in the kits of the present disclosure are described below.

[0128] In certain embodiments, the kit can include at least one labeled nucleotide or nucleoside, together with labeled or unlabeled nucleotides or nucleosides. For example, nucleotides labeled with a dye can be supplied in combination with unlabeled or natural nucleotides, and / or fluorescently labeled nucleotides, or any combination thereof. Combinations of nucleotides can be provided as separate individual components (e.g., one nucleotide type per container or tube) or as nucleotide mixtures (e.g., two or more nucleotides mixed within the same container or tube). In some embodiments, one or more dyes are selected from the photoswitchable dyes disclosed herein.

[0129] When the kit contains a plurality, in particular two, or three, or more specifically four, labeled nucleotides each labeled with a dye compound, different nucleotides may be labeled with different dye compounds, or one may be dark-colored without a dye compound. When different nucleotides are labeled with different dye compounds, it is a feature of the kit that the dye compounds are fluorescent dyes that are spectrally distinguishable through one or more imaging events. When two nucleotides labeled with a fluorescent dye compound are provided in the form of a kit, it is a feature of some embodiments that spectrally distinguishable fluorescent dyes can be excited at the same wavelength, for example by the same laser or the like. When four nucleotides labeled with a fluorescent dye compound are provided in the form of a kit, it is a feature of some embodiments that two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes are not excited at such a wavelength. In some embodiments, one of the four different types of nucleotides is unlabeled.

[0130] Although the kit is exemplified herein with respect to configurations having different nucleotides labeled with different dye compounds, it will be understood that the kit can include two, three, four or more different nucleotides having the same dye compound.

[0131] In certain embodiments, the kit can include a polymerase enzyme that can catalyze the incorporation of nucleotides into a polynucleotide. Other components included in such a kit include buffers and the like. The nucleotides of the present disclosure, and any other nucleotide components including mixtures of different nucleotides, can be provided in the kit in a concentrated form that is diluted before use. In such embodiments, a suitable dilution buffer can also be included. Again, one or more of the components specified by the methods described herein can be included in the kits of the present disclosure.

[0132] Some embodiments of the present disclosure relate to kits comprising at least one type of labeled nucleotide, the nucleotide being labeled with a photoswitchable dye as described herein. In some further embodiments, the kit comprises two or more different types of nucleotides, the first type of nucleotide being labeled with a first photoswitchable label as described herein, and the second type of nucleotide being labeled with a second photoswitchable label as described herein. In further embodiments, the kit comprises a third type of nucleotide comprising a label that emits at the same or substantially the same wavelength as the first photoswitchable label, or a photoswitchable label comprising a label that can be excited using the same or substantially the same excitation wavelength as the first 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.

[0133] In any embodiment of the kits described herein, they can be used in an automated sequencing apparatus, which comprises two lasers operating at different excitation wavelengths and a detection system having a single detection channel set to a fixed emission wavelength.

[0134] In addition to the fluorescent moieties disclosed for the optically switchable label, other exemplary fluorescent moieties or derivatives thereof for use as fluorescent moieties include, but are not limited to, fluorescein and carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein derivatives such as fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lysamine rhodamine, B1, rhodamine, TMR-iodoacetamide, lysamine rhodamine B sulfonyl chloride, lysamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA and other coumarin and coumarin derivatives, AMCE-hydrazide, BODIPY and BODIPYFL C3-SE, BODIPY and other derivatives such as 530 / 550C3, BODIPY530 / 550C3-SE, BODIPY530 / 550C3 hydrazide, BODIPY493 / 503C3 hydrazide, BODIPYFL C3 hydrazide, BODIPYFL IA, BODIPY5IA4-1 BODIPY530 / 530 cascade blue acetyl azide, cascade blue cadaverine, cascade blue ethylenediamine, cascade blue hydrazide, lucifer yellow and derivatives thereof, and lucifer yellow iodoacetamide, lucifer yellow CH, cyanine and indolium-based cyanine dyes, benzoindolium-based cyanine dye derivatives, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelate, terbium chelate, AlexaFluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CAL flour dyes,JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycobiliprotein and phycobilin dyes, malachite green, stilbene, DEG dyes described in US 2010 / 0009353, which is hereby incorporated by reference in its entirety, NR dyes, near-infrared dyes, and other known ones in the art such as those described in the 6th Edition of the Handbook of Fluorescent Probes and Research Chemicals (Eugene, Oregon), Molecular Probes; Synthegen Catalog (Houston, Texas), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Edition, Plenum Press New York (1999), Hermanson, Bioconjugate Techniques, 2nd Edition, US 2010 / 0009353 or WO 98 / 59066, which are hereby incorporated by reference in their entirety. In some embodiments, the third label described herein can also be selected from any of the exemplary fluorescent moieties or derivatives thereof described herein.

[0135] (Sequencing application) The labeled nucleotides or nucleosides according to the present disclosure can be used in any analytical method such as a method involving the detection of a fluorescent label attached to a nucleotide or nucleoside. In this context, the term "incorporated into a polynucleotide" means that the 5'-phosphate is linked by a phosphodiester bond to the 3'-hydroxy group of a second (modified or unmodified) nucleotide, which itself may form part of a long polynucleotide chain. The 3'-end of the nucleotides described herein may or may not be linked by a phosphodiester bond to the 5'-phosphate of a further (modified or unmodified) nucleotide. Thus, in one non-limiting embodiment, the present disclosure provides a method for detecting a nucleotide incorporated into a polynucleotide, comprising (a) incorporating at least one nucleotide of the present disclosure into a polynucleotide, and (b) detecting the incorporated nucleotide. By detecting the fluorescent signal from the dye compound attached to the nucleotide, it is inserted into the polynucleotide.

[0136] This method can include a synthesis step (a) in which one or more nucleotides according to the present disclosure are incorporated into a polynucleotide, and a detection step (b) in which one or more nucleotides incorporated into the polynucleotide are detected by detection. Or quantitatively measure their fluorescence.

[0137] Some embodiments of the present application are directed to sequencing methods that include: (a) incorporating at least one labeled nucleotide described herein into a polynucleotide; (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting the fluorescent signal from a new fluorescent dye attached to the nucleotide.

[0138] In one embodiment, at least one nucleotide is incorporated into a polynucleotide in a synthesis step by the action of a polymerase enzyme. However, other methods of attaching nucleotides to polynucleotides can be used, such as chemical oligonucleotide synthesis or ligation of a labeled oligonucleotide to an unlabeled oligonucleotide. Thus, the term "incorporated" can encompass polynucleotide synthesis by chemical and enzymatic methods when used with respect to nucleotides and polynucleotides.

[0139] In certain embodiments, a synthesis step is performed, optionally including incubating a template polynucleotide strand with a reaction mixture comprising a fluorescently labeled nucleotide of the present disclosure. A polymerase can also be provided under conditions that allow formation of a phosphodiester bond between the free 3' hydroxy group on the polynucleotide strand annealed to the template polynucleotide strand and the 5' phosphate group on the nucleotide. Thus, the synthesis step can include formation of a polynucleotide strand directed by complementary base pairing of nucleotides to the template strand.

[0140] In all embodiments of the method, a detection step can be performed while the polynucleotide strand into which the labeled nucleotide is incorporated is annealed to the template strand, or after a denaturation step in which the two strands are separated. Additional steps, such as chemical or enzymatic reaction steps or purification steps, can be included between the synthesis step and the detection step. In particular, the target strand incorporating the labeled nucleotide can be isolated or purified and further processed or used for subsequent analysis. By way of example, a target polynucleotide labeled with a nucleotide described herein in a synthesis step can subsequently be used as a labeled probe or primer. In other embodiments, the product of the synthesis step described herein can be subjected to further reaction steps, and optionally, the products of these subsequent steps can be purified or isolated.

[0141] The conditions suitable for the synthesis step are well known to those skilled in standard molecular biology techniques. In one embodiment, the synthesis step is similar to a standard primer extension reaction using nucleotide precursors containing the nucleotides described herein, and in the presence of a suitable polymerase enzyme, an extended target strand complementary to the template strand can be formed. In other embodiments, the synthesis step itself forms part of an amplification reaction, generating a labeled double-stranded amplification product consisting of annealed complementary strands derived from copies of the target and template polynucleotide strands. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random prime DNA labeling, and the like. Polymerase enzymes particularly useful for the synthesis step are those that can catalyze the incorporation of the nucleotides described herein. A variety of natural or modified polymerases can be used. By way of example, thermostable polymerases can be used in synthesis reactions carried out using thermal cycling conditions, although thermostable polymerases may not be desirable for isothermal primer extension reactions. Suitable thermostable polymerases capable of incorporating the nucleotides according to the present disclosure include those described in WO2005 / 024010 or WO06120433, which are hereby incorporated herein by reference. For synthesis reactions carried out at low temperatures such as 37°C, the polymerase enzyme does not necessarily have to be a thermostable polymerase. Thus, the choice of polymerase will depend on a number of factors such as reaction temperature, pH, strand displacement activity, and the like.

[0142] In certain non-limiting embodiments, the present disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, and other applications including the detection of the labeled nucleotides or nucleosides described herein when incorporated into a polynucleotide. Any of a variety of other uses that benefit from the use of polynucleotides labeled with nucleotides containing a fluorescent dye can be carried out using the nucleotides or nucleosides labeled with the dyes described herein.

[0143] In certain embodiments, the present disclosure provides for use in a polynucleotide sequencing reaction by synthesis of labeled nucleotides according to the present disclosure. Sequencing by synthesis generally uses a polymerase or ligase to sequentially add one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction to form an extended polynucleotide chain complementary to a template nucleic acid. The identity of the base(s) present in one or more of the ordered added nucleotides can be determined in a detection or "imaging" step. The identity of the added base can be determined after each nucleotide incorporation step. Next, the sequence of the template can be inferred using conventional Watson-Crick base pairing rules. The use of the labeled nucleotides described herein for determining the identity of a single base can be useful, for example, in scoring single nucleotide polymorphisms, and such single base extension reactions are within the scope of the present disclosure.

[0144] 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 being sequenced through detection of a fluorescent label attached to the incorporated polynucleotide. Nucleotide. Sequencing of the template polynucleotide can be primed with an appropriate primer (or prepared as a hairpin construct that includes the primer as part of the hairpin), and the nascent strand is extended stepwise by addition of nucleotides to the 3' end. Amount of primer in the polymerase-catalyzed reaction.

[0145] In certain embodiments, each of the different nucleotide triphosphates (A, T, G, and C) can be labeled with a unique fluorophore and also contain a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides can be unlabeled (dark). The polymerase enzyme incorporates nucleotides into the nascent strand complementary to the template polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescence signal from each incorporated nucleotide can be optically "read" by appropriate means such as a charge-coupled device using laser excitation and an appropriate emission filter. Next, the 3'-blocking group and the fluorescent dye compound can be removed (deprotected) simultaneously or sequentially to expose the nascent strand for further nucleotide incorporation. Usually, the identity of the incorporated nucleotide is determined after each incorporation step, although this is not strictly necessary. Similarly, U.S. Patent No. 5,302,509 (incorporated herein by reference) discloses a method for sequencing polynucleotides immobilized on a solid support.

[0146] This method utilizes the incorporation of fluorescently labeled 3'-blocked nucleotides A, G, C, and T into a growing strand complementary to an immobilized polynucleotide in the presence of a DNA polymerase. The polymerase incorporates bases complementary to the target polynucleotide, but further addition is blocked by the 3'-blocking group. Next, the label of the incorporated nucleotide is determined, and the protecting group is removed by chemical cleavage to allow further polymerization. The nucleic acid template to be sequenced in a sequencing reaction by synthesis can be any polynucleotide for which sequencing is desired. The nucleic acid template for the sequencing reaction typically includes a double-stranded region having a free 3'-hydroxy group that functions as a primer or starting point for the addition of further nucleotides in the sequencing reaction. The region of the template to be sequenced overhangs this free 3'-hydroxy group on the complementary strand. The overhang region of the template to be sequenced may be single-stranded, but can also be double-stranded provided that there is a "nick" on the strand complementary to the template strand to be sequenced, providing a free 3'OH group for initiation. Sequencing reaction. In such embodiments, sequencing can proceed by strand displacement. In certain embodiments, a primer having a free 3'-hydroxy group can be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and template strands to be sequenced can each form part of a partially self-complementary nucleic acid strand that can form an intramolecular double-strand, such as a hairpin loop structure. Hairpin polynucleotides and methods for attaching them to a solid support are disclosed in PCT Publication Nos. WO01 / 57248 and WO2005 / 047301, which are incorporated herein by reference. Nucleotides are added sequentially to the growing primer, and a polynucleotide strand is synthesized in the 5' to 3' direction. The nature of the added base is determined, although not necessarily so, particularly after each nucleotide addition, to provide sequence information of the nucleic acid template.Thus, nucleotides are incorporated into a nucleic acid strand (or polynucleotide) by binding the nucleotide to the free 3'-hydroxyl group of the nucleic acid strand via formation of a phosphodiester bond with the 5'-phosphate group of the nucleotide.

[0147] 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 include natural and / or non-natural nucleotides and natural or non-natural backbone linkages, so long as they do not interfere with copying of the template in the sequencing reaction.

[0148] In certain embodiments, the nucleic acid template to be sequenced can be attached to a solid support by any suitable attachment method known in the art, such as by covalent bonding. In certain embodiments, the template polynucleotide can be directly attached to a solid support (e.g., a silica-based support). However, in other embodiments of the disclosure, the surface of the solid support can be modified in some way such that it allows for direct covalent bonding of the template polynucleotide or immobilizes the template polynucleotide through a hydrogel or polyelectrolyte multilayer that may or may not be covalently attached to the solid support.

[0149] Arrays in which polynucleotides are directly attached to silica-based supports are disclosed, for example, in WO00 / 06770 (incorporated herein by reference), where the polynucleotide is an internal amino group on the polynucleotide and a pendant epoxide group on the glass. Further, polynucleotides can be attached to solid supports by reaction of a sulfur-based nucleophile with the solid support, as described, for example, in WO2005 / 047301 (incorporated herein by reference). Yet another example of a template polynucleotide supported on a solid is found in, for example, WO00 / 31148, WO01 / 01143, WO02 / 12566, WO03 / 014392, U.S. Patent No. 6,465,178, and WO00 / 53812. Each of these is incorporated herein by reference.

[0150] A particular surface to which a template polynucleotide can be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO2005 / 065814, which is incorporated herein by reference. Particular hydrogels that can be used include WO2005 / 065814 and U.S. Pub. No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)).

[0151] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Patent No. 5,253,003. U.S. Patent No. 6,172,218 (incorporated herein by reference). Attachment to beads or microparticles serves sequencing applications. A bead library can be prepared such that each bead contains a different DNA sequence. Examples of libraries and methods for their creation are described in Nature, 437, 376 - 380 (2005). Science, 309, 5741, 1728 - 1732 (2005), which are each incorporated herein by reference. Sequencing of such an array of beads using the nucleotides described herein is within the scope of the present disclosure.

[0152] 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 any type of high - density array, including single - molecule arrays, clustered arrays, and bead arrays. The labeled nucleotides of the present disclosure can be used to sequence templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support.

[0153] However, the labeled nucleotides of the present disclosure are particularly advantageous in the context of sequencing clustered arrays. In a clustered array, different regions on the array (often referred to as sites or features) comprise multiple polynucleotide template molecules. Generally, multiple polynucleotide molecules cannot be individually resolved by optical means and are instead detected as a population. Depending on the method of forming the array, each site on the array may contain multiple copies of a single individual polynucleotide molecule (e.g., the site is homogeneous with respect to a particular single-stranded or double-stranded nucleic acid species) or a small number of multiple copies. Of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules can be generated using techniques generally known in the art. By way of example, WO98 / 44151 and WO00 / 18957 (each incorporated herein by reference) describe methods of amplifying nucleic acids in which both the template and the amplification product remain immobilized on a solid support to form an array of clusters. Or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using the nucleotide labeled with the dye compound of the present disclosure.

[0154] The labeled nucleotides of the present disclosure are also useful for sequencing templates on single molecule arrays. As used herein, the term "single molecule array" or "SMA" refers to a population of polynucleotide molecules dispersed (or arrayed) on a solid support, and the spacing between individual polynucleotides and all other populations is such that the individual polynucleotide molecules can be separated individually. Thus, the target nucleic acid molecule immobilized on the surface of the solid support can, in some embodiments, be resolved by optical means. This means that one or more different signals, each representing one polynucleotide, occur within the resolvable area of the particular imaging device used.

[0155] Single molecule detection can be achieved where the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more particularly at least 250 nm, even 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 from the single molecule fluorescence spot also exhibits single-step photobleaching.

[0156] As used herein, the terms "resolved individually" and "individual resolution" are used to specify that, when visualized, one molecule on the array can be distinguished from its adjacent molecules. The separation between individual molecules on the array is determined in part by the particular technique used to resolve the individual molecules. General features of single molecule arrays will be understood by reference to the published applications WO00 / 06770 and WO01 / 57248, which are hereby incorporated by reference in their entirety. One use of the nucleotides of the present disclosure is in sequencing by synthesis reactions, but the utility 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.

[0157] 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 Sanger and co-workers' chain termination sequencing method. Such methods typically use an enzyme 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 randomized chain termination by labeled dideoxynucleotides.

[0158] Accordingly, the present disclosure also encompasses labeled nucleotides that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, and such dideoxynucleotides are suitable for use in Sanger-type sequencing methods and the like.

[0159] The labeled nucleotides of the present disclosure incorporating 3'-blocking groups may also be useful in the Sanger method and related protocols. This is because the same effect achieved by using dideoxynucleotides can be achieved by using nucleotides having a 3'-OH blocking group. Both prevent the incorporation of subsequent nucleotides. When the nucleotides according to the present disclosure are used in the Sanger-type sequencing method, it will be understood that the dye compound or detectable label attached to the nucleotide need not be attached via a cleavable linker. Each example in which the labeled nucleotides of the present disclosure are incorporated. Since there is no need to incorporate nucleotides later, there is no need to remove the label from the nucleotides.

[0160] Additional embodiments disclosed herein provide a method for determining a plurality of nucleic acid sequences, including providing a sample containing a plurality of different nucleic acids. Each nucleic acid includes a template and a primer. Performing cycles of a sequencing reaction, where a cycle includes extending the primers of the nucleic acids in the sample to form a plurality of extended primers having at least four different labeled nucleotide types as described herein, thereby forming an extended sample, wherein at least two of the different nucleotide types in the first extended primer are in a signal state and at least one of the different nucleotide types in the extended primer is in a dark state, collecting signals from the extended sample. Irradiating the polynucleotide with a light source to cause a change in the emission signal of a specific nucleotide label and obtaining a second collection of signals from the sample. Here, two different nucleotide types are in different states in the first collection of signals. The second collection of signals. Determining the sequences of the plurality of different nucleic acids by evaluating the first collection of signals from the cycle and the second collection of signals.A number of embodiments, the plurality of different nucleic acids are attached to a substrate. In some embodiments, the extension of the primer includes polymerase-catalyzed addition of different nucleotide types. In some embodiments, the different nucleotide types include reversible blocking moieties, whereby a single nucleotide type is added to each of the extended primers in each cycle. In some embodiments, the extension of the primer includes ligase-catalyzed addition of oligonucleotides containing different nucleotide types. In some embodiments, two of the different nucleotide types in the extended primer are in a dark state during the acquisition of the first collection of signals from the extended sample. In a preferred embodiment, the aforementioned sequencing reaction cycle is repeated one or more times.

Example

[0161] Additional embodiments are disclosed in more detail in the following examples, which are in no way intended to limit the scope of the claims.

[0162] (Example 1) In this example, a simplified approach for obtaining the identification of dyes incorporated based on a modified photoswitchable red-emitting phosphor is described, and the workflow for one cycle of incorporation is shown in FIG. 2.

[0163] The structures and functions of each base-binding fluorescent tag required for this approach are shown in Table 1. The "A" and "C" dyes have a photoswitching function. The "T" dye can be a standard red-emitting dye, for example, the same red dye that is part of a photoswitchable dyad for an "A" nucleotide. The linker that binds the dye to the nucleotide is the LN3 linker described herein and is shown only partially in Table 1 and the following photochemical reaction scheme. Other linkers disclosed herein can also be used. All of these dyes can function in biologically relevant media.

[0164] [Table 1]

[0165] The new A dye is designed to be photoswitchable using a blue laser or LED (405 nm). Photoactivation leads to a ring-opening reaction that converts the lower part of the dyad into a quencher that can suppress the emission from the Si-containing rhodamine dye (Scheme 1). [Chemical Structure]

[0166] The photoactivatable C dye can be switched to the "on" fluorescent state upon exposure to blue laser or LED irradiation (405 nm) while the A dye is converted to the quenched "off" state. In the case of the C dye, a similar ring-opening reaction reversibly opens the oxazine ring, generating a highly fluorescent state with a maximum absorption concentrated around 550 - 650 nm (Scheme 2). Thus, all of the dyes proposed here can be excited with the same red LED or laser (633 nm). [Chemical]

[0167] In Figure 2, four different types of nucleotides according to Table 1 are exposed to the flow cell for incorporation into the polynucleotide. During the first imaging step, the luminescence from each cluster is recorded. In this first imaging event, fluorescence signals emitted from both 'A' and 'T' nucleotides are detected. Next, a blue laser at a wavelength of 405 nm is used to quench the 'A' dye and activate the 'C' dye. Next, a second imaging step is performed and the luminescence from each cluster is recorded again. In this second imaging event, fluorescence signals emitted from 'C' and 'T' nucleotides are detected. In both imaging events, the 'G' nucleotide is dark (unlabeled). The nucleotides are identified by analysis of the different luminescence patterns of each base across the two images. The combination of Image 1 and Image 2 is processed with image analysis software to identify which base is incorporated at each cluster position. After the second imaging event, the incorporated nucleotides are unblocked according to standard procedures that allow the incorporation of another nucleotide. This sequencing cycle is repeated 'n' times to create a read length of 'n' bases.

[0168] In this example, a 405 nm blue laser or LED is used. This can be further adjusted by introducing additional substituents into the coumarin moiety of the 'C' dye or the spiro-naphthothiopyran moiety of the 'A' dye. This allows for full compatibility with the current iSeq™ system that uses a 450 nm laser. The proposed changes would also reduce the likelihood of DNA damage that could occur as a result of repeated exposure to higher energy irradiation over several 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 cycles of: (i) incorporating four different types of nucleotide conjugates into a plurality of polynucleotides complementary to the target polynucleotide to produce an extended polynucleotide, wherein the first type of nucleotide conjugate comprises a first photoswitchable label, the second type of nucleotide conjugate comprises a second photoswitchable label, and the third type of nucleotide conjugate comprises a third label; (ii) detecting a first collection of signals from the extended polynucleotide in each cycle via a first imaging event; (iii) irradiating the extended polynucleotide with a light source to cause a change in the emission signals of the first photoswitchable label and the second photoswitchable label; (iv) detecting a second collection of signals from the extended polynucleotide via a second imaging event; (b) determining the sequence of the target polynucleotide based on the consecutively incorporated nucleotide conjugates.

2. The method of claim 1, wherein incorporation of the first type of nucleotide conjugate is determined from a signal state in the first imaging event and a dark state in the second imaging event.

3. The method of claim 1 or 2, wherein incorporation of the second type of nucleotide conjugate is determined from a dark state in the first imaging event and a signal state in the second imaging event.

4. The method of any one of claims 1 to 3, wherein incorporation of the third type of nucleotide conjugate is determined from signal states in the first imaging event and the second imaging event.

5. The method of any one of claims 1 to 4, wherein incorporation of the fourth type of nucleotide conjugate is determined from dark states in the first imaging event and the second imaging event.

6. The method of any one of claims 1 to 5, wherein the four different types of nucleotide conjugates are present simultaneously and compete for incorporation during each cycle.

7. The method of any one of claims 1 to 6, wherein step (a) is repeated at least 50 times. Claim 8 The method according to any one of claims 1 to 7, wherein the incorporation of the nucleotide conjugate is effected by a polymerase. Claim 9 The method according to any one of claims 1 to 8, wherein the different types of nucleotide conjugates comprise reversible terminator moieties. Claim 10 The method according to claim 9, wherein step (a) further comprises cleaving the reversible terminator moiety from the incorporated nucleotide conjugate prior to the next incorporation cycle. Claim 11 The method according to any one of claims 1 to 10, wherein the nucleotide conjugate comprises a nucleotide type selected from the group consisting of dATP, dTTP, dUTP, dCTP, dGTP, and non-natural nucleotide analogs thereof. Claim 12 The label of the first type of nucleotide conjugate and the third type of nucleotide conjugate comprises the same fluorescent moiety; or 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, The method according to any one of claims 1 to 11. Claim 13 The method according to any one of claims 1 to 12, wherein the fourth type of nucleotide conjugate is not labeled with a fluorescent moiety. Claim 14 The method according to any one of claims 1 to 13, wherein the irradiation in step (a)(iii) does not substantially change the signal detected from the third label of the third type of nucleotide conjugate. Claim 15 The method according to any one of claims 1 to 14, wherein the irradiation light source in step (a)(iii) comprises a laser, a light emitting diode (LED), or a combination thereof. Claim 16 The method according to any one of claims 1 to 15, wherein the irradiation light source in step (a)(iii) has a wavelength different from the excitation wavelength used in the first imaging event. Claim 17 The method according to any one of claims 1 to 16, wherein the irradiation light source in step (a)(iii) has a wavelength in the range of 350 nm to 450 nm. Claim 18 The method according to claim 17, wherein the irradiation light source in step (a)(iii) has a wavelength of 405 nm. Claim 19 The method according to any one of claims 1 to 18, wherein the first imaging event and the second imaging event have substantially the same excitation wavelength.

20. The method according to claim 19, wherein the first imaging event and the second imaging event have an excitation wavelength of 550 nm to 650 nm.

21. The method according to claim 20, wherein the first imaging event and the second imaging event have an excitation wavelength of 633 nm.

22. The method according to any one of claims 1 to 21, wherein the first photoswitchable label comprises a first fluorescent moiety covalently attached to a first photochromic moiety.

23. The method according to claim 22, wherein the first fluorescent moiety is covalently attached to the first photochromic moiety via a first linker.

24. The method according to claim 22 or 23, wherein the first fluorescent moiety comprises a fluorescent moiety that emits red light.

25. The method according to claim 24, wherein the first fluorescent moiety comprises a silicon rhodamine moiety and / or a spiropyran or spirothiopyran moiety.

26. The method according to any one of claims 22 to 25, wherein the first photochromic moiety comprises a structure of formula (I): 【Chemical 1】 Wherein: X is O (oxygen) or S (sulfur); R １ａ and R ２ａ are each independently selected from the group consisting of H, halo, 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 is selected from the group consisting of H, C １－６ alkyl, and -(CH ２ ) ｎ -R ４ ; R ４ is selected from the group consisting of C ６－１０ aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic, and 3- to 7-membered heterocyclic, each of which may be substituted; Ring A is C ６－１０ aryl or 5-10 membered heteroaryl, each substituted with at least one electron-withdrawing group; n is an integer from 1 to 6; and Irradiation step (a)(iii) causes cleavage of the spiro C-X bond.

27. The method according to claim 26, wherein X is S.

28. Each R １ａ and R ２ａ is C １－６ alkyl, the method according to claim 26 or 27.

29. The method according to any one of claims 26 to 28, wherein ring A is phenyl or naphthyl substituted with at least one electron-withdrawing group.

30. The electron-withdrawing group is nitro, cyano, fluoro, bromo, -S(O) ２ OH, -S(O) ２ CF ３ , ammonium, alkylammonium, and C substituted with one or more fluoros or bromos １－６ alkyl, the method according to claim 29

31. The first photochromic moiety has a structure of formula (Ia): 【Chemical 2】 The method according to any one of claims 26 to 30, comprising.

32. 【Chemical Formula 3】 , where L １ is the first linker. The method according to any one of claims 22 to 31, wherein the first photoswitchable label comprises the following structure:

33. The method according to any one of claims 1 to 32, wherein the second photoswitchable label comprises a second fluorescent moiety covalently attached to a second photochromic moiety.

34. The method according to claim 33, wherein the second fluorescent moiety is covalently attached to the second photochromic moiety via a second linker.

35. The method according to claim 33 or 34, wherein the second fluorescent moiety comprises a coumarin moiety.

36. The method of any one of claims 33 to 35, wherein the second photochromic moiety comprises an oxazine or thiazine moiety.

37. The method of any one of claims 33 to 36, wherein the second photochromic moiety comprises the structure of formula (II): 【Chemical 4】 During the ceremony: Y is O (oxygen) or S (sulfur); R １ｂ and R ２ｂ are each independently selected from the group consisting of H, halo, 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 is substituted with at least one electron-withdrawing group; and * indicates the point of attachment to the fluorescent moiety, and the irradiation step (a)(iii) causes cleavage of the carbon *-Y bond.

38. 38. The method of claim 37, wherein Y is O.

39. R １ｂ and R ２ｂ are each C １－６ alkyl, the method according to claim 37 or 38.

40. 40. The method of any one of claims 37 to 39, wherein Ring B is phenyl or naphthyl substituted with at least one electron-withdrawing group.

41. The electron-withdrawing group is nitro, cyano, fluoro, bromo, -S(O) ２ OH, -S(O) ２ CF ３ , ammonium, alkylammonium, and C substituted with one or more fluoros or bromos １－６ alkyl, the method according to claim 40, selected from the group consisting of.

42. The second photochromic moiety has the structure of Formula (IIa): 【Chemical Formula 5】 42. The method of any one of claims 37 to 41, comprising:

43. 43. The method of any one of claims 37 to 42, wherein the second photoswitchable label comprises the structure: 【Chemical Formula 6】 , where L2 is a second linker.

44. The method of any one of claims 1 to 43, wherein a plurality of polynucleotides are attached to a substrate.

45. 45. The method of claim 44, wherein detecting the first set of signals and the second set of signals comprises acquiring an image of the substrate.

46. A kit comprising four types of nucleotides, i) the first type of nucleotide is a first labeled nucleotide conjugate that includes a first photoswitchable label; the first photoswitchable label comprises a first fluorescent moiety covalently linked to a first photochromic moiety; the first fluorescent moiety comprises a silicon rhodamine moiety; and The first photochromic moiety comprises the structure of formula (I): 【Chemical Formula 7】 During the ceremony: X is O (oxygen) or S (sulfur); R １ａ and R ２ａ are each independently selected from the group consisting of H, halo, 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 is selected from the group consisting of H, C １－６ alkyl, and -(CH ２ ) ｎ -R ４ and is selected from the group consisting of R ４ is selected from the group consisting of C ６－１０ aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclic, and 3- to 7-membered heterocyclic, each of which may be substituted; Ring A is C ６－１０ aryl or 5- to 10-membered heteroaryl, each substituted with at least one electron-withdrawing group; and n is an integer from 1 to 6; ii) the second type of nucleotide is a second labeled nucleotide conjugate that includes a second photoswitchable label; the second photoswitchable label comprises a second fluorescent moiety covalently linked to a second photochromic moiety; the second fluorescent moiety comprises a coumarin moiety, an oxazine moiety, or a thiazine moiety; and The second photochromic moiety comprises the structure of formula (II): [Chemical Formula 8] During the ceremony: Y is O (oxygen) or S (sulfur); R １ｂ and R ２ｂ each independently is selected from the group consisting of H, halo, 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; and Ring B is C ６－１０ aryl or 5- to 10-membered heteroaryl; each substituted with at least one electron-withdrawing group; and * indicates the point of attachment to the fluorescent moiety; iii) The third type of nucleotide contains a label that emits light at the same wavelength as the first type of nucleotide, iv) The fourth type of nucleotide is unlabeled, Kit.

47. The kit according to claim 46, wherein the first fluorescent moiety is covalently bonded to the first photochromic moiety via a first linker.

48. The kit according to claim 46 or 47, wherein X is S.

49. Each R １ａ and R ２ａ is C １－６ alkyl, the kit according to any one of claims 46 to 48.

50. The kit according to any one of claims 46 to 49, wherein the second fluorescent moiety is covalently bonded to the second photochromic moiety via a second linker.

51. The kit according to any one of claims 46 to 50, wherein Y is O.

52. Each R １ｂ and R ２ｂ is C １－６ alkyl, the kit according to any one of claims 46 to 51.

53. Ring A is substituted with at least one electron-withdrawing group selected from the group consisting of nitro, cyano, fluoro, bromo, -S(O ２ )OH, -S(O) ２ CF ３ , ammonium, alkylammonium, and phenyl or naphthyl substituted with C １－６ alkyl substituted with one or more fluoros or bromos. The kit according to any one of claims 46 to 52

54. Ring B is phenyl or naphthyl substituted with at least one electron-withdrawing group selected from the group consisting of nitro, cyano, fluoro, bromo, -S(O ２ )OH, -S, (O) ２ CF ３ , ammonium, alkylammonium, and C １－６ alkyl, the kit according to any one of claims 46 to 53.

55. The kit according to any one of claims 46 to 54, wherein the first type of nucleotide, the second type of nucleotide, and the third type of nucleotide are detectable at the same emission wavelength.

56. The kit according to any one of claims 46 to 55, wherein the photochromic moiety comprises the structure of formula (Ia): 【Chemical Formula 9】 。

57. The kit according to any one of claims 46 to 56, wherein the light-switchable label comprises the following structure: 【Chemical 10】 , where L １ is a linker.

58. The kit according to any one of claims 46 to 57, wherein the photochromic moiety comprises the structure of formula (IIa): 【Chemical 11】 。

59. The kit according to any one of claims 46 to 58, wherein the light-switchable label comprises the following structure: 【Chemical 12】 , where L ２ is a linker.

60. i) The light-switchable label is bonded to the C5 position of the pyrimidine base or the C7 position of the 7-deazapurine base; or ii) The light-switchable label of the first type of nucleotide and / or the second type of nucleotide is bonded to the nucleobase via a cleavable linker. The kit according to claim 59.

Images