Sequencing of DNA by sequential addition / incorporation of 3' unprotected labeled nucleotides

Abstract

To provide DNA sequencing by sequential addition / incorporation of non 3'capped and fluorescently labeled nucleotides.SOLUTION: Each incorporation of individual nucleotides (A, or T or G or C) is separated by washing. After all incorporation using all four bases, a substrate is imaged, dyes are then cleaved, and a following cycle of incorporation, washing, imaging and cleaving is resumed.SELECTED DRAWING: None

Description

Technical Field

[0001] Background Art Next-generation sequencing (NGS) is a very powerful tool for studying clinically relevant target sequences, and gene-based panel tests are the first choice for studying DNA variants. For example, sequencing by hybridization (Drmanac et al. (1998) Nat Biotechnol 16:54-58), sequence-specific detection of single-stranded DNA using engineered nanopores (Kasianowicz et al. (1996) Proc Natl Acad Sci USA 93:13770-13773), pyrosequencing (Ronaghi et al. (1998) Science 281:363-365), sequencing of single DNA molecules (Braslavsky et al. (2003) Proc Natl Acad Sci USA 100:3960-3964), sequencing by ligation (Shendure et al. (2005) Science 309:1728-1732), and rolling circle / polony (Mitra et al. (2003) Anal Biochem 320:55-65) and other approaches have been widely studied.

[0002] However, currently, synthetic DNA sequencing (SBS) is the method selected for sequencing. In this approach, each sequencing cycle involves at least 1) addition of a protected nucleotide with a cleavable fluorescent label, 2) addition of polymerase to DNA or RNA primed with an oligonucleotide and incorporation of a nucleotide, 3) detection of the fluorescent label, 4) removal of the fluorescent label, 5) deprotection of the incorporated nucleotide, where each step may be followed by a washing step.

[0003] DNA sequencing (SBS) chemistry using reversibly terminated nucleotides has already been incorporated into several DNA sequencing systems with considerable performance, utilizing polymerase as the primary enzyme. However, many challenges remain to improve sequencing speed and cost, including the efficient recognition and integration of modified nucleotides containing chemically reversible linkers that cleave fluorophores to bases and cap the 3'-OH group of the deoxyribose phosphate backbone by DNA polymerase. To date, no successful integration of such nucleotides with conventional DNA polymerases has been reported. The main reason is that the 3' position of deoxyribose is located near the amino acid residues in the polymerase's active site, and modifications in this region of the ribose ring impair or reduce the integration of these modified nucleotides (Pelletier et al. (1994) Science 264:1891-1903).

[0004] Conversely, proprietary fluorescent dyes linked to the 5th position (T and C) of pyrimidines and the 7th position (G and A) of purines via cleavable linkers are more acceptable because they are not located in close proximity to the active site residues. Furthermore, capping groups must be chemically removed between each cycle to allow for subsequent reversibly terminated nucleotides. Consequently, the overall process becomes very slow; for example, typical sequencing of DNA / RNA with 100 nucleotide units takes approximately 9–15 hours.

[0005] Furthermore, to allow for a very wide range of modifications, including the small chemical moieties required for the SBS approach used to cap the 3'-OH group, and to enable the incorporation of these nucleotide analogs into the growing DNA strand, it is necessary to use a modified DNA polymerase. Therefore, the objective of the present invention was to provide a remarkably rapid and simple process for sequencing DNA or RNA libraries. Summary of the Invention

[0006] In this invention, the inventors describe a method for sequencing DNA or RNA by an approach that uses the sequential addition / incorporation of non-3' capping fluorescently labeled nucleotides.

[0007] Therefore, the present invention relates to a method for detecting at least a portion of the sequence of an RNA or DNA molecule, a) A step of sequentially supplying nucleotides A, T, C, and G, each equipped with at least one fluorescent dye, to DNA or a DNA molecule via a linker that can be cleaved in the presence of polymerase, thereby incorporating the nucleotides into a DNA or RNA molecule, wherein the fluorescent dyes of the nucleotides have different luminescence maximums, and the nucleotides have an unprotected 3'OH position; b) A step of removing nucleotides that have not been incorporated, c) A step of detecting the incorporated nucleotides by excitation, detecting the fluorescence emission of a fluorescent dye, and thereby obtaining sequence information, d) The step of removing the fluorescent dye from the incorporated nucleotide by cleaving a cleavable linker. The method is characterized by including [a certain element].

[0008] In this approach, individual nucleotides that are not protected (also called uncapped) at the 3'OH position of the deoxyribose phosphate backbone are used sequentially in a predetermined order, and the extended DNA or RNA strand is imaged individually after all four nucleotides have been added by polymerase.

[0009] This invention utilizes multiple embedded cycles and a reduction in the number of images that need to be captured. [Brief explanation of the drawing]

[0010] [Figure 1]The general formulas for nucleotides A, T, C, and G having unprotected ("uncapped") and protected ("capped") 3'OH positions used in the present invention are shown below. [Figure 2] This shows one cycle of the method of the present invention. [Figure 3] The present invention includes embodiments that provide RNA / DNA molecules as Rorony immobilized on a surface. [Figure 4] This demonstrates a calibration step using single incorporation during the first four cycles of sequencing, utilizing the last four nucleotides of a library adapter (calibration sequence) containing T, G, A, and C.

[0011] Detailed explanation Using uncapped nucleotides allows the use of conventional polymerases, eliminating the need for the high-temperature and genetically engineered thermophilic DNA polymerases currently used by SBS. These enzymes share the same catalytic mechanism as their mesothermic counterparts but are optimally activated at high temperatures.

[0012] Preferably, the nucleotides are incorporated into the DNA or RNA molecule in the presence of polymerase at a temperature of 10 to 80°C, and preferably in the presence of polymerase at a temperature of 10 to 40°C.

[0013] During SBS, labels and terminators are chemically removed after incorporation and imaging to prepare the complementary chain for the next sequencing cycle. If no bases are incorporated during the cycle, the chain begins to lag. On the other hand, if multiple bases are added in a single cycle, the chain advances (leads). This is called phasing. Phasing will make the actual base calling more difficult and will increase the error rate. The difficulty in incorporating highly modified nucleotides, especially nucleotides with modifications at the 3'OH position of the deoxyribose phosphate backbone located near the polymerase active site, will be advantageous against the phasing phenomenon. By not using modifications at the 3'OH position, the phasing problem is eliminated or greatly reduced.

[0014] Optionally, before the sequencing process, provide RNA or DNA molecules with a sequence of 4-50 nucleotides to serve as primers or adapters for polymerase.

[0015] For further identification purposes, it is possible to optionally provide RNA or DNA molecules with a sequence of 4-50 nucleotides as a unique identifier (UMI) as part of an adapter sequence prior to the sequencing process. Such sequences are known in the field of single-cell sequencing.

[0016] In another embodiment of the present invention, an RNA or DNA molecule is provided with a sequence of 4 to 50 A, T, C, and G nucleotides, each equipped with at least one fluorescent dye as a calibration sequence for fluorescence emission, and the fluorescence emission of the incorporated nucleotides is detected by its relative intensity to the fluorescence emission of the calibration sequence.

[0017] Calibration of each DNA / RNA molecule or colony or cluster to be sequenced can be achieved by measuring the signals of known calibration sequences. In the simplest variant form, a single incorporation (one each) of A, T, C, and G nucleotides during the first 4 cycles of sequencing is used for calibration. To increase the calibration efficiency, 2 to 4 of each of the A, T, C, and G nucleotides may be provided as the calibration sequence.

[0018] A general workflow of the calibration process is shown in Figure 4, and the measured intensities are equal to one nucleotide, and these values are used to adjust the intensity of each nucleotide during the sequencing process of the present invention.

[0019] The signals of these single incorporations performed for each colony or cluster before actually sequencing the target sequence can determine the signal for single nucleotide incorporation and predict the expected signals for multiple base incorporations found in homopolymers.

[0020] In step a), nucleotides A, T, C, and G each provided with at least one fluorescent dye via a cleavable linker are provided continuously, that is, there are other nucleotides after a certain type of nucleotide, and optionally, a washing procedure for removing unincorporated nucleotides follows.

[0021] As shown in Figure 1, the nucleotides used in the method of the present invention have an unprotected 3'OH position.

[0022] The nucleotide may have the general formula (I) NP-(CL-D) x (I), wherein, N: natural or artificial nucleic acid P: 3 or more phosphate groups CL: cleavable linker D: fluorescent dye x: an integer from 1 to 10 It is.

[0023] Nucleotides are provided to a DNA or RNA molecule in the presence of a polymerase such that the nucleotides are incorporated into the DNA or RNA molecule.

[0024] To distinguish incorporated nucleotides for detection, the fluorescent dyes of the nucleotides have different emission maxima.

[0025] Each of nucleotides A, T, C, and G is provided as a mixture containing a species with a fluorescent dye via a cleavable linker and a species without a fluorescent dye, and both species are further capable of having an unprotected 3’OH position. In this embodiment, quenching of adjacent dyes can be reduced.

[0026] Suitable fluorescent dyes and detection means are known from published sequencing techniques. Nucleotides can be labeled using suitable fluorescent dyes known in the field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. For example, the fluorescent dye is a xanthene dye such as fluorescein or rhodamine dye, a coumarin dye, a cyanine dye, a pyrene dye, an oxazine dye, a pyridyloxazole dye, a pyromethene dye, an acridine dye, an oxadiazole dye, a carbopyronin dye, a benzopyrylium dye, a fluorene dye, or an organometallic complex such as Ru, Eu, Pt complex. Not only single molecule entities, but also clusters of small organic molecule dyes, fluorescent oligomers, or fluorescent polymers such as polyfluorene can be used as the fluorescent moiety. Furthermore, the fluorescent dye can be a protein-based such as phycobiliprotein, nanoparticles such as quantum dots, upconverting nanoparticles, gold nanoparticles, stained polymeric nanoparticles. These can have modifications at the 3’ or 5’ end, or the modification can be any base on the backbone, such as dUTP with a PA linker. The labeling molecule can be a fluorescent molecule (such as R6G, ROX, Cy3, Cy5, Alexa dye, ATTO dye, etc.) or an energy transfer dye.

[0027] DNA or RNA molecules can be provided in several modified forms in this method. In the first modified form, the DNA or RNA molecule can be provided as RNA or DNA loroney containing multiple concatemers of the RNA or DNA molecule. In the second modified form, the RNA or DNA molecule can be provided as a single-stranded RNA or DNA molecule, and / or as a mixture of sense and antisense single-stranded DNA. In the third modified form, the RNA or DNA molecule is fragmented before nucleotide incorporation.

[0028] Furthermore, RNA or DNA molecules (optionally in the form of Rollonie) can be immobilized on solid surfaces. This immobilization can be achieved by interacting with the surface via electrostatic charge or an NHS ester-activated crosslinking agent.

[0029] RNA or DNA molecules / Rolonys / nanoballs / clusters can be generated from libraries optionally containing calibration sequences, adapters, primers, UMIs, or further barcodes. These have several copies of a desired region on a given genome and are bound to a solid surface or embedded in a gel.

[0030] In step a), uncapped fluorescent nucleotide (T, A, G, or C) triphodes are added to a solid surface in a specific known order in the presence of DNA polymerase. For example, a solution containing T is added first, followed by A, G, and then C. After each solution is added, the surface is washed to remove any trace amounts of unintegrated nucleotides.

[0031] In step c, after all integration, the extended sequencing primers corresponding to the region to be sequenced are imaged in their respective nucleotide fluorescence channels using a solution containing all four nucleotides.

[0032] In step d), the fluorescent dye bound to the nucleotide base is removed via a cleavable linker (e.g., a linker containing a disulfide or azide bond), and the cycle, which begins again in step a), is repeated.

[0033] The cleavable linker may be cleaved enzymatically, chemically, or by radiation.

[0034] Removal of the fluorescent dye from each nucleotide after the integration cycle depends on a cleavable linker and can be appropriately selected from the following: - Disulfide linkers can be selectively cleaved by treatment with thiols that do not impair other linkers.

[0035] - Oxymethine azide (-OCH(N3)-) can be selectively cleaved in the presence of a photocleavable or other enzymatically cleavable linker when treated with phosphine (e.g., TCEP).

[0036] Alternatively, TCEP can be used to cleave both the disulfide and the oxymethine azid linker.

[0037] - The cleavage agent may be a thiol (e.g., dithiothreitol-DDT, dimercaptopropanesulfonate-DMPS, etc.), a phosphine (e.g., TCEP, THPP, etc.), a mild reducing agent (e.g., Na2S2O4), light of a specific wavelength, or an enzyme (e.g., peptidase, dextranase, esterase, phosphatase, proteinase, etc.).

[0038] Enzymatically cleavable linkers can be any molecule that can be cleaved by a specific enzyme, such as a hydrolase. Suitable enzymatically cleavable spacer P includes, for example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids, and their derivatives. Suitable polysaccharides include, for example, dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan or glucomannan, pectin, chitosan, or chitin, which can be derivatized to provide functional groups for the covalent or non-covalent bonds of linker L; proteins, peptides, and depsipeptides used as enzymatically cleavable linkers can be functionalized via amino acid side-chain functional groups; and polyesters and polyesteramides used as enzymatically cleavable linkers can be synthesized with comonomers providing side-chain functional groups or functionalized afterward. In the case of branched polyesters, functionalization may be via carboxyl or hydroxyl terminal groups. Post-polymerization functionalization of polymer chains may occur, for example, through addition to unsaturated bonds, i.e., thiolene reactions or azide-alkyne reactions, or through the introduction of functional groups via radical reactions.

[0039] Steps b) through e) can be repeated until all the nucleotides necessary for sequencing at least some or all of the DNA or RNA molecules to be sequenced are completed. For example, steps b) through e) can be repeated for 5 to 1000 cycles.

Claims

1. 1. A method for detecting at least a portion of a sequence of an RNA or DNA molecule, comprising: a) providing the RNA or DNA molecule with successive nucleotides A, T, C, and G, each of which is equipped with at least one fluorescent dye, via a linker that is cleavable in the presence of a polymerase, thereby incorporating the nucleotides into the RNA or DNA molecule, wherein the fluorescent dyes of the nucleotides have different emission maxima and the nucleotides have an unprotected 3' OH position; b) removing unincorporated nucleotides; c) detecting the incorporated nucleotide by exciting it and detecting the fluorescence emission of the fluorescent dye, thereby obtaining sequence information; d) removing the fluorescent dye from the incorporated nucleotide by cleaving the cleavable linker; A method comprising:

2. 2. The method of claim 1, wherein steps b) to e) are repeated until all nucleotides necessary for sequencing at least a portion of the RNA or DNA molecule to be sequenced are completed.

3. 2. The method of claim 1, wherein steps b) to e) are repeated for 5 to 1000 cycles.

4. The method of claim 1, wherein each of the nucleotides A, T, C, and G is provided as a mixture containing a species having a fluorescent dye attached via a cleavable linker and a species without a fluorescent dye, both species having an unprotected 3' OH position.

5. 2. The method of claim 1, wherein the cleavable linker is cleaved enzymatically or chemically, or by radiation.

6. 2. The method of claim 1, wherein the RNA or DNA molecule is provided with a sequence of 4 to 50 nucleotides as a unique identifier (UMI) as part of an adapter sequence.

7. 2. The method of claim 1, wherein the RNA or DNA molecule is provided with a sequence of 4 to 50 A, T, C, and G nucleotides, each of which is provided with at least one fluorescent dye as a calibration sequence for fluorescence emission, and the fluorescence emission of the incorporated nucleotides is detected by its relative intensity to the fluorescence emission of the calibration sequence.

8. The nucleotide is represented by the general formula (I): NP-(CL-D) ｘ (I) wherein N: natural or artificial nucleic acid P: 3 or more phosphate groups CL: cleavable linker D: fluorescent dye X: an integer from 1 to 10 2. The method of claim 1, wherein:

9. 2. The method of claim 1, wherein the nucleotides are incorporated into the DNA or RNA molecule in the presence of a polymerase at a temperature between 10 and 80°C.

10. The method of claim 1, wherein the RNA or DNA molecule is provided as an RNA or DNA lone molecule comprising multiple concatemers of the RNA or DNA molecule.

11. 2. The method of claim 1, wherein the RNA or DNA molecule is provided as a single-stranded RNA or DNA molecule and / or as a mixture of sense and antisense DNA single strands.

12. 2. The method of claim 1, wherein the RNA or DNA molecule is fragmented prior to incorporation of nucleotides.

13. 2. The method of claim 1, wherein the RNA or DNA molecule is immobilized on a solid surface.

14. 14. The method of claim 13, wherein the RNA or DNA molecules are immobilized by interacting with the surface via electrostatic charges or NHS ester-activated crosslinkers.

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