Nucleic acid sequencing using raman spectroscopy

Abstract

This disclosure is directed to methods and systems to perform accurate, long-read sequencing of nucleic acids using Raman spectroscopy with nanophotonic metasurfaces. This technology can be utilized for various nucleic acids (e.g., DNA or RNA) using different potential biochemical approaches with solid substrates, nanochannels or nanopores.

Description

[0001] NUCLEIC ACID SEQUENCING USING RAMAN SPECTROSCOPY

[0002] RELATED CASES

[0003] [1] This International PCT application claims priority to U.S. Serial No. 63 / 660,112, filed 14 June 2024 and is incorporated by reference in its entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] [2] The present disclosure relates to systems and methods for determining the sequence of nucleic acids.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] [3] Existing methods for nucleic acid sequencing, based on detection of fluorescently labeled nucleic acids that have been separated by size, generally focus on generating massive numbers of short base reads or generating small numbers of more informative longer base reads, forcing the use to prioritize speed and cost versus information gained from the sequencing operation. For example, next-gen sequencing-by-synthesis requires that many copies of the gene be produced, cut into overlapping fragments and sequenced, after which the overlapping DNA sequences may be assembled into a complete sequence using massively-parallel assembly techniques. Alternatively, sequencing methods that provide more informative long reads generally result in small numbers of more informative long reads, in a process that is laborious, expensive, inefficient and timeconsuming.

[0008] [4] The physical process of Raman scattering and the potential utility of Raman spectroscopy to nucleic acid sequencing has been appreciated in the scientific community, but it suffers from a couple of key issues that have limited its applicability in molecular detection applications. In particular, Raman scattering is orders of magnitude less efficient than fluorescence, resulting in a weak signal in comparison, and Raman scattering interrogates all molecular bonds of a target molecule at once, producing a highly complex emission spectrum that can be difficult to interpret. The present invention overcomes these limitations to provide novel methods and systems for improved long-read sequencing of individual nucleic acids. SUMMARY OF ILLUSTRATIVE EMBODIMENTS

[0009] [5] This disclosure is directed to methods and systems to perform accurate, high- throughput long-read sequencing of nucleic acids. More specifically, this disclosure is directed to devices and methods of utilizing spectroscopy (e.g., Raman spectroscopy) with nanophotonic metasurfaces to determine the base sequence of nucleic acid templates.

[0010] [6] This technology can be utilized for various nucleic acids (e.g., DNA, RNA or nucleic acids with modified sugar or backbone structures) using different potential biochemical approaches and solid substrates as set forth herein in more detail.

[0011] [7] The nanophotonic metasurface substrates used with the methods and systems of the disclosure are improved over conventional substrates, and in particular display electric field enrichment and quality factors capable of amplifying light in local regions by as much as 10A8-r, which enables the pragmatic use of Raman scattering for single molecule analysis. The use of such metasurfaces combined with the solutions for optical detection and informatics enables massively parallelized single-molecule Raman spectroscopy capable of real-time nucleic acid sequencing. These methods and systems include polymerase-based or pore-based sequencing approaches to generate signals for nucleotides as the DNA translocates through the resonators on the metasurface in real time. The methods and systems disclosed herein allow the generation of long sequencing reads (e.g., lOOOs / lOOOOs bp) at read counts that far exceed what is currently achievable (e.g., 250M+ reads per run).

[0012] [8] The methods and systems described herein can be compatible with labeled and / or unlabeled nucleotides that generate distinguishable Raman signatures. The technology enables single-molecule real-time DNA sequencing at a scale and accuracy that was previously unachievable using conventional technologies. Combined with the described improvements to end-to-end optics that enable imaging at the required resolution, field of view (FOV), spectrum, and speed provide real-time sequencing at scale for determining the base sequence of longer nucleic acid molecules.

[0013] [9] The excitation used for detection in the presently disclosed methods and systems include, but are not limited to, spontaneous Raman and stimulated Raman microscopy. A nanophotonic metasurface combined with optical detection strategies leverage the advantages of Raman spectroscopy, allowing broad compatibility with various molecular biology strategies for base detection in individual molecules on a solid substrate.

[0010] In some aspects, the disclosure provides methods of identifying a base sequence of a nucleic acid template by Raman spectroscopy by providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized polymerase, exposing a single-stranded nucleic acid template to be sequenced to the solid substrate under conditions to allow the polymerase to create a complementary strand from the template, and detecting the base sequence of the nucleotides incorporated into the strand complementary to the template using Raman spectroscopy with a wide field of view optics solution, thereby determining the base sequence of the nucleic acid template.

[0014]

[0011] In some aspects, the disclosure provides methods of identifying a base sequence of an RNA template by Raman spectroscopy by providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized reverse transcriptase, exposing an RNA template to be sequenced to the solid substrate under conditions to allow the reverse transcriptase to create a complementary strand from the template, and detecting the base sequences of the nucleotides incorporated into the strand complementary to the template using Raman spectroscopy with a wide field of view optics solution, thereby determining the base sequence of the RNA.

[0015]

[0012] In some aspects, the disclosure provides methods of identifying a base sequence of a nucleic acid template by Raman spectroscopy by providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized exonuclease, exposing a single-stranded nucleic acid template to be sequenced to the solid substrate under conditions to allow the exonuclease to remove single nucleotides from an unattached end of the nucleic acid template, and detecting the base sequences of the nucleotides removed from the template using Raman spectroscopy with a wide field of view optics solution, thereby determining the base sequence of the nucleic acid template.

[0016]

[0013] In some aspects, the disclosure provides methods of sequencing a single stranded nucleic acid template by passing the template through a nanochannel comprising a Raman resonator region, wherein the resonator region in actively monitored in real time, repeating this process to produce at least 2X coverage of the template through the resonator region, and inferring the base sequence of the template based on the change of the Raman signal produced. In some aspects, the process produces at least 5X coverage of the template. In some aspects, the process produces at least at least 10X coverage of the template.

[0014] In some aspects, the disclosure provides methods of sequencing a single stranded nucleic acid template by passing the template through a nanochannel comprising a Raman resonator region, wherein the resonator region in actively monitored in real time, identifying the nucleotides of a first section of the template present in the nanochannel at a first period in time by a Raman signal, and identifying the nucleotides of a second section of the DNA strand present in the nanochannel at a second period in time by a second Raman signal, and inferring the base sequence of the template based on the Raman signals produced by the first and second sections of the template. As will be appreciated by one skilled in the art, a nanochannel may have two or more Raman resonator regions that simultaneously or sequentially measure the Raman signal of the nucleic acids passing through them in real time.

[0017]

[0015] In some aspects, the disclosure provides methods of sequencing a single stranded nucleic acid template by modifying the template to incorporate Raman labels, passing the modified template through a solid state nanopore, wherein the opening of the pore contains a resonator region that is actively monitored in real-time, and inferring the base sequence of the template based on the Raman signals produced by the template.

[0018]

[0016] These and other embodiments, features, and advantages will be set forth in the present disclosure.

[0019]

[0017] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. These and various other features and advantages will be apparent from a reading of the following detailed description.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate one or more embodiments and, together with the description, explain these embodiments. The accompanying drawings have not necessarily been drawn to scale. Any values or dimensions illustrated in the accompanying graphs and figures are for illustration purposes only and may or may not represent actual or preferred values or dimensions. Where applicable, some or all features may not be illustrated to assist in the description of underlying features.

[0019] Figures 1-3 illustrate an aspect of the disclosure for DNA sequencing using polymerase immobilized on a nanophotonic metasurface solid substrate.

[0022]

[0020] Figure 4 illustrates an aspect of the disclosure for RNA sequencing using reverse transcriptase immobilized on a nanophotonic metasurface solid substrate.

[0023]

[0021] Figure 5 illustrates an aspect of the disclosure for sequencing using an exonuclease immobilized on a nanophotonic metasurface solid substrate.

[0024]

[0022] Figures 6-8 illustrate an aspect of the disclosure for sequencing using Raman spectroscopy with nanochannel technology.

[0025]

[0023] Figures 9-11 illustrate an aspect of the disclosure for sequencing using Raman spectroscopy with nanopore technology.

[0026]

[0024] Figures 12-14 illustrate an aspect of the disclosure for sequencing using Raman spectroscopy with immobilized nucleic acids and template degradation.

[0027]

[0025] Figure 15 illustrates an optical detection strategy using Skip-seq imaging to detect nucleic acid sequences on a rectangular chip.

[0028]

[0026] Figure 16 illustrates an optical detection strategy using Skip-seq imaging to detect nucleic acid sequences on a circular disc.

[0029]

[0027] Figure 17 illustrates exemplary coverage for Skip-seq imaging for a 2 kb template.

[0030]

[0028] Figure 18 illustrates exemplary coverage for Skip-seq imaging for a 5 kb template.

[0031]

[0029] Figure 19 illustrates exemplary coverage for Skip-seq imaging for a 10 kb template.

[0032]

[0030] Figure 20 illustrates exemplary coverage for Skip-seq imaging for a 20 kb template.

[0033] DETAILED DESCRIPTION

[0034]

[0031] The following detailed description of preferred embodiments of the disclosure will be better understood when read in conjunction with the appended drawings.

[0032] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0035]

[0033] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0036] Definitions

[0037]

[0034] As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

[0038]

[0035] A “nanophotonic metasurface” as used herein refers to the surface of a solid substrate that can manipulate light at subwavelength scales to enable highly parallelizable single-molecule detection. As Raman spectroscopy uses the intrinsic absorbance spectra of molecular bonds to build identifiable chemical signatures, use of nanophotonic metasurfaces allows highly efficient electric field enrichment and addresses the inherent inefficiencies of Raman scattering. Examples of nanophotonic metasurfaces can be found in US20220364982; US20230350266; US20230341384; WO2022213092; W02023097050 and Hu J. et a; Nat Commun. 2023 Jul 26; 14(1):4486.

[0039]

[0036] “Nucleic acid” encompasses deoxyribonucleic acids, ribonucleic acids, nucleic acids with modified sugar structures (e.g., 2-0-Me nucleic acids), single-stranded, doublestranded or triple stranded and any chemical modifications thereof. Virtually any modification of the nucleic acid is contemplated, including modifications to the base, the sugar molecule, or the backbone. Modifications can also include labels that are added to the nucleic acid to enhance Raman spectroscopy detection or modifications to allow epigenetic analysis.

[0040]

[0037] In an embodiment the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).

[0041]

[0038] A “nucleoside” is a molecule comprising a purine or pyrimidine base or any chemical modification or structural analog thereof, covalently attached to a pentose sugar such as deoxyribose or ribose or derivatives or analogs of pentose sugars.

[0042]

[0039] A “nucleotide” refers to a nucleoside further comprising at least one phosphate group covalently attached to the pentose sugar. The nucleotides to be detected may be ribonucleoside monophosphates or deoxyribonucleoside monophosphates although nucleoside diphosphates or triphosphates might be used. Alternatively, nucleosides may be released from the nucleic acid and detected. In other alternatives, purines or pyrimidines may be released, for example by acid treatment, and detected by Raman spectroscopy. Various substitutions or modifications may be made in the structure of the nucleotides, so long as they are still capable of being released from the nucleic acid, for example by exonuclease activity. For example, the ribose or deoxyribose moiety may be substituted with another pentose sugar or a pentose sugar analog. The phosphate groups may be substituted by various analogs. The purine or pyrimidine bases may be substituted or covalently modified. In embodiments involving labeled nucleotides, the label may be attached to any portion of the nucleotide so long as it does not interfere with the methods and systems disclosed herein.

[0043]

[0040] A “Raman label” may be any organic or inorganic molecule, atom, complex or structure capable of producing a detectable Raman signal, including but not limited to synthetic molecules, dyes, naturally occurring pigments such as phycoerythrin, organic nanostructures such as Ceo, buckyballs and carbon nanotubes, metal nanostructures such as gold or silver nanoparticles or nanoprisms and nano-scale semiconductors such as quantum dots. Numerous examples of Raman labels are disclosed below. The skilled artisan will realize that such examples are not limiting, and that “Raman label” encompasses any organic or inorganic atom, molecule, compound or structure known in the art that can be detected by Raman spectroscopy. Examples of Raman labels that can be used with the methods and systems of the disclosure include, e.g., Hu F et al, .Nat Methods. 2018 Mar;15(3):194-200.

[0044]

[0041] “Solid substrate” shall mean any suitable medium present in the solid phase to which a nucleic acid and / or an agent used for determining the sequence of a nucleic acid may be affixed for use in the methods and systems of the disclosure. Non-limiting examples include chips, discs, beads, nanopore structures and columns.

[0042] A “template” as used herein refers to any molecule used for determining the base sequence of a nucleic acid. This includes but is not limited to, nucleic acids themselves, modified or unmodified, native or amplified. The templates for use herein may be modified to enhance Raman spectroscopy detection, or the template molecule itself may have Raman labels incorporated therein.

[0045] Spectroscopy Model

[0046]

[0043] Raman spectroscopy has become an important tool for researchers to understand biological processes at the cellular level in medicine and biotechnology. Allakhverdiev ES et ah, Cells. 2022 Jan 24; 11(3) :386. Raman spectroscopy is a form of spectroscopy that can be used to determine vibrational attributes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy relies upon inelastic scattering of photons, known as Raman scattering. A source of monochromatic light, usually from a laser, in the visible, near infrared or near ultraviolet range is used, although X-rays can also be used. The laser light interacts with molecular vibrations or other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrational modes in the system.

[0047]

[0044] There are two primary modes of Raman spectroscopy, each with their own pros and cons. Although either spontaneous Raman spectroscopy or stimulated Raman spectroscopy can be used with the methods and systems of the disclosure, stimulated Raman spectroscopy is a preferred spectroscopy mode for most (although not all) uses for determining the sequence of individual nucleic acids on a solid substrate.

[0048]

[0045] Spontaneous Raman spectroscopy can be used in conjunction with emission spectrum analysis via confocal imaging to improve signal detection. Resonance Raman spectroscopy, which is performed using a single laser light source to excite the sample, and preferably this is a tunable laser that can be used to generate many possible excitation wavelengths to match different samples. (Efremov et al., (2008). Analytica Chimica Acta. 606 (2): 119-134.) By using multiple lasers, pulsed lasers, and / or certain sample preparation techniques, a range of more sophisticated variants of resonance Raman spectroscopy can also be utilized.

[0049]

[0046] Stimulated Raman spectroscopy has an added advantage of using two lasers (a static excitation laser and a tunable pump laser) which increases the efficiency of Raman scattering by 2-3 orders of magnitude. The pump laser also allows a single emission wavelength to be used at a time, eliminating the need for a confocal imaging setup for emission spectrum analysis and enabling rapid widefield microscopy. For example, four wavelengths can be used with the pump sensor, each corresponding to a different base of the nucleic acid template (e.g., one each for G-A-T-C for DNA, or G-A-U-C for RNA).

[0050]

[0047] Other variations of Raman spectroscopy can be used with the methods and systems of the disclosure. These include, but are not limited to, surface-enhanced Raman, tip-enhanced Raman, polarized Raman, transmission Raman, spatially-offset Raman, and hyper Raman.

[0051] Sequencing Approaches

[0052]

[0048] A number of different biochemical sequencing approaches are compatible with the enhanced nanophotonic metasurfaces and optical detection strategies as set forth herein.

[0053]

[0049] Figures 1-3 illustrate a first sequencing strategy in which a DNA polymerase is tethered to a nanophotonic metasurface for sequencing-by-synthesis approaches of nucleic acid (e.g., DNA) sequencing. In this approach, a ternary complex of a polymerase plus DNA template is immobilized to the surface of a chip in the amplification region of the resonator features. Native nucleotides or Raman labeled nucleotides are added to the reaction. As the polymerase incorporates the native or Raman labeled nucleotides, the Raman signal is amplified and captured either through optics or an integrated sensor.

[0054]

[0050] In one specific aspect, shown in Figure 2, a ternary complex of a polymerase plus DNA template is immobilized to the surface of a chip in the amplification region of the resonator features. Native nucleotides or Raman labeled nucleotides with a 3’ blocker are added to the solution. A complementary base is incorporated generating a signal but extension cannot happen because of the 3’ blocker. Free nucleotides are washed away, the chemical blocker is removed, and the process is repeated. After incorporation, the signal is captured either through optics or an integrated sensor.

[0055]

[0051] In an aspect shown in Figure 3, a ternary complex of a polymerase plus DNA template is immobilized to the surface of a chip in the amplification region of the resonator features. Native nucleotides or Raman labeled nucleotides are added along with nucleotides that contain a 3’ blocker or other blocker that prevents incorporation into the growing strand and are added to solution in a mixed ratio. A complementary base enters the polymerase pocket to generate a signal but if it contains the blocker it cannot fully be incorporated and eventually diffuses into solution. This process continues until a non-blocked nucleotide is incorporated. The signal is captured either through optics or an integrated sensor.

[0056]

[0052] The timing of the sequencing reaction may be controlled via the ratio of nucleotides having 5’ blockers versus unblocked nucleotides. This may be useful in certain aspects in which the timing of the biochemical reaction may be tuned to the detection techniques, e.g., to more efficiently provide for real time detection of the signal.

[0057]

[0053] Figure 4 illustrates a variation on the methods shown in Figures 1-3, in which a reverse transcriptase is tethered to the nanophotonic metasurface for determining the sequence of an RNA molecule. This method mirrors the method shown in Figures 1-3 but instead of immobilizing a polymerase to a solid substrate a reverse transcriptase is used to perform native RNA sequencing.

[0058]

[0054] Figure 5 illustrates an aspect in which an exonuclease is immobilized on a solid substrate having a nanophotonic metasurface, and the sequence of nucleic acids are inferred via change in signal as nucleotides are removed from their terminal end. In this approach there is no need for the use of nucleotide reagents because there is no nucleic acid synthesis. As part of preparing the nucleic acids for sequencing, they can be modified to include the Raman-enhanced nucleotide analogues or other Raman labels.

[0059]

[0055] In each of the above-described aspects, the solid substrate used optionally has an integrated sensor. As the enzyme incorporates or removes the native or Raman labeled nucleotides of the template during the sequencing operation, the Raman signal can be amplified and captured using the integrated sensor. The use of the integrated sensor can be in place of or in addition to detection of Raman signal using optical lenses or other strategies.

[0060]

[0056] Nanochannel approaches to sequencing using Raman spectroscopy and nanophotonic metasurfaces are shown in Figures 6-8. In these approaches, single stranded nucleic acid template molecules are pulled through a nanochannel, passing through a resonator region that is actively monitored in real-time. The base sequence is inferred by the change of the Raman signal produced by the k-mer of the template that is in the amplification region. After processing one nucleic acid template, the nanochannel is free to process another. In this approach there is no polymerase, transcriptase or nucleotides required. The basic approach is shown in Figure 6.

[0057] In certain aspects using the nanochannel detection, accuracy can be improved by enabling multiple passes over the same region of the nucleic acid using alternating forces to “floss” the template back and forth in the resonator region. The results obtained from the multiple passes and signal capture of the same region can be combined to improve accuracy of the sequencing operation. See Figure 7.

[0061]

[0058] In other aspects, each nanochannel can contain multiple resonator regions so that each region of the template is measured multiple times, after which the signals can be combined to improve accuracy. See Figure 8. This aspect can be used alone or combined with the prior “flossing” aspect shown in Figure 7 to further improve accuracy.

[0062]

[0059] Nanopore approaches to sequencing using Raman spectroscopy and nanophotonic metasurfaces are shown in Figures 9-12. In these aspects, single-stranded nucleic acids are pulled through a solid state nanopore where the opening of the pore contains a resonator region that is actively monitored in real-time. The base sequence of the template is inferred by the change of the Raman signal produced by the k-mer of the template that is in the amplification region. After processing one DNA template, the nanopore is free to process another. As with the nanochannel approaches, no polymerase, transcriptase or nucleotides are required. The basic concept is illustrated in Figure 9.

[0063]

[0060] In certain aspects, the nucleic acid template is modified prior to the use of the nanopore detection. In a first such aspect, Raman labels that signify individual nucleotides are incorporated into the templates as part of template preparation, e.g., using PCR amplification to incorporate nucleotides that have Raman labels that each correspond to an individual base. See Figure 10.

[0064]

[0061] In another aspect, a secondary strand is created from the template (e.g., using ligation) and the Raman label corresponds to a combination of nucleotides rather than individual nucleotides. This allows for more space between the labels in the nucleic acid template, potentially increasing the accuracy of the spectroscopy read out. See Figure 11. The number of labels needed is greater in this approach than for the aspect shown in Figure 10; for example, if each label corresponds to three bases then 64 unique labels would be required.

[0065]

[0062] Figures 12-14 illustrate specific aspects of the disclosure that utilize template degradation, In these approaches the nucleic acid template is immobilized in the sensing region of the resonator and a global fingerprint is captured via Raman spectroscopy. The nucleic acid template is immobilized on a nanophotonic metasurface, and exonucleases are introduced to the substrate which will remove the terminal nucleotide from the template fragment, resulting in a modified Raman signal fingerprint. The removed template sequence is inferred by the signal change. This process is repeated until the whole template has been sequenced. The basic concept is illustrated in Figure 12.

[0066]

[0063] In certain aspects, the signal may be amplified on the solid substrate to increase the signal produced by the exonuclease. See Figure 13. As increased template length increases the probability that the template will form secondary structures and / or fall outside of the signal amplification region, in certain aspects it is beneficial to provide a cathode source with a positive charge that is above the amplification region, which acts to “stretch” and hold straight the nucleic acid template. See Figure 14.

[0067] Sequencing and Detection Reagents

[0068]

[0064] Various reagents can also be optimized for the sequencing operation used, and in particular certain sequencing reagents can be optimized for the use and detection of Raman spectroscopy for determining the base sequence of a nucleic acid depending on the format.

[0069]

[0065] For example, any suitable polymerase enzyme can be used in the systems and methods of the invention that utilize sequencing-by-synthesis techniques. Suitable polymerases include DNA- dependent DNA polymerases, DNA-dependent RNA polymerases, reverse transcriptases, and RNA dependent RNA polymerases. These enzymes can be immobilized to a solid substrate using various surface binding technologies well- known in the art, as described in more detail below.

[0070] DNA Polymerases

[0071]

[0066] Wild-type or modified DNA polymerases can be used with the present methods and systems of the disclosure, and include polymerases from any families of polymerases as shown below in Tabe 1:

[0072] Chen CY. Front Microbiol. 2014 Jun 24;5:305.

[0073]

[0067] See also “Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol. 71 : 133-163; Alba (2001); “Protein Family Review: Replicative DNA Polymerases” Genome Biology 2(l):reviews 3002.1-3002.4; and Steitz (1999) J Biol Chem 274:17395- 17398.

[0074]

[0068] In addition to wild-type polymerases, chimeric polymerases made from a mosaic of different sources can be used. Chimeras can be produced, e.g., using consideration of similarity regions between the polymerases to define consensus sequences that are used in the chimera, or using gene shuffling technologies (e.g., via “family gene shuffling”; see Crameri et al. (1998) Nature 391 :288-291; Clackson et al. (1991) Nature 352:624-628). Available DNA polymerase enzymes have also been modified in any of a variety of ways, e.g., to reduce or eliminate exonuclease activities or to confer improvements in specificity, processivity, and improved retention time of labeled nucleotides in polymerase-DNA-nucleotide complexes (e.g., WO 2007 / 076057 and WO 2008 / 051530). Examples of modified polymerases that may be used in the methods and systems of the invention include those disclosed in US9650672; US9719073; US9951321; US10597643; US10731141; US11198906; and USRE47476E1.

[0075] RNA Polymerases

[0076]

[0069] In some embodiments, the polymerase enzyme that is used for sequencing is an RNA polymerase. Any suitable RNA polymerase can be used including RNA polymerases from bacteria, eukaryotes, viruses, or archea. Suitable RNA polymerases include RNA Pol I, RNA Pol II, RNA Pol III, RNA Pol IV, RNA Pol V, T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. The use of RNA polymerases allows for the direct sequencing of any form of RNA, including but not limited to messenger RNA, transfer RNA, non-coding RNA, ribosomal RNA, micro RNA, pre-mRNA, synthetic RNA, bacterial RNA, archaeal RNA, circular RNA, viral RNA or catalytic RNA. Where RNA polymerases are used, the polymerizing reagents will generally include NTPs or their analogs rather than the dNTPs used for DNA synthesis.

[0077] Reverse Transcriptases

[0078]

[0070] The polymerase enzyme used in the methods and systems of the invention includes RNA dependent DNA polymerases or reverse transcriptases. Suitable reverse transcriptase enzymes include, but are not limited to, HIV-1, M-MLV, AMV, and Telomere Reverse Transcriptase. See, e.g., Tang S. et al., bioRxiv, 2024 May 8:2024.05.08.593200. Reverse transcriptases also allow for the direct sequencing of any RNA substrates including but not limited to messenger RNA, transfer RNA, non-coding RNA, ribosomal RNA, micro RNA pre-mRNA, synthetic RNA, bacterial RNA, archaeal RNA, circular RNA, viral RNA or catalytic RNA.

[0079] Immobilization of Enzymes to the Solid Substrates

[0080]

[0071] Various methods can be used with the methods and systems of the disclosure to immobilize the enzymes to a solid substrate. Preferably, the immobilization is carried out in a manner that ensures appropriate enzyme orientation to minimize negative effects of conformational changes that may occur during the immobilization process. (Hernandez K. et al., Enzyme Microb. Technol. 2011, 48 (2), 107-122). One of the key factors for successful immobilization is enzyme orientation, as undirected immobilization can result in the binding of enzymes in unfavorable orientations or excessively rigid conformations (Bolivar J. M. et al., Catal. Today 2016, 259, 66-80; Zhang Y. et al., ACS Catal. 2015, 5 (8), 4503-4513.

[0081]

[0072] For example, the surface binding can be accomplished using coupling groups attached to the surface, and having molecules to which the coupling groups bind on the polymerase. Accordingly, in some aspects, traditional fusion partners can be used for immobilization of the enzymes onto the substrate, e.g., biotin with avidin or streptavidin. For example, the polymerase will have a biotin binding protein such as avidin or streptavidin, and the surface will have biotin coupling groups attached to it. The strong chemical interaction between the biotin and the binding protein will result in the immobilization of the polymerase.

[0082]

[0073] In other aspects, oriented immobilization can be achieved using “peptide tags”, which typically consist of a small peptide fused at either the N- or C-terminus of the target protein. See, e.g., Costa S. et al., Front. Microbiol. 2014, 5, 63; Young C. L. et al., Biotechnol. J. 2012, 7 (5), 620-634; Paraskevopoulou V. et al., Microorganisms 2018, 6 (2), 47. Various small peptides have been reported in the literature for this purpose, including FLAG tag, c-myc, S-tag, strep ILtag, poly-arginine (poly-Arg), and poly-histidine tag (poly- His).

[0083] Nucleotides for Use with Enzymatic Reactions

[0084]

[0074] Unlabeled nucleotides are often preferable to minimize the costs, workflow challenges, and development complexity of the sequencing approach. However, labeled nucleotides provide some significant advantages. Nucleotides can be labeled with chemical adducts that can help increase the Raman scattering efficiency and provide exogenous signals that are clearly differentiable between the 4 bases.

[0085]

[0075] For example, existing fluorescent detection reagents can be used in the systems and methods of the disclosure. Select fluorescent dyes contain elaborate chemical structures that can be distinguished by their Raman spectra independently of fluorescence. Leveraging existing dyes and / or dye-conjugated nucleotide analogues (Colombo, J. et al, Antkowiak, A., Kogan, K. et al. Nat Commun 12, 548 (2021) for use in Raman spectroscopy would allow the repurposing of existing detection technologies for use in the methods and systems of the disclosure.

[0086]

[0076] Other chemical detection reagents have been developed that are specifically designed to be highly Raman-active and / or differentiable within a narrow Raman shift range (e.g., Hu F et al., Nat Methods, 2018 Mar; 15(3): 194-200). These will enhance the signal-to- noise of Raman scattering and make the 4 nucleobases more straightforward to differentiate. In particular, these detection reagents can be optimized for compatibility with specific optical detection solutions e.g., uses compatible with a metalens objective) to utilize Raman labels within a defined wavelength range for transmission of signal. Examples of such Raman detection reagents are disclosed in, e.g., US11408894; US11225687; US11473135;

[0087] WO2015148402A1, US 20210404958; W02014205074; US20180372632; and US20230341376.

[0077] In specific aspects, one or more nucleotides can be tethered to one or more reagents, e.g., a quantum dot or a metallic bead, in order to achieve a boost in signal efficiency and / or improve nucleotide incorporation rates.

[0088]

[0078] The specific reagents and lasers used for detection of the Raman labels can be selected based on the biochemistry and optical solutions utilized in the sequence determination of the individual nucleic acid molecules on the nanophotonic metasurface, as will be apparent to one of ordinary skill in the art upon reading the present disclosure. Examples of Raman labels for use with the methods and system of the present disclosure include those described in, e.g., Wang Y et al., Chem Rev. 2013 ;113:1391-428; Wang Z et al., Chem Rev. 2017;117:7910-63; Laing S et al., Riat Rev Chem. 2017; 1 :0060; Smith BR et al., Jiang C et al., Anal Bioanal Chem. 2019;411 :3993-4006.

[0089] Solid Substrates and Optical Detection

[0090]

[0079] Various optics and solid substrate solutions can be used with the methods and systems of the disclosure. For example, objective lenses for emission capture that can be used with the methods and systems of the present disclosure include traditional objectives, mesolenses, metalenses, or microlens array lenses.

[0091]

[0080] For example, the optics solution uses a metalens, which is a nanophotonic material that uses a metasurface etched on flat glass to manipulate transmitted light in ways that recapitulate what traditional lenses can do with a much thinner footprint. Metalenses can be fabricated to increase the FOV beyond what traditional lenses are capable of achieving, and in specific aspects the methods and systems of the disclosure can use metalenses that are arranged in a 2D array format to expand the FOV. When using a metalens, it may be preferable to use nucleotide tags that are distinguishable within a narrow range of Raman shift wavenumbers, as metalenses only allow a narrow band of wavelengths to pass through (-20-50 nm width, depending on the central wavelength). The metalens can be used in conjunction with components that optimize its use with detectors depending upon the source of monochromatic light and wavelengths used. In some aspects, the optics solution can use a “microlens” array. This can be built using traditional lenses or metalenses as the unit lens element, and can be used in conjunction with components that optimize the compatibility of the microlens with the detector depending upon the source of monochromatic light used. The array can be built to a wide range of sizes and projects many individual images onto a camera sensor. Skip-seq imaging

[0092]

[0081] In some preferred aspects, the disclosure provides methods and systems using informatics to expand the field of view (FOV) of the metasurface substrates for detection of the sequencing reaction data. In this approach, one section of a substrate (e.g., chip, disc or bead) is imaged for a block of time before the imaging area shifts to another region. See, e.g., US10830703B1; US11591651B2; US11747323B2; and WO2023192403 A3. This process can happen multiple times to cover a large area before circling back to the first region. Since each molecule queried is sequenced multiple times, individual “snapshots” of data are collected throughout the run to create a final consensus sequence of each molecule at the end of the sequencing operation. This allows the expansion of the FOV and thus increases the total number of molecules that can be sequenced in a single run. Figure 15 shows an example application of this “Skip-seq” imaging strategy to a rectangular (e.g., 1 mm X 2 mm) nanophotonic metasurface chip.

[0093]

[0082] This same approach can be applied to a system where the chip is a circular shape where the optics continuously capture data from a region as the disc spins. See Figure 16. Like before, once the run is complete, the data can be integrated into a consensus sequence for each position on the chip.

[0094] Detection of Epigenetic Modifications

[0095]

[0083] The present disclosure also provides methods and systems to determine epigenetic modification of individual nucleic acid molecules by detecting DNA epigenetic markers that play a key role in DNA methylation and demethylation. For example, four modified cytosines have been discovered in mammalian genomes that are regarded as DNA epigenetic markers: 5 -methylcytosine (5mC), 5 -hydroxy methylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxycytosine (5caC). The methods and systems of the present disclosure can provide simultaneous determination of all four modified cytosines using Raman spectroscopy-based methods for directly sensing the four DNA modifications using spectral features resulting from DNA base modifications, as 5mC, 5hmC, 5fC, and 5caC exhibit distinct Raman spectroscopic signatures at 785, 660, 1450, and 1680 cm-1, respectively. Luo X et al., Anal. Chem. 2019, 91, 11, 7304-7312.

[0096]

[0084] Alternatively, the methods and systems of the invention can utilize Raman spectroscopy detection for determination of the sequence of individual nucleic acids in conjunction with techniques for epigenetic determination that are complementary with long- read sequencing techniques. For example, nanopore sequencing utilizes the ionic current changes that occur when different nucleotides transit the nanopore channel to discriminate various DNA and RNA modifications. Specifically, nanopore-based methods have demonstrated the ability to detect various nucleic acid modifications, such as 5mC, 5hmC in DNA and m6A, m5C in RNA (see, e.g., Simpson JT et al., Nat Methods. 2017;14:407-10; Liu H et al., Nat Commun. 2019;10:4079; Schadt EE et al., Genome Res. 2013;23:129-41; Begik 0 et al., Nat Biotechnol. 2021 ;39: 1278-91. Polymerase kinetic-based sequencing enables the identification of RNA modification (m6A) through the analysis of fluorescent signal alterations during reverse transcription. Vilfan ID et al. J Nanobiotechnol. 2013 ; 11:8.

[0097] EXAMPLES

[0098]

[0085] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0099] Example 1: Construction of Nanophotonic Metasurface Substrates for Use with Skip- Seq Imaging and Informatics

[0100]

[0086] A series of nanophotonic metasurface chips are constructed containing resonator elements with the following general specifications:

[0101] • 500 nm x 500 nm pitch between resonator sensors in a 2D array substrate format

[0102] • 1 cm2 total chip area

[0103] • Approximately 500M sensors per chip

[0104]

[0087] A chip with these specifications is capable of sensing 500M individual molecular reactions, a very significant advancement from conventional long-read sequencing technologies.

[0105]

[0088] Ordinary objective lenses used in many short-read sequencing platforms are not sufficient for capture of data in the entire area of the substrate, as traditional objectives capable of imaging at the required resolution are generally constrained to ~1 mm2FOV, which only could cover about 1% of the chip. Moreover, it is impractical to use a blocking agent for long-read sequencing applications using cyclic sequencing approach because the individual reads are lOx-lOOOx longer and sequencing times would become intractable.

[0089] To achieve real-time sequencing data on the metasurface chips, informatics in conjunction with the Raman spectroscopy detection is used to interrogate one section of the substrate for a defined period of time, and this takes place multiple times for each individual section (see, e.g., Figure 15). Since each molecule on the substrate is sequenced multiple times, snapshots of data are collected throughout the operation to create a final consensus sequence of each molecule on the substrate. This allows expansion of the FOV and increases the total number of molecules that are sequenced in a single sequencing operation.

[0106]

[0090] This same approach is applied to a system where the metasurface substrate is a circular disc, with optics continuously capturing data from a region as the disc spins. Figure 16 shows an example application of Skip-seq to a circular nanophotonic metasurface chip. Similar to the approach with the rectangular ship, this results in each section of the substrate being interrogated multiple times at various times during the sequencing operation, allowing the data to be integrated into a consensus sequence for each position on the disc.

[0107] Example 2: Use of Skip-Seq Imaging with Nucleic Acid Templates of Different Length

[0108]

[0091] To demonstrate the feasibility of using Skip-seq for real-time sequencing, a simulation was performed using templates of fixed size, e.g. lOkb. The detection can be iterated over the template at a fixed speed e.g., 1-3 bases / second). If a particular point in time during the run is within a "Capture Window" for identifying the Raman signal, this will increase the coverage of those positions. If it occurs during a "Dark Window", the detection coverage over this region will not be increased as it represents a time when the camera would be imaging another area of the chip. At the “end” of the simulated run, the fraction of the bases that have sufficient coverage to deliver a high-quality consensus and which have zero coverage are determined. Zero coverage bases would directly impact error rate as they would appear as deletions to the user. Low coverage bases are those that don't meet the sufficient coverage cutoff; a base can still be called but the quality of the consensus may be impacted.

[0109]

[0092] The following parameters were used for the simulation:

[0110] • Run times (hours): 12, 24, 36, 48

[0111] • Template sizes (kb): 2, 5, 10, 20

[0112] • Capture window times (hours): 2, 4, 8, 16

[0113] • Dark window times (hours): 2, 4, 8, 16

[0114] • High coverage: 10 - if a position has 10X or more coverage a high-quality consensus base can be called

[0115] • Iterations: 10 - for each combination of parameters do 10 simulations and average across the runs both the fraction of bases that have sufficient coverage and zero coverage.

[0116]

[0093] Figures 17-20 show the results when a Skip-seq imaging simulation is applied to various DNA template sizes. Figure 17 is the results summary for a template of 2kb, Figure 18 is the results summary for a template of 5 kb, Figure 19 is the results summary for a template of 10 kb and Figure 20 is the results summary for a template of 20 kb.

[0117]

[0094] Within a given template size, the rows presented in Figures 18-21 are the capture times in the Capture Period and columns are dark times in the Dark Period. Each heatmap shows the fraction of high coverage bases (> lOx), low coverage bases (lx - 9x), and no coverage. For example, if you have a 2kb template the “2 Cap I 8 Dark I 5X Thpt” plot means that each section is imaged for 2 hours followed by 8 hours of dark time, implying you have 5 regions you are rotating between. This means one can increase the FOV effectively by 5X. With just one cycle, with one capture piece on each of the subsections, the run would take 10 hours and only 8.5% of the bases would be expected to have high coverage.

[0118] However, by performing 3 cycles and 30 hour runs, 100% of the bases would be expected to be high coverage.

[0119]

[0095] In addition, certain applications may benefit from mixed Capture and Dark Periods that deliver better performance, e.g. a first imaging round is 4 hour capture and 4 hour dark, a second imagine round 2 hour capture and 2 hour dark, etc. Depending on the applications and performance requirements, this method could deliver the equivalent of a 2X - 9X increase in active sequencing area.

[0120] Equivalents

[0121]

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0122]

[0097] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0123]

[0098] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0124]

[0099] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0125]

[0100] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0126]

[0101] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0127]

[0102] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0128]

[0103] Other aspects are set forth within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a base sequence of a nucleic acid template by Raman spectroscopy, comprising: providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized polymerase; exposing a single-stranded nucleic acid template to be sequenced to the solid substrate under conditions to allow the polymerase to create a complementary strand from the template; and detecting the base sequences of the nucleotides incorporated into the strand complementary to the template using Raman spectroscopy using a wide field of view optics solution; thereby determining the base sequence of the nucleic acid template.

2. A method of identifying a base sequence of an RNA template by Raman spectroscopy, comprising: providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized reverse transcriptase; exposing an RNA template to be sequenced to the solid substrate under conditions to allow the reverse transcriptase to create a complementary strand from the template; and detecting the base sequences of the nucleotides incorporated into the strand complementary to the template using Raman spectroscopy using a wide field of view optics solution; thereby determining the base sequence of the RNA.

3. A method of identifying a base sequence of a nucleic acid template by Raman spectroscopy, comprising: providing a solid substrate comprising a nanophotonic metasurface, Raman resonators and an immobilized exonuclease; exposing a single-stranded nucleic acid template to be sequenced to the solid substrate under conditions to allow the exonuclease to remove single nucleotides from an unattached end of the nucleic acid template; anddetecting the base sequences of the nucleotides removed from the template using Raman spectroscopy using a wide field of view optics solution; thereby determining the base sequence of the nucleic acid template.

4. A method of sequencing a single stranded nucleic acid template, comprising: passing the template through a nanochannel comprising a Raman resonator region, wherein the resonator region in actively monitored in real time; and inferring the base sequence of the template based on the change of the Raman signal produced.

5. A method of sequencing a single stranded nucleic acid template, comprising: passing the template through a nanochannel comprising a Raman resonator region, wherein the resonator region in actively monitored in real time; identifying the nucleotides of a first section of the template present in the nanochannel at a first period in time by a Raman signal, and identifying the nucleotides of a second section of the DNA strand present in the nanochannel at a second period in time by a second Raman signal; and inferring the base sequence of the template based on the Raman signals produced by the first and second sections of the template.

6. A method of sequencing a single stranded nucleic acid template, comprising: modifying the template to incorporate Raman labels; passing the modified template through a solid state nanopore, wherein the opening of the pore contains a resonator region that is actively monitored in real-time; and inferring the base sequence of the template based on the Raman signals produced by the template.

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