Methods for cell addressable nucleic acid sequencing

Abstract

A method and system for analyzing nucleic acids in a biological sample in a manner that preserves the spatial and / or cellular origin of the nucleic acids in the biological sample is provided. Compositions and kits that enable the methods and systems of the present disclosure are also provided.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 904,623, filed September 23, 2019, which is incorporated herein by reference in its entirety. Background Technology

[0003] Emerging diagnostic approaches for cancer, infectious diseases, ecological imbalances, and other diseases and conditions rely on next-generation sequencing (NGS) methods to provide high-resolution genetic and genomic data, enabling robust and personalized diagnosis, treatment planning, and ultimately, curing previously intractable diseases. While NGS methods are powerful, they are limited by the methods available for providing nucleic acid samples to the instruments that perform the actual sequencing. For example, identifying the precise nature of mutations present in a specific tumor requires multiple steps, including isolating tumor tissue, isolating nucleic acids, and preparing samples for a particular sequencing method before using the instrument to obtain the actual sequence data. Furthermore, the deconvolution and processing of sequence data in a manner that allows specific sequences to be associated with specific cells or tissues is complicated by the nature of NGS technology, often requiring sample aggregation during which spatial and cellular identity information is lost.

[0004] Various approaches have been proposed to address the loss of cell addressability in NGS methods, aiming to provide molecular diagnostics with higher spatial or tissue resolution. For example, some methods rely on cell isolation, followed by applying unique barcoding to the nucleic acids from each individual cell, and then performing batch sequencing. After the sequencing run is complete, the unique barcoding is used to identify the sequence associated with each individual cell. This can be achieved, for example, by exposing individual cells to a mixture of lysis and hybridization in an isolated environment (e.g., beads or emulsions). These methods may also require enrichment or treatment of target cell subpopulations, such as through cell sorting of circulating cells or through tissue harvesting followed by dissociation and protease treatment of solid tumor cells.

[0005] While these methods can provide cell-addressable information, they face significant limitations, such as difficulties in handling solid tissues and throughput rates limited by the ability to isolate, label, and prepare nucleic acids for sequencing. Similarly, there are limitations associated with the need to transfer prepared libraries to separate instruments, systems, or locations to perform sequencing steps. This imposes a practical limitation on sequencing throughput of approximately 50,000 cells per sequencing run, which severely restricts the sensitivity and practicality of these assays given the large number of cells present in diagnostically relevant samples such as tissues, secretions, excretions, exudates, or microbiome samples. A degree of addressability can be achieved simply by physically separating samples and performing isolation, library preparation, and sequencing reactions in a known order. However, this process is labor-intensive and time-consuming, making it impractical as a means of screening large numbers of patients or as a means of deploying systematic screening methods.

[0006] Therefore, there is a need for compositions and methods that can improve the accuracy and throughput of cell-addressable sequencing methods and cell or spatially addressable sequencing methods that overcome the aforementioned limitations of the prior art. Summary of the Invention

[0007] The aspects disclosed herein provide methods for analyzing biological samples, the methods comprising: (a) detecting a multivalent binding complex formed between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate in the presence of the biological sample or a derivative thereof; and (b) determining the origin of the target nucleic acid sequence in the biological sample or the derivative thereof. In some embodiments, the determination in (b) is performed at least in part by analyzing the relative three-dimensional relationship between the target nucleic acid sequence and a reference point of the biological sample or the derivative thereof. In some embodiments, the method further comprises contacting the biological sample or the derivative thereof with the detectable polymer-nucleotide conjugate in the presence of the biological sample. In some embodiments, the method further comprises coupling at least a portion of the target nucleic acid sequence to a trapping oligonucleotide molecule coupled to a surface of a substrate. In some embodiments, the surface has a water contact angle of less than or equal to 45 degrees. In some embodiments, coupling includes hybridization in the presence of a hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4-9; and (ii) a second polar aprotic solvent having a dielectric constant less than or equal to 115. In some embodiments, the method further includes immobilizing the biological sample or a derivative thereof on the surface in a manner sufficient to fix the relative three-dimensional relationship. In some embodiments, the method further includes optionally amplifying the target nucleic acid sequence on the surface of the substrate using rolling circle amplification. In some embodiments, in the presence of the biological sample or a derivative thereof, the image of the surface exhibits a contrast-to-noise ratio greater than or equal to about 5, which is measured by: (a) contacting the surface with fluorescently labeled nucleotide molecules containing a nucleic acid sequence complementary to at least a portion of a capture oligonucleotide immobilized on the surface; and (b) after (a), imaging the surface using an inverted microscope and camera under non-signal saturation conditions while immersing the surface in the buffer. In some embodiments, the method further includes a nucleotide binding reaction between the nucleotide moiety coupled to the polymer-nucleotide conjugate and the target nucleic acid molecule or a derivative thereof. In some embodiments, the target nucleic acid molecule or its derivative is a deoxyribonucleic acid (DNA) molecule. In some embodiments, the biological sample or its derivative comprises a fluid biological sample. In some embodiments, the source is cancerous tissue.

[0008] The aspects disclosed herein provide a method for in situ identification of at least a portion of a subcellular component within a cell or tissue, the method comprising: (a) detecting a signal from a multivalent binding complex between the subcellular component or a derivative thereof and a detectable polymer-nucleotide conjugate; and (b) processing at least the signal detected in (a) to identify at least the portion of the subcellular component or a derivative thereof. In some embodiments, the subcellular component or a derivative thereof is a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the method further comprises: (c) immobilizing the cell or tissue on a surface of a substrate. In some embodiments, the method further comprises: (d) coupling at least a portion of the subcellular component to a capture molecule coupled to the surface. In some embodiments, the method further comprises: (e) permeabilizing the tissue or lysing the cell prior to detection in (a). In some embodiments, the surface has a water contact angle of less than or equal to 45 degrees. In some embodiments, the coupling in (d) includes hybridizing the capture molecule with at least the said portion of the subcellular component in the presence of a hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4-9; and (ii) a second polar aprotic solvent having a dielectric constant less than or equal to 115. In some embodiments, the image of the surface exhibits a contrast-to-noise ratio greater than or equal to about 5, which is measured by: (a) contacting the surface with fluorescently labeled nucleotide molecules containing a nucleic acid sequence complementary to at least a portion of the capture oligonucleotide immobilized on the surface; and (b) after (a), imaging the surface using an inverted microscope and camera under non-signal saturation conditions while immersing the surface in the buffer. In some embodiments, detecting the signal from the multivalent binding complex in (a) includes a nucleotide binding reaction between the nucleotide moiety coupled to the polymer-nucleotide conjugate and the subcellular component or a derivative thereof. In some embodiments, the tissue is derived from a tumor.

[0009] A system for analyzing biological samples, the system comprising: a substrate including a surface coupled with a polymer layer adapted to immobilize the biological sample to the surface, wherein: the biological sample or a derivative thereof comprises a target nucleic acid molecule or a derivative thereof; the polymer layer is configured to be coupled to (i) the biological sample or a derivative thereof, or (ii) the target nucleic acid molecule or a derivative thereof; the target nucleic acid molecule or a derivative thereof is configured to be coupled to a nucleotide moiety comprising a detectable label; and the image of the surface exhibits a contrast-to-noise ratio greater than or equal to about 5 when the image of the surface is acquired using an inverted microscope and a camera under non-signal saturation conditions while immersing the surface in a buffer solution, and wherein the detectable label is a fluorescent dye. In some embodiments, the polymer layer is hydrophilic. In some embodiments, the system further includes a fixative that immobilizes the biological sample to the surface when the biological sample comes into contact with the fixative while adjacent to the surface. In some embodiments, the fixative comprises formaldehyde or glutaraldehyde. In some embodiments, the target nucleic acid molecule is a polynucleotide. In some embodiments, the target nucleic acid molecule includes a universal sequence region, which includes a spatial barcode sequence or a sample barcode sequence configured to retain the source of the target nucleic acid molecule in the biological sample. In some embodiments, when an image of the surface is obtained, the image of the surface exhibits a contrast-to-noise ratio greater than or equal to about 10. In some embodiments, the substrate is a flow cell device including a first flow channel and an optional second flow channel. In some embodiments, the substrate is a reflective, transparent, or translucent planar substrate. In some embodiments, the flow cell device is a capillary flow cell device.

[0010] The aspects disclosed herein include a system for analyzing nucleic acid sequence information in a biological sample or a derivative thereof, the system comprising: one or more computer processors programmed to: (a) detect a signal from a multivalent binding complex formed in the presence of the biological sample or a derivative thereof between a target nucleic acid sequence of a target nucleic acid molecule or a derivative thereof and a detectable polymer-nucleotide conjugate, wherein the signal indicates the identity of a nucleotide in the target nucleic acid sequence; and (b) determine the origin of the target nucleic acid sequence in the biological sample. In some embodiments, the one or more computer processors are programmed to determine the origin of the target nucleic acid sequence in (b) by analyzing the relative three-dimensional relationship between the target nucleic acid molecule or a derivative thereof and the biological sample or a derivative thereof. In some embodiments, the system further includes a database configured to store three-dimensional data associated with the origin of the target nucleic acid sequence. In some embodiments, the database is further configured to store sequencing data including the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, (b) is performed by correlating the sequencing data and the three-dimensional data. In some embodiments, the one or more computer processors are programmed to identify the target nucleic acid sequence in less than 60 minutes by repeating (a) to (b). In some embodiments, the one or more computer processors are programmed to perform (a) to (b) with a base determination accuracy characterized by a Q score greater than 25 for at least 80% of the identified nucleotides. In some embodiments, the detectable polymer-nucleotide conjugate comprises: (a) a polymer core; and (b) two or more nucleotide moieties attached to the polymer core, wherein the polymer-nucleotide conjugate is configured to form a multivalent binding complex between the two or more nucleotide moieties and the target nucleic acid molecule or a derivative thereof. In some embodiments, the one or more nucleotide moieties comprise nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs. In some embodiments, the polymer core comprises a polymer having a star-shaped, comb-shaped, cross-linked, bottle-brush-like, or dendritic macromolecular configuration. In some embodiments, the polymer core comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the system further includes an optical imaging system comprising a diameter greater than 1.0 mm. 2 Field of view (FOV).

[0011] The aspects disclosed herein provide a kit comprising: (a) a detectable polymer-nucleotide conjugate comprising: (i) a polymer core; and (ii) two or more nucleotide moieties attached to the polymer core; and (b) for the following specification: to identify at least a portion of a subcellular component within a cell or tissue in situ by contacting the detectable polymer-nucleotide conjugate with the subcellular component under conditions sufficient to form a multivalent binding complex between the two or more nucleotide moieties and the subcellular component. In some embodiments, the kit comprises four types of the detectable polymer-nucleotide conjugate, each of which has a different nucleotide moieties attached thereto.

[0012] The aspects disclosed herein include a kit comprising: (a) a substrate including a surface having a polymer layer coupled thereto, the polymer layer being adapted to immobilize a biological sample or a derivative thereof to the surface; and (b) instructions for determining a target nucleic acid sequence in the biological sample or derivative on the surface and the source of the target nucleic acid sequence. In some embodiments, the kit further includes: (a) a hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4-9; and (ii) a second polar aprotic solvent having a dielectric constant less than or equal to 115; and (b) instructions for hybridizing at least a portion of the target nucleic acid sequence with at least a portion of a capturing oligonucleotide coupled to the surface.

[0013] By incorporating references

[0014] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent or patent application is specifically and individually incorporated herein by reference in its entirety. Attached Figure Description

[0015] The novel features of the invention are specifically set forth in the appended claims. The novel features and advantages of the invention can be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, in conjunction with the accompanying drawings, in which:

[0016] Figure 1This is a schematic diagram of one embodiment of a low-binding carrier according to embodiments of the present disclosure, the low-binding carrier comprising alternating layers of a glass substrate and a hydrophilic coating, the hydrophilic coating being covalently or non-covalently adhered to the glass, and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., trapping oligonucleotides and cyclized oligonucleotides). In alternative embodiments, the carrier may be made of any material, such as glass, plastic, or polymer materials.

[0017] Figure 2 This is a schematic diagram illustrating a vector comprising a capture oligonucleotide and a cyclic oligonucleotide immobilized thereon, according to an embodiment of the present disclosure. In some embodiments, the vector comprises a plurality of capture oligonucleotides and a plurality of cyclic oligonucleotides immobilized thereon.

[0018] Figure 3 This is a schematic diagram illustrating a vector according to an embodiment of the present disclosure and a biological sample (e.g., a tissue sample) placed on the vector (see schematic diagram on the left), the vector comprising a plurality of capture oligonucleotides and cyclic oligonucleotides immobilized thereon. Figure 3 A magnified cross-section of a vector with a series of features is shown, each feature having a circular shape and being labeled for spatial identification on the vector (see schematic diagram on the right). Each feature includes multiple immobilized capture oligonucleotides and cyclic oligonucleotides.

[0019] Figure 4 This is a schematic diagram illustrating a carrier comprising a capture oligonucleotide immobilized thereon and a soluble cyclized oligonucleotide according to an embodiment of the present disclosure. In some embodiments, the carrier comprises a plurality of capture oligonucleotides immobilized thereon.

[0020] Figure 5A This is a schematic diagram illustrating the nucleotide arm of a polymer-nucleotide conjugate according to an embodiment of the present disclosure.

[0021] Figure 5B This is a schematic diagram of a polymer-nucleotide conjugate according to an embodiment of the present disclosure, the polymer-nucleotide conjugate comprising a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer, (iii) a linker and (iv) a nucleotide unit.

[0022] Figure 5C This is a schematic diagram of a polymer-nucleotide conjugate in the form of a dendritic macromolecule according to an embodiment of the present disclosure, the polymer-nucleotide conjugate comprising a branched polymer radiating from a central attachment point or central portion, wherein a plurality of nucleotide arms radiate from the central attachment point.

[0023] Figure 5DIt is a nucleotide arm of a polymer-nucleotide conjugate according to an embodiment of the present disclosure, comprising a biotin core attachment portion, a spacer, an aliphatic linker, and a nucleotide attached to the linker via a propargyl group at a base.

[0024] Figure 6A The structures of spacers and linkers of polymer-nucleotide conjugates according to embodiments of the present disclosure are shown.

[0025] Figure 6B The structure of an additional linker for a polymer-nucleotide conjugate according to an embodiment of the present disclosure is shown.

[0026] Figure 7 The workflow of an embodiment according to this disclosure is shown.

[0027] Figures 8A-8B A non-limiting example of imaging a dual-surface carrier structure is schematically shown for presenting sample sites for imaging by the imaging system disclosed herein. Figure 8A : Illustration of the images of the inner surfaces before and after the flow cell. Figure 8B Illustration of the imaging of the front and rear outer surfaces of the substrate.

[0028] Figures 9A-9B A non-limiting example of a multi-channel fluorescence imaging module including a dichroic beam splitter is shown. The dichroic beam splitter is used to transmit an excitation beam to a sample and to receive the generated fluorescence emission and redirect the generated fluorescence emission to four detection channels via reflection. The four detection channels are configured to detect fluorescence emission at four different corresponding wavelengths or bands. Figure 9A Isometric top view. Figure 9B Isometric top view.

[0029] Figures 10A-10B It shows Figure 10A and Figure 10B The optical path within the multi-channel fluorescence imaging module includes a dichroic beam splitter, which transmits the excitation beam to the sample and receives the generated fluorescence emission, redirecting the emission to four detection channels via reflection. The four detection channels are used to detect fluorescence emission at four different corresponding wavelengths or bands. Figure 10A Top view. Figure 10B Side view.

[0030] Figures 11A-11B The modulation transfer function (MTF) of an exemplary dual-surface imaging system with a numerical aperture (NA) of 0.3 disclosed herein is shown. Figure 11A : First surface. Figure 11B : Second surface.

[0031] Figures 12A-12B The MTF of an exemplary dual-surface imaging system with an NA of 0.5 disclosed herein is shown. Figure 12A : First surface. Figure 12B : Second surface.

[0032] Figures 13A-13B The MTF of an exemplary dual-surface imaging system with an NA of 0.7 disclosed herein is shown. Figure 13A : First surface. Figure 13B : Second surface.

[0033] Figures 14A-14B A plot of the calculated Strell ratio for imaging the surface of the second flow cell through the surface of the first flow cell is provided. Figure 14A : A plot of Strelby ratio for imaging a second flow cell surface through a first flow cell surface as a function of the intermediate fluid layer thickness (fluid channel height) for different objective lenses and / or optical system numerical apertures. Figure 14B The graph shows the Strell ratio as a function of numerical aperture for imaging the surface of the second flow cell through the surface of the first flow cell and the intermediate water layer with a thickness of 0.1 mm.

[0034] Figure 15 A ray tracing diagram is provided for an objective lens design designed to image the surfaces of opposite sides of a 0.17 mm thick coverslip.

[0035] Figure 16 Provided for imaging the surfaces of opposite sides of a 0.17 mm thick coverslip, Figure 15 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0036] Figure 17 Provided for imaging the surfaces of opposite sides of a 0.3 mm thick coverslip, Figure 19 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0037] Figure 18 Provided for imaging surfaces separated from the 0.1 mm thick aqueous fluid layer by a 0.3 mm thick coverslip, Figure 15 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0038] Figure 19 Provided for imaging the surfaces of opposite sides of a 1.0 mm thick coverslip, Figure 15 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0039] Figure 20Provided for imaging surfaces separated from the surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer. Figure 15 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0040] Figure 21 Provides ray tracing diagrams for lens barrel design, if compared with... Figure 15 When used in combination with the objective lenses shown, the tube lens design provides improved double-sided imaging through a 1 mm thick coverslip.

[0041] Figure 22 Provided for imaging the surfaces of opposite sides of a 1.0 mm thick coverslip, Figure 15 The figure shows a graph of the modulation transfer function of the combination of objective lens and tube lens as a function of spatial frequency.

[0042] Figure 23 Provided for imaging surfaces separated from the surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer. Figure 15 The figure shows a graph of the modulation transfer function of the combination of objective lens and tube lens as a function of spatial frequency.

[0043] Figure 24 The illustration shows a non-limiting example of a single capillary flow cell with two fluid adapters.

[0044] Figure 25 The illustration shows a non-limiting example of a flow cell box that includes a base, a fluid adapter, and optional other components, and is designed to accommodate two capillaries.

[0045] Figure 26 The illustration shows a non-limiting example of a system comprising a single capillary flow cell connected to various fluid flow control components, wherein the single capillary is compatible with mounting on a microscope stage or in a custom imaging instrument for a variety of imaging applications.

[0046] Figure 27 The diagram illustrates a vector on which capturing oligonucleotides and cyclic oligonucleotides are immobilized according to various embodiments described herein, and an exemplary method for capturing nucleic acids from cellular biological samples located on the vector.

[0047] Figure 28 This is a schematic diagram illustrating a carrier on which a capture oligonucleotide is immobilized according to various embodiments described herein, and an exemplary method for capturing nucleic acids from a cellular biological sample located on the carrier, wherein the method includes using soluble cyclized oligonucleotides. Detailed Implementation

[0048] This document provides spatially addressable and cellularly addressable sequencing methods and systems, as well as compositions, apparatus, and kits for performing the methods and systems described herein. The methods and systems described herein can utilize polymer-nucleotide conjugates in in situ nucleotide binding reactions. Nucleotide binding reactions can be performed on hydrophilic surfaces, which provides many of the advantages described herein. Hybridization buffers containing polar and aprotic solvents in combination with pH buffers are also provided. Optical systems for spatially resolving sequencing data are also provided. In some embodiments, the optical systems described herein have a diameter greater than 1.0 mm. 2 The field of view.

[0049] like Figure 7 As shown, in some embodiments, the method described herein includes: (a) providing a surface (e.g., a low-nonspecific binding surface) having a plurality of capture oligonucleotides coupled thereto (701); immobilizing a biological sample containing a target nucleic acid molecule to the surface and optionally permeating the biological sample (702); (c) contacting the plurality of capture oligonucleotides to the target nucleic acid molecule under conditions sufficient to allow at least a portion of the plurality of capture oligonucleotides to hybridize with the target nucleic acid molecule (703); (d) amplifying the target nucleic acid molecule to produce an amplified target nucleic acid molecule or a derivative thereof (704); (e) contacting the amplified target nucleic acid molecule or a derivative thereof with one or more polymerases and one or more primer nucleic acid molecules having primer sequences complementary to one or more regions of the amplified target nucleic acid molecule or a derivative thereof to produce an initiated target nucleic acid molecule or a derivative thereof (705); (f) contacting the primers to produce an initiated target nucleic acid molecule or a derivative thereof. (706) The initiated target nucleic acid molecule or its derivative is contacted with a polymer-nucleotide conjugate comprising two or more nucleotide moieties coupled to a polymer (e.g., PEG) core labeled with a detectable marker (e.g., a fluorophore); (g) a multivalent binding complex is detected between the initiated target nucleic acid molecule or its derivative and the polymer-nucleotide conjugate; (h) the surface is washed with a buffer sufficient to remove the polymer-nucleotide conjugate from the initiated target nucleic acid molecule or its derivative; (i) a nucleotide is incorporated, the nucleotide being free of the detectable marker and optionally containing a blocking group (e.g., azidomethyl) that blocks the incorporation of a second nucleotide at the N+1 position on the initiated target nucleic acid molecule or its derivative; (j) steps (f)-(j) are optionally repeated (710).

[0050] Existing spatially addressable sequence identification methods (also referred to herein as spatial transcriptomics techniques) suffer from low sensitivity, nonspecificity, and inaccurate spatial localization of target transcripts. In contrast, the methods, systems, compositions, and kits described herein overcome these challenges by, for example, utilizing low-nonspecificity binding surfaces, highly efficient hybridization buffers, methods for preparing nanospheres with high copy numbers, and multivalent molecules.

[0051] Compared to existing methods, the low nonspecific binding and improved signal of this disclosure provide a significantly improved contrast-to-noise (CNR) ratio. The CNR is at least partially improved by utilizing highly compact reaction focal points (e.g., highly compacted nucleic acid clusters with high copy numbers), efficient surface hybridization (allowing for precise localization of nucleic acid capture), and very low background, while achieving highly efficient capture, amplification, and target nucleic acid clustering. Sequencing reactions can be performed in the presence of a biological sample (e.g., tissue, cell suspension) coupled to a substrate. Analysis of the sequencing reactions can be performed in a manner that provides cell addressability and / or spatial addressability, allowing sequence data to be correlated with its originating tissue, cell type, physiological location, or spatial location.

[0052] The high-efficiency hybridization buffers described in this article promote the high rigor (e.g., specificity), speed, and power of nucleic acid hybridization reactions, and improve the efficiency of subsequent amplification and sequencing steps. High-efficiency hybridization buffers can significantly shorten nucleic acid hybridization time and reduce sample input requirements. High-efficiency hybridization buffers can be used in isothermal nucleic acid annealing workflows, eliminating the need for a cooling step during annealing. High-efficiency hybridization buffers provide precise localization of nucleic acid capture on surfaces for the precise spatial localization of nucleic acids derived from cells or tissues (e.g., transcripts).

[0053] The rolling circle amplification (RoBA) methods described in this paper include a two-stage approach that uses a non-catalytic divalent cation followed by a catalytic divalent cation to synchronize RoBA events on the surface and generate multiplexes. Following the RoBA reaction can be relaxation conditions and flexing amplification reactions, which generate new multiplexes from existing ones. In summary, these amplification methods produce highly compacted nanospheres containing high copy numbers of the target sequence, which improves sequencing signal intensity.

[0054] The nucleic acid analysis method described herein can achieve higher throughput than existing methods, allowing analysis of 50,000, 100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000 or more cells per run, thereby significantly improving diagnostic sensitivity by allowing the detection of mutations in as few as one cell per million cells in principle. Another advantage of the nucleic acid method disclosed herein is that the required reaction can be carried out at a single temperature (e.g., isothermal conditions), such as 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 42°C, 50°C, 60°C, 65°C, 70°C, or 72°C or higher, or within any two of the above definitions.

[0055] Multivalent molecules used during sequencing reactions offer many advantages that free nucleotides lack. Multivalent molecules contain a core attached to multiple arms, each tethered to a nucleotide. Multivalent molecules increase the local concentration of nucleotides near the polymerase / template binding site. They also exhibit increased duration of action when forming a stable ternary complex with the polymerase and nucleic acid template. Therefore, labeled multivalent molecules provide shorter imaging times and increased signal intensity during sequencing reactions.

[0056] The cellular and spatial resolution of sequencing data generated using the methods and systems described herein is achieved through the imaging methods and systems described herein, which provide increased optical resolution and improved image quality for genomics applications.

[0057] This paper discloses optical components and system designs for high-performance fluorescence imaging methods and systems, which can provide any one or more of the following: a larger field of view, improved optical resolution (including high-performance optical resolution), improved contrast, improved image quality, faster transition between image captures when repositioning the sample plane to capture a series of images (e.g., images with different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

[0058] In some cases, such as for two-sided (flow cell) imaging applications (including the use of thick flow cell walls (e.g., wall (or coverslip) thickness > 700 μm) and fluid channels (e.g., fluid channel height or thickness is 50-200 μm)), improvements in imaging performance can be achieved using novel objective designs that correct for optical aberrations introduced by imaging the surfaces of the thick coverslip and / or fluid channels on the opposite side of the objective.

[0059] In some cases, such as for double-sided (flow cell) imaging applications (including the use of thick flow cell walls (e.g., wall (or coverslip) thickness > 700 μm) and fluid channels (e.g., the height or thickness of the fluid channel is 50-200 μm)), improved imaging performance can be achieved even when using commercially available off-the-shelf objectives by using novel tube lens designs (different from tube lenses in conventional microscopes that only form images on the intermediate image plane), which, in combination with the objectives, correct for optical aberrations caused by the thick flow cell walls and / or intermediate fluid layer.

[0060] In some cases, such as for multi-channel (e.g., two-color or four-color) imaging applications, improved imaging performance can be achieved by using multiple lens barrels, one lens barrel for one imaging channel, wherein each lens barrel is designed to be optimized for a specific wavelength range used in that imaging channel.

[0061] In some cases, such as for two-sided (flow cell) imaging applications, improved imaging performance can be achieved by using an electro-optic phase plate in combination with the objective lens to compensate for optical aberrations caused by the fluid layer separating the upper (near) inner surface and the lower (far) inner surface of the flow cell. In some cases, this design approach can also compensate for vibrations introduced by, for example, a motion-actuated compensator that moves into or out of the optical path depending on which surface of the flow cell is being imaged.

[0062] Further advantageous features of the disclosed imaging optics design may include the position and orientation of one or more excitation sources and one or more detection optical paths relative to the objective lens and the dichroic filter receiving the excitation beam. The excitation beam may also be linearly polarized, and the linear polarization orientation may allow s-polarized light to be incident on the dichroic reflective surface of the dichroic filter. Such features can potentially improve excitation beam filtering and / or reduce wavefront errors introduced into the emitted beam due to, for example, surface deformation of the dichroic filter.

[0063] Although this article is primarily discussed in the context of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc.), those skilled in the art will understand that many of the disclosed optical design methods and features can be applied to other imaging modalities, such as bright-field imaging, dark-field imaging, phase-contrast imaging, etc.

[0064] In addition to the optical components and imaging system designs disclosed herein, flow cell devices and systems for performing various genomic analysis methods, including cell-addressable nucleic acid sequencing, are also disclosed. These may include various combinations of the disclosed optical, mechanical, fluidic, thermal, electrical, and computational modules or subsystems. The advantages offered by the disclosed flow cell devices, cartridges, and analysis systems include, but are not limited to: (i) reduced fabrication complexity and cost of the devices and systems; (ii) significantly reduced consumable costs (e.g., compared to the consumable costs of currently available nucleic acid sequencing systems); (iii) compatibility with typical flow cell surface functionalization methods; (iv) flexible flow control when combined with microfluidic components such as syringe pumps and diaphragm valves; and (v) flexible system throughput.

[0065] In some cases, the disclosed capillary flow cell devices and capillary flow cell cartridges may be constructed from off-the-shelf, disposable, single-cavity (e.g., single fluid flow channel) or multi-cavity capillaries, and may also include a fluid adapter, a cartridge base, one or more integrated fluid flow control components, or any combination thereof. In some cases, the disclosed flow cell-based systems may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof. Some publicly disclosed capillary flow cell devices, cartridges, and systems have design features including, but not limited to, (i) a uniform flow channel configuration, (ii) sealed, reliable, and repeatable switching between reagent flows, which can be achieved through: a simple loading / unloading mechanism that reliably seals the fluid interface between the system and the capillary, thereby facilitating capillary replacement and system reuse, and enabling precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) replaceable single fluid flow channel devices or capillary flow cell cartridges comprising multiple interchangeable flow channels to provide flexible system throughput; and (iv) compatibility with a variety of detection methods, such as fluorescence imaging.

[0066] Although the disclosed capillary flow cell devices and systems, as well as microfluidic devices and systems, are described primarily in the context of their application in nucleic acid sequencing, various aspects of the disclosed devices and systems can be applied not only to nucleic acid sequencing but also to any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis application. It should be understood that the different aspects of the disclosed methods, devices, and systems can be understood individually, collectively, or in combination with each other.

[0067] The implementation described herein offers significant advantages for: diagnosing cancer (including circulating tumors and solid tumors), analyzing biopsy samples (e.g., for diagnosing genetic disorders), analyzing microbiome samples (e.g., for diagnosing disorders associated with microbial dysbiosis), diagnosing disorders accompanied by secretions or exudates, or assessing general health or disease risk (where such risk can be assessed based on the presence or identity of specific gene sequences in specific cells, tissues, or locations). For example, using high-resolution cell-addressable sequencing technology to identify the presence of low levels of circulating tumor cells can be useful for diagnosing blood cancers or early metastases.

[0068] In some embodiments, cells or single cells within a tissue may be exposed to a surface under conditions optimized for binding (capture) to the target nucleic acid, for example, by using high-density poly-T (polythymidine) or poly-dT oligonucleotides to capture RNA transcripts followed by reverse transcription, or by using random-sequence capture oligonucleotides for hybridization with genomic, circulating, or organelle DNA. In some embodiments, this capture process may be followed by one or more library preparation steps, such as attaching at least one adaptor to the captured nucleic acid, wherein the adaptor may contain an index sequence, a barcode sequence, and / or a unique molecular identifier (UMI). The adaptor attachment step may be performed by ligation (e.g., blunt-end ligation) or using "splint" oligonucleotides. These library preparation steps may result in or may also include circularization of the captured nucleic acid. In some embodiments, the circularized nucleic acid molecule may be amplified, for example, by rolling circle amplification (RCA), to produce a large, multi-copy nucleic acid molecule (e.g., a polymer) containing multiple tandem repeats of the target sequence. In some embodiments, the large multicopy nucleic acid can form a condensed state, for example by using buffer conditions that favor a tight DNA state, a surface with a high density of capture oligonucleotides, using a divalent or bispecific oligonucleotide (“clustered oligonucleotide” or “clustered oligonucleotide”) that bridges two or more sites in the large multicopy nucleic acid, or by any combination of the foregoing, or by any method known or to be known in the art to produce a tight cluster containing the large multicopy nucleic acid.

[0069] In some embodiments, surfaces for capturing nucleic acids from tissues or cells can be configured to retain highly active nucleic acids while maintaining low levels of binding to other components such as unwanted proteins, lipids, carbohydrates, or cellular debris. Therefore, the surfaces considered herein are capable of binding to nucleic acids from cells in tissues or from single cells that lyse upon contact with or adjacency to the surface. Furthermore, the surfaces do not retain cellular debris and do not exhibit significant nonspecific binding to added proteins (e.g., nucleic acid polymerases) or other molecules, portions, particles, or articles (e.g., dye molecules or fluorophores).

[0070] In some embodiments, cell lysis (and optional nucleic acid fragmentation) occurs upon contact with or proximity to a surface, such that a large amount, such as a representative amount or substantially all of the DNA, RNA, or other target nucleic acids released from the cell or tissue sample, is captured by the surface. The surface can be configured such that cells can flow across it to reach capture sites on the surface. Alternatively, the capture surface can be configured such that tissue (e.g., a tissue slice) can be placed in contact with or in fluid communication with the surface, wherein reagents can then flow through the tissue in a manner that facilitates in-situ capture of nucleic acids from the tissue, such that nucleic acids from one cell or region of the tissue are captured in the same location and orientation relative to nucleic acids from other cells or regions of the tissue, because the nucleic acids are oriented or localized within the intact tissue.

[0071] In some implementations, the capture, adaptor attachment, circularization, amplification, and clustering of the target nucleic acid can be performed simultaneously with attachment to or immediately adjacent to the surface. Alternatively, one or more of the aforementioned preparation steps can be performed in a free solution or during attachment to the beads.

[0072] Spatially resolved binding of cell-specific complementary nucleic acid sequences (e.g., cell genomes or cell transcriptomes), followed by adaptor attachment, circularization, amplification, and clustering, enables the use of sequencing technologies, such as affinity-based sequencing methods, as described in U.S. Applications 62 / 897,172 and 16 / 579,794 (incorporated herein by reference in their entirety); and as described elsewhere herein. Advances in low-binding surfaces disclosed in U.S. Patent Application 16 / 363,842, hybridization methods disclosed in U.S. Patent Application 16 / 543,351, and library preparation methods disclosed in U.S. Application 62 / 767,943 and related international application WO 2020 / 102766 have further enabled cell or tissue-addressable sequencing, the contents of which are hereby expressly incorporated herein by reference for all purposes. Thus, in some embodiments, sequence data can be obtained in a manner spatially mapped to the cells or tissues from which the genomic or transcriptome nucleic acids are derived. In some implementations, sequence data can be obtained in a substantially one-to-one correspondence with the cell locations of the sample source. In other implementations, sequence data can be obtained in a manner different from a one-to-one spatial correspondence with cell locations in the original sample, but with substantially the same location relative to other cells or sources in the tissue's genetic, genomic, or transcriptomic sample.

[0073] Solid support surfaces. This document provides solid supports that include surfaces (e.g., low nonspecific binding). In some cases, solid supports include non-hydrophilic surfaces. In other cases, solid supports include hydrophilic surfaces. Generally, the disclosed supports may include a substrate (or support structure), one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers), a polymer film, and one or more covalently or non-covalently attached primer sequences that can be used to tether single-stranded template oligonucleotides to the support surface. Figure 1 In some cases, the formulation of the surface, such as the chemical composition of one or more layers, the coupling chemistry for crosslinking the one or more layers with and / or with each other on the carrier surface, and the total number of layers, can be modified to minimize or reduce nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the carrier surface relative to a comparable monolayer. Typically, the surface formulation can be modified to minimize or reduce nonspecific hybridization on the carrier surface relative to a comparable monolayer. The formulation composition of the surface can be modified to minimize or reduce nonspecific amplification on the carrier surface relative to a comparable monolayer. The surface formulation can be modified to maximize the specific amplification rate and / or yield on the carrier surface. In some cases disclosed herein, a suitable amplification level is achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles.

[0074] Examples of materials that can be used to manufacture substrate or carrier structures include, but are not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of glass and plastic substrates are considered.

[0075] The substrate or carrier structure can be endowed with any of a variety of geometries and dimensions known to those skilled in the art, and can comprise any of a variety of materials known to those skilled in the art. For example, in some cases, the substrate or carrier structure can be partially planar (e.g., comprising a microscope slide or the surface of a microscope slide). Generally, the substrate or carrier structure can be cylindrical (e.g., comprising a capillary or the inner surface of a capillary), spherical (e.g., comprising the outer surface of a non-porous bead), or irregular (e.g., comprising the outer surface of an irregularly shaped non-porous bead or particle). In some cases, the surface of the substrate or carrier structure used for nucleic acid hybridization and amplification can be a solid, non-porous surface. In some cases, the surface of the substrate or carrier structure used for nucleic acid hybridization and amplification can be porous, such that the coating described herein penetrates the porous surface, and the nucleic acid hybridization and amplification reactions performed thereon can occur within the pores.

[0076] A substrate or carrier structure comprising one or more chemically modified layers (e.g., a low-nonspecificity-binding polymer layer) can be standalone or integrated into another structure or component. For example, in some cases, the substrate or carrier structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or carrier structure may include one or more surfaces within a microplate form, such as the bottom surface of a hole in a microplate. As described above, in some preferred embodiments, the substrate or carrier structure includes the inner surface of a capillary (e.g., an inner lumen surface). In alternative embodiments, the substrate or carrier structure includes the inner surface of a capillary etched into a planar chip (e.g., an inner lumen surface).

[0077] The chemically modified layer can be uniformly coated on the surface of a substrate or support structure. Alternatively, the surface of the substrate or support structure can be non-uniformly distributed or patterned, such that the chemically modified layer is confined to one or more discrete regions of the substrate. For example, photolithography can be used to pattern the substrate surface to generate an ordered array or random pattern of chemically modified regions on the surface. Alternatively or in combination, contact printing and / or inkjet printing techniques can be used to pattern the substrate surface. In some cases, the ordered array or random pattern of chemically modified discrete regions may contain at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions, or any intermediate number of discrete regions across the scope of this document.

[0078] To obtain surfaces with low nonspecific binding (also referred to herein as “low-binding” or “passivated” surfaces), hydrophilic polymers can be nonspecifically adsorbed or covalently grafted onto the substrate or support surface. Typically, passivation is performed using hydrophilic polymers with different molecular weights and end groups (attached to the surface using, for example, silane chemistry). End groups away from the surface can include, but are not limited to, biotin, methoxyethers, carboxylic esters, amines, NHS esters, maleimides, and bissilanes. In some cases, two or more layers of hydrophilic polymers, such as linear, branched, or multibranched polymers, can be deposited on a surface. In others, two or more layers can be covalently coupled or internally cross-linked to improve the stability of the resulting surface. In still others, oligonucleotide primers (or other biomolecules, such as enzymes or antibodies) with different base sequences and base modifications can be tethered to the resulting surface layer at various surface densities. In some cases, for example, both the surface functional group density and the oligonucleotide concentration can be varied to target a specific primer density range. Furthermore, primer density can be controlled by diluting the oligonucleotide with other molecules containing the same functional groups. For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol that reacts with an NHS ester-coated surface to reduce the final primer density. Primers with different length linkers between the hybridization region and the surface attachment functional groups can also be used to control surface density. Examples of suitable adapters include poly-T and poly-A (polyadenylate) chains (e.g., 0 to 20 bases) at the 5' end of the primer, PEG adapters (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be tethered to a surface, and the fluorescence readings can then be compared to the fluorescence readings of a dye solution of known concentration.

[0079] In some embodiments, the hydrophilic polymer may be a crosslinked polymer. In some embodiments, the crosslinked polymer may include a type of polymer crosslinked with another type of polymer. Examples of crosslinked polymers may include poly(ethylene glycol) crosslinked with another polymer selected from: polyethylene oxide (PEO) or polyethylene oxide, poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucosides, streptavidin, dextran, or other hydrophilic polymers. In some embodiments, the crosslinked polymer may be poly(ethylene glycol) crosslinked with polyacrylamide.

[0080] Due to the surface passivation techniques disclosed herein, proteins, nucleic acids, and other biomolecules do not “adhere” to the substrate; that is, they exhibit low nonspecific binding (NSB). Examples of standard monolayer surface preparation using different glass preparation conditions are shown below. Hydrophilic surfaces that have been passivated to achieve ultra-low NSB for proteins and nucleic acids require novel reaction conditions to improve primer deposition efficiency, hybridization performance, and induced efficient amplification. All of these processes require oligonucleotide attachment and subsequent protein binding and delivery to the low-binding surface. As described below, the combination of novel primer surface conjugation formulations (Cy3 oligonucleotide graft titration) and the resulting ultra-low nonspecific background (NSB functional assay using red and green fluorescent dyes) demonstrates the feasibility of the disclosed method. Some surfaces disclosed herein exhibit both specific binding to fluorophores (e.g., hybridization with tethered primers or probes) and nonspecific binding (e.g., B... inter The ratio is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1 or any intermediate value across the scope of this document. Some surfaces disclosed herein exhibit specific fluorescence signals of fluorophores (e.g., Cy3) and non-specific fluorescence signals (e.g., for specifically hybridized labeled oligonucleotides and non-specifically bound labeled oligonucleotides, or for specifically amplified labeled oligonucleotides and non-specifically bound (B)). inter ) or nonspecific amplification (B intra Labeled oligonucleotides or combinations thereof (B)inter +B intra The ratio is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1 or greater than 100:1 or any intermediate value across the scope of this document.

[0081] To scale primer surface density and add another dimension to hydrophilic or amphoteric surfaces, substrates incorporating multilayer coatings of PEG and other hydrophilic polymers have been developed. Primer loading density on surfaces can be significantly increased by using hydrophilic and amphoteric surface layering methods (including, but not limited to, the polymer / copolymer materials described below). Conventional PEG coating methods, using monolayer primer deposition, have been generally reported for single-molecule applications but fail to achieve high copy numbers for nucleic acid amplification applications. As described herein, “layering” can be accomplished using any compatible polymer or monomer subunit with conventional crosslinking methods, allowing for the sequential construction of surfaces comprising two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and copolymers of polylysine and PEG. In some cases, different layers can be attached to each other via any of a variety of conjugation reactions, including but not limited to biotin-streptavidin binding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged and negatively charged polymers. In some cases, high primer density materials can be constructed in solution and then stacked on a surface through multiple steps.

[0082] The attachment chemistry used to graft a first chemically modified layer onto a carrier surface typically depends on the material used to fabricate the carrier and the chemical properties of the layer. In some cases, the first layer may be covalently attached to the carrier surface. In others, the first layer may be non-covalently attached to the surface, for example, through non-covalent interactions such as electrostatic interactions, hydrogen bonds, or van der Waals interactions between the surface and the molecular components of the first layer, such as adsorption. In either case, the substrate surface may be treated prior to attachment or deposition of the first layer. Any of a variety of surface preparation techniques known to those skilled in the art can be used to clean or treat the carrier surface. For example, glass or silicon surfaces may be acid-washed using a Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or cleaned using oxygen plasma treatment methods.

[0083] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, Cl2, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) onto the surface. Examples of suitable silanes that can be used to generate any of the disclosed low-binding carrier surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), and any of a variety of PEG-silanes (e.g., having molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., having free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, etc.

[0084] Any of a variety of molecules known to those skilled in the art, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers or combinations thereof, can be used to generate one or more chemically modified layers on a carrier surface, wherein the choice of components used can vary to alter one or more properties of the carrier surface, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the carrier surface, or the three three-dimensional properties (i.e., “thickness”) of the carrier surface. Examples of preferred polymers that can be used to generate one or more layers of low-nonspecific binding material on any of the disclosed carrier surfaces include, but are not limited to, polyethylene glycol (PEG), streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof, of various molecular weights and branched structures. Examples of conjugation chemistry methods that can be used to graft one or more layers of material (e.g., polymer layers) onto a carrier surface and / or crosslink the layers to each other include, but are not limited to, biotin-streptavidin interactions (or variations thereof), His-tagged – Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylic acid ester conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxides, azides, hydrazides, alkynes, isocyanates, and silanes.

[0085] One or more layers of a multilayer surface may contain a branched polymer or may be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligomeric (ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

[0086] In some cases, the branched polymers used to generate any of the multilayer surfaces disclosed herein may contain at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches. Molecules typically exhibit a power of 2 number of branches, such as 2, 4, 8, 16, 32, 64, or 128 branches.

[0087] Exemplary PEG multilayers include PEG(8,16,8) (8 arms, 16 arms, 8 arms) on PEG-amine-APTES. Similar concentrations were observed for 3-layer multi-arm PEGs (8 arms, 16 arms, 8 arms) and (8 arms, 64 arms, 8 arms) on PEG-amine-APTES exposed to 8 μM primers, as well as for 3-layer multi-arm PEGs (8 arms, 8 arms, 8 arms) using star-shaped PEG-amines instead of the 16 and 64 arms. PEG multilayers with comparable first, second, and third PEG layers were also considered.

[0088] The molecular weight of the linear, branched, or multibranched polymer used to generate one or more layers of any multilayer surface disclosed herein may be at least 500 Daltons, at least 1,000 Daltons, at least 1,500 Daltons, at least 2,000 Daltons, at least 2,500 Daltons, at least 3,000 Daltons, at least 3,500 Daltons, at least 4,000 Daltons, at least 4,500 Daltons, at least 5,000 Daltons, at least 7,500 Daltons, at least 10,000 Daltons, at least 12,500 Daltons, at least 15,000 Daltons, at least 17,500 Daltons, at least 20,000 Daltons, at least 25,000 Daltons, at least 30,000 Daltons, at least 35,000 Daltons, at least 40,000 Daltons, at least 45,000 Daltons, or at least 50,000 Daltons. In some cases, the molecular weight of the linear, branched, or multibranched polymer used to generate one or more layers of any multilayer surface disclosed herein may be up to 50,000 Daltons, up to 45,000 Daltons, up to 40,000 Daltons, up to 35,000 Daltons, up to 30,000 Daltons, up to 25,000 Daltons, up to 20,000 Daltons, up to 17,500 Daltons, up to 15, The molecular weights of the linear, branched, or multibranched polymers used to generate any one or more layers of any multilayer surface disclosed herein may range from about 1,500 Daltons to about 20,000 Daltons. The lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure. For example, in some cases, the molecular weight of the linear, branched, or multibranched polymer used to generate any one or more layers of any multilayer surface disclosed herein may be in the range of about 1,500 Daltons to about 20,000 Daltons. Those skilled in the art will recognize that the molecular weight of the linear, branched, or multibranched polymer used to produce one or more layers of any of the multilayer surfaces disclosed herein can have any value within that range, for example, about 1,260 Daltons.

[0089] In some cases, for example, where at least one layer of the multilayer surface comprises a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the previous layer can range from about 1 covalent bond per molecule to about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, or more than 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be up to 32, up to 30, up to 28, up to 26, up to 24, up to 22, up to 20, up to 18, up to 16, up to 14, up to 12, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any lower and upper limits described in this paragraph can be combined to form the ranges covered by this disclosure; for example, in some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can range from about 4 to about 16. Those skilled in the art will recognize that the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can have any value within this range, for example, about 11 in some cases, or an average of about 4.6 in others.

[0090] Any reactive functional groups remaining after a material layer is coupled to the support surface can be selectively blocked by coupling with small, inert molecules using high-yield coupling chemistry. For example, in the case of attaching a new material layer to a previous layer using amine coupling chemistry, any residual amine groups can subsequently be acetylated or deactivated by coupling with small amino acids (e.g., glycine).

[0091] The number of layers of low-nonspecific binding material, such as hydrophilic polymer material, deposited on the surface of the disclosed low-binding support can range from 1 to about 10. In some cases, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the number of layers can range from about 2 to about 4. In some cases, all layers can contain the same material. In some cases, each layer can contain a different material. In some cases, multiple layers can contain multiple materials. In some cases, at least one layer can contain a branched polymer. In some cases, all layers can contain a branched polymer.

[0092] In some cases, one or more layers of low-nonspecific binding materials can be deposited on and / or coupled to a substrate surface using polar protic solvents, polar aprotic solvents, nonpolar solvents, or any combination thereof. In some cases, solvents used for layer deposition and / or coupling may include alcohols (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, aqueous buffer solutions (e.g., phosphate buffer, phosphate-buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.) or any combination thereof. In some cases, the organic component of the solvent mixture used may constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any percentage across or close to the scope of this document, with the balance consisting of water or an aqueous buffer solution. In some cases, the aqueous component of the solvent mixture used may constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any percentage across or close to the scope of this document, with the balance consisting of an organic solvent. The pH of the solvent mixture used may be less than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or greater than 10, or any value across or close to the range described herein.

[0093] In some cases, a mixture of organic solvents can be used to deposit one or more layers of low-nonspecific binding materials onto and / or conjugate them to a substrate surface, wherein at least one component has a dielectric constant of less than 40 and constitutes at least 50% of the total mixture by volume. In some cases, the dielectric constant of at least one component may be less than 10, less than 20, less than 30, or less than 40. In some cases, at least one component constitutes at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the total mixture by volume.

[0094] As noted, the low nonspecific binding vectors of this disclosure exhibit reduced nonspecific binding to proteins, nucleic acids, and other components of the hybridization and / or amplification formulations used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given vector surface can be assessed qualitatively or quantitatively. For example, in some cases, exposing the surface to fluorescent dyes (e.g., Cy3, Cy5, etc.), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, followed by a specified rinsing procedure, and fluorescence imaging can be used as qualitative tools to compare nonspecific binding on vectors containing different surface formulations. In some cases, under a standardized set of conditions, exposing the surface to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases), followed by a specified rinsing procedure and fluorescence imaging, can be used as a quantitative tool to compare nonspecific binding on carriers containing different surface formulations—provided that fluorescence imaging is performed using appropriate calibration criteria under conditions where the fluorescence signal is linearly correlated (or predictably correlated) with the number of fluorophores on the carrier surface (e.g., under conditions where signal saturation and / or fluorophore self-quenching are not problematic). In some cases, the degree of nonspecific binding exhibited by the different carrier surface formulations of this disclosure can be quantitatively assessed using other techniques known to those skilled in the art, such as radioisotope labeling and counting methods.

[0095] Some surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of fluorophores such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or greater than 100, or any intermediate value across the scope of this document. Some surfaces disclosed herein may exhibit a ratio of specific to nonspecific fluorescence of fluorophores such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or greater than 100, or any intermediate value across the scope of this document.

[0096] As noted, in some cases, a standardized procedure can be used to contact the surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a set of standard incubation and rinsing conditions, followed by detection of the amount of label remaining on the surface, and comparison of the resulting signal with appropriate calibration criteria to assess the degree of nonspecific binding exhibited by the disclosed low nonspecific binding carrier. In some cases, the label may include a fluorescent label. In some cases, the label may contain a radioactive isotope. In some cases, the label may include any other detectable label known to those skilled in the art. In some cases, the degree of nonspecific binding exhibited by a given carrier surface formulation can thus be assessed based on the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some cases, the low nonspecific binding carrier of this disclosure may exhibit less than 0.001 molecules / μm. 2 Less than 0.01 molecules / μm 2 Less than 0.1 molecules / μm 2 Less than 0.25 molecules / μm 2 Less than 0.5 molecules / μm 2 Less than 1 molecule / μm 2 Less than 10 molecules / μm 2 Less than 100 molecules / μm 2 Or less than 1,000 molecules / μm 2 Non-specific protein binding (or non-specific binding to other specific molecules, such as Cy3 dyes). Those skilled in the art will recognize that a given carrier surface of this disclosure can exhibit any number of non-specific binding values ​​falling within this range, e.g., less than 86 molecules / μm. 2For example, after contacting the surface with 1 μM of Cy3-labeled streptavidin (GE Amersham) solution in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by rinsing three times with deionized water, some of the modified surfaces disclosed herein exhibited nonspecific protein binding of less than 0.5 molecules / µm. 2 The modified surfaces disclosed in this paper exhibit non-specific binding of Cy3 dye molecules to less than 0.25 molecules / µm. 2In independent nonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM 7-propynylamino-7-denitro-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propynylamino-7-denitro-dGTP–Cy3 (Jena Biosciences) were incubated in 384-well plates at 37 °C for 15 min on a low-binding substrate. Each well was washed 2–3 times with 50 μL of deionized RNase / DNase-free water and rinsed with 25 mM ACES buffer (pH 10). 7.4) Rinse 2-3 times. Image the 384-well plate on a GETyphoon (GE Healthcare Lifesciences, Pittsburgh, PA) instrument using the manufacturer-specified Cy3, AF555, or Cy5 filter set (depending on the dye tests performed) at a PMT gain of 800 and a resolution of 50-100 μm. For higher resolution imaging, images were acquired on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) equipped with total internal reflection fluorescence (TIRF) objectives (20x, 0.75NA or 100x, 1.5NA, Olympus), an sCMOS Andor camera (Zyla 4.2), and excitation wavelengths of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX). Health & Science, LLC, Rochester, New York), for example, 405, 488, 532, or 633 nm dichroic mirrors / beam splitters, with bandpass filters selected at 532 LP or 645 LP, consistent with the appropriate excitation wavelength. Some of the modified surfaces disclosed herein show nonspecific binding of dye molecules below 0.25 molecules / μm. 2 .

[0097] In some cases, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of a fluorophore, such as Cy3, to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or greater than 100, or any intermediate value across the scope of this document. In some cases, the surfaces disclosed herein may exhibit a ratio of specific to nonspecific fluorescence of a fluorophore, such as Cy3, to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or greater than 100, or any intermediate value across the scope of this document.

[0098] Low background surfaces consistent with those disclosed herein can exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to non-specific dye adsorption (e.g., Cy3 dye adsorption) of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 of the attached specific dye molecules to the non-specific adsorption molecules. Similarly, when subjected to excitation energy, low-background surfaces with attached fluorophores such as Cy3, consistent with the disclosure herein, can exhibit a ratio of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1 specific fluorescence signals (e.g., generated by Cy3-labeled oligonucleotides attached to the surface) to non-specifically adsorbed dye fluorescence signals.

[0099] In some cases, the degree of hydrophilicity (or "wetting" in the case of aqueous solutions) of the disclosed carrier surface can be assessed, for example, by measuring the water contact angle, wherein a small droplet of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, a static contact angle can be determined. In some cases, a forward or backward contact angle can be determined. In some cases, the water contact angle of the hydrophilic, low-binding carrier surface disclosed herein can be in the range of about 0 degrees to about 50 degrees. In some cases, the water contact angle of the hydrophilic, low-binding carrier surface disclosed herein can not exceed 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed any value within this range, for example, not exceeding 40 degrees. Those skilled in the art will recognize that the given hydrophilic, low-binding carrier surface of this disclosure can exhibit a water contact angle with any value within that range, such as about 27 degrees.

[0100] In some cases, the hydrophilic surfaces disclosed herein help reduce washing time for bioassays, typically due to reduced nonspecific binding of biomolecules to low-binding surfaces. In some cases, a sufficient washing step can be performed in less than 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, in some cases, a sufficient washing step can be performed in less than 30 seconds.

[0101] Some of the low-binding surfaces disclosed herein exhibit significantly improved stability or durability in the face of prolonged exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups or tethered biomolecules (e.g., oligonucleotide primers) on the surface and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. In some cases, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours during exposure to solvents and / or elevated temperatures. In some cases, within 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 cycles of repeated exposure to solvent and / or temperature changes, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured within this cycle range).

[0102] In some cases, the surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces may exhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times, or greater than 100 times, that of adjacent unfilled regions of the surface. Similarly, some surfaces may exhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times, or greater than 100 times, that of adjacent regions of amplified nucleic acid populations of the surface.

[0103] The fluorescence excitation energy varies between specific fluorophores and schemes and can be consistent with other parameters used in the fluorophore selection or the surface disclosed herein, within an excitation wavelength range from less than 400 nm to more than 800 nm.

[0104] Therefore, the low nonspecific binding surfaces disclosed herein exhibit low background fluorescence signals or high contrast-to-noise (CNR) ratios compared to surfaces known in the art. For example, in some cases, the background fluorescence at locations spatially different on the surface or removed from labeled features (e.g., labeled points, clusters, discrete regions, sub-parts, or subsets of the surface) on the surface (containing hybridized nucleic acid clusters, or cloned amplified nucleic acid clusters generated, for example, via 20 cycles of thermal cycling) can be no more than 20x, 10x, 5x, 2x, 1x, 0.5x, 0.1x, or less than 0.1x greater than the background fluorescence measured at the same location before performing the hybridization or the 20 cycles of nucleic acid amplification.

[0105] In some cases, the fluorescence images of the disclosed low background surface (when used in nucleic acid hybridization or amplification applications to generate clusters of nucleic acid molecules for hybridization or clonal amplification (e.g., already directly or indirectly labeled with fluorophores)) exhibit a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or greater than 250.

[0106] Typically, at least one layer of one or more low-nonspecific binding materials may contain functional groups for covalent or non-covalent attachment of oligonucleotide molecules, such as adaptors or primer sequences, or at least one layer may already contain covalent or non-covalent attachment of oligonucleotide adaptors or primer sequences when it is deposited on the support surface. In some cases, oligonucleotides tethered to polymer molecules in at least one third layer may be distributed at multiple depths throughout the layer.

[0107] In some cases, oligonucleotide intransitive or primer molecules are covalently coupled to the polymer in solution before polymer coupling or deposition on the surface. In some cases, oligonucleotide intransitive or primer molecules are covalently coupled to the polymer after polymer coupling or deposition on the surface. In some cases, at least one hydrophilic polymer layer contains multiple covalently attached oligonucleotide intransitive or primer molecules. In some cases, at least two, at least three, at least four, or at least five hydrophilic polymer layers contain multiple covalently attached intransitive or primer molecules.

[0108] In some cases, any of a variety of suitable conjugation chemistry methods known to those skilled in the art can be used to couple oligonucleotide adapters or primer molecules to one or more hydrophilic polymers. For example, the oligonucleotide adapter or primer sequence may contain a portion that reacts with amine, carboxyl, thiol, etc. Examples of suitable amine-reactive conjugation chemistry methods that can be used include, but are not limited to, reactions involving isothiocyanate groups, isocyanate groups, acyl azide groups, NHS ester groups, sulfonyl chloride groups, aldehyde groups, glyoxal groups, epoxide groups, ethylene oxide groups, carbonate groups, aryl halide groups, imide ester groups, carbodiimide groups, anhydride groups, and fluorophenyl ester groups. Examples of suitable carboxyl-reactive conjugation chemistry methods include, but are not limited to, reactions involving carbodiimide compounds, such as water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl). Examples of suitable thiol-reactive conjugation chemistry methods include maleimides, haloacetyl groups, and pyridyl disulfides.

[0109] One or more types of oligonucleotide molecules can be attached to or tethered to the surface of a vector. In some cases, one or more types of oligonucleotide adaptors or primers may comprise a spacer sequence, an adaptor sequence for hybridization with a template library nucleic acid sequence linked to the adaptor, a forward amplification primer, a reverse amplification primer, a sequencing primer, and / or a molecular barcoding sequence, or any combination thereof. In some cases, one primer or adaptor sequence can be tethered to at least one layer of the surface. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adaptor sequences can be tethered to at least one layer of the surface.

[0110] The length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some cases, the tethered oligonucleotide adaptor and / or primer sequence can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides long. In some cases, the tethered oligonucleotide adaptor and / or primer sequence can be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides long. Any lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some cases, the length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 20 nucleotides to about 80 nucleotides. Those skilled in the art will recognize that the length of the tethered oligonucleotide adaptor and / or primer sequence can have any value within that range, such as about 24 nucleotides.

[0111] In some cases, the tethered adaptor or primer sequence may contain modifications designed to enhance the specificity and efficiency of nucleic acid amplification, such as on low-binding vectors. For example, in some cases, primers may contain polymerase termination sites, such that the primer sequence between the surface conjugate and the modification site is always in single-stranded form, and serves as the 5' to 3' helicase loading site in some helicase-dependent isothermal amplification methods. Other examples of primer modifications that can be used to generate polymerase termination sites include, but are not limited to, inserting a PEG chain toward the 5' end between two nucleotides of the primer backbone, inserting a baseless nucleotide (i.e., a nucleotide without either purine or pyrimidine bases), or a lesion site that can be bypassed by helicase.

[0112] As will be further discussed in the following examples, it may be necessary to vary the surface density of the oligonucleotide adaptors or primers tethered to the vector surface and / or the spacing of the tethered adaptors or primers away from the vector surface (e.g., by varying the length of the adapter molecules used to tether the adaptors or primers to the surface) to “adjust” the vector for optimal performance when using a given amplification method. As described below, adjusting the surface density of the tethered oligonucleotide adaptors or primers may affect the level of specific and / or nonspecific amplification observed on the vector in a manner that varies depending on the selected amplification method. In some cases, the surface density of the tethered oligonucleotide adaptors or primers can be varied by adjusting the proportion of the molecular components used to generate the vector surface. For example, in the case of using oligonucleotide primer-PEG conjugates to generate the final layer of a low-binding vector, the ratio of the oligonucleotide primer-PEG conjugate to unconjugated PEG molecules can be varied. The surface density of the tethered primer molecules can then be estimated or measured using any of a variety of techniques known to those skilled in the art. Examples include, but are not limited to, the use of radioisotope labeling and counting methods; covalent coupling of cleavable molecules comprising optically detectable tags (e.g., fluorescent tags) that can be cleaved from a defined area of ​​a carrier surface, collected in a fixed volume of a suitable solvent, and then quantified by comparing the fluorescence signal with the fluorescence signal of a calibration solution with a known concentration of the optical tag or by using fluorescence imaging techniques (provided that attention has been paid to the labeling reaction conditions and image acquisition settings to ensure that the fluorescence signal is linearly correlated with the number of fluorophores on the surface) (e.g., the fluorophores on the surface do not exhibit obvious self-quenching).

[0113] In some cases, the resulting surface density of oligonucleotide adaptors or primers on the low-binding vector surface of this disclosure can be as low as approximately 100 primer molecules / μm. 2 Approximately 1,000,000 primer molecules / μm 2Within the range. In some cases, the surface density of oligonucleotide adapters or primers can be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45, 000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, to At least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules / μm 2In some cases, the surface density of oligonucleotide adaptors or primers can be up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, or up to 450,000. 0, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 9,500, up to 9,000, up to 8,500, up to 8,000, up to 7,500, up to 7,000, up to 6,500, up to Up to 6,000, up to 5,500, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200 or up to 100 molecules / μm 2 Any lower and upper limits described in this paragraph may be combined to form the range included in this disclosure; for example, in some cases, the surface density of the adaptor or primer may be approximately 10,000 molecules / μm. 2 Approximately 100,000 molecules / μm 2 Within this range. Those skilled in the art will recognize that the surface density of the adaptor or primer molecule can have any value within this range, for example, in some cases about 3,800 molecules / μm. 2 Or, in other cases, approximately 455,000 molecules / μm 2In some cases, as will be further discussed below, the surface density of the template library nucleic acid sequence (e.g., the sample DNA molecule) initially hybridized with the adaptor or primer sequence on the vector surface may be less than or equal to the surface density indicated by the surface density of the tethered oligonucleotide primer. In some cases, as will also be further discussed below, the surface density of the cloned and amplified template library nucleic acid sequence hybridized with the adaptor or primer sequence on the vector surface may span the same or different ranges as the surface density indicated by the surface density of the tethered oligonucleotide adaptor or primer.

[0114] The local surface density of the linker or primer molecules listed above does not preclude density variations across the entire surface, such that the surface may include, for example, a density of 500,000 / μm. 2 The region of oligonucleotide density also includes at least a second region with significantly different local densities.

[0115] Solid carriers for capturing and analyzing DNA. In some embodiments, a surface thereon is bound with multiple oligonucleotides for capturing target nucleic acids, such as DNA molecules (e.g., capturing oligonucleotides; (200)). Figure 2 As shown. In some embodiments, each capturing oligonucleotide comprises a single-stranded oligonucleotide. The capturing oligonucleotides can be anchored to the passivated surface via their 5' ends, or the internal portion of the capturing oligonucleotide can be anchored to the passivated surface. Each capturing oligonucleotide may contain an extendable 3' end. (See figure.) Figure 2As shown, each capturing oligonucleotide may include a cleavable region (250) located near the end of the surface immobilized to a passivated surface. For example, each capturing oligonucleotide may include a cleavable region near the 5' end. The cleavable region may be cleaved by enzymes, compounds, light, or heat. In some embodiments, each capturing oligonucleotide includes a target capturing region (210) and a universal sequence region (220, 230, 240). In some embodiments, the target capturing region of the capturing oligonucleotide includes a sequence that can hybridize with at least a portion of the target nucleic acid. The target capturing region may include, for example, a random nucleotide sequence or a target-specific sequence corresponding to a known sequence of the target nucleic acid. In some embodiments, the universal sequence region includes a sample barcode sequence (220) that can be used to distinguish target nucleic acids from different sample sources in multiplex assays. In some embodiments, the universal sequence region includes a spatial barcode sequence (230) that conveys location information of the capturing oligonucleotide on the vector, which in turn conveys location information of cells or single cells within a tissue sample. In some embodiments, the sample barcode sequence (220) may be upstream or downstream of the spatial barcode sequence (230). In some embodiments, the universal sequence region of the capture oligonucleotide includes a cyclization anchoring region (240) that hybridizes to a portion of a second type of oligonucleotide that promotes the cyclization of the captured nucleic acid (300). In some embodiments, the universal sequence region of the capture oligonucleotide includes at least one sequence that binds to / hybridizes to a universal primer sequence (e.g., a sequencing primer sequence and / or an amplification primer sequence). In some embodiments, the cyclization anchoring region (240) includes any combination of any one or two or more of the sequencing primer sequence, the amplification primer sequence, the sample barcode sequence, and / or the spatial barcode sequence. In some embodiments, the cyclization anchoring region (240) includes a separate sequence that hybridizes to a portion of a second type of oligonucleotide that promotes the cyclization of the captured nucleic acid. In some embodiments, the universal sequence region includes a cleavable region that can be cleaved by enzymes, compounds, light, or heat.

[0116] Still referencing Figure 2 In some embodiments, the surface has been bound thereto with multiple type II oligonucleotides (e.g., cyclic oligonucleotides (300)) that facilitate the cyclization of the target nucleic acid for capture. In some embodiments, each cyclic oligonucleotide comprises a single-stranded oligonucleotide. Cyclic oligonucleotides may be anchored to the passivated surface via their 5' ends, or internal portions of the cyclic oligonucleotides may be anchored to the passivated surface. Each cyclic oligonucleotide may contain an extendable 3' end. Each cyclic oligonucleotide contains a homopolymer region (310) and a universal sequence region (320), such as... Figure 3As shown in the diagram. The homopolymer region can be selected from poly-T tail, poly-dT tail, poly-A tail, poly-dA tail, poly-C tail, poly-dC tail, poly-G tail, and poly-dG tail. The homopolymer region can be located at or near the 3' end of the cyclic oligonucleotide. In some embodiments, the universal sequence region of the cyclic oligonucleotide hybridizes with the cyclic anchoring region of the capture oligonucleotide. In some embodiments, the universal sequence region of the cyclic oligonucleotide includes at least one sequence that binds / hybridizes with a universal primer sequence (e.g., a sequencing primer sequence for the capture oligonucleotide). In some embodiments, the universal sequence region of the cyclic oligonucleotide includes at least one sequence that binds / hybridizes with a universal primer sequence (e.g., an amplification primer sequence for the capture oligonucleotide). In some embodiments, the universal sequence region of the cyclic oligonucleotide includes at least one sequence that binds / hybridizes with a sample barcode sequence and / or spatial barcode sequence of the capture oligonucleotide. In some embodiments, the cyclic oligonucleotide includes a separate sequence (e.g., a cyclic anchoring binding sequence) that binds / hybridizes with a portion of the cyclic anchoring region of the capture oligonucleotide.

[0117] In some implementations, oligonucleotides are captured ( Figure 2 ,200) and cyclic oligonucleotides ( Figure 3 (300) can be immobilized on the passivated surface prior to contacting the target nucleic acid molecule with the passivated surface for the target molecule capture step. In an alternative embodiment, the capturing oligonucleotide is immobilized on the passivated surface prior to contacting the passivated surface with the target nucleic acid molecule for the target molecule capture step, and subsequently, multiple cyclic oligonucleotides (e.g., in soluble form) can be provided in solution and allowed to flow onto the passivated surface to immobilize the cyclic oligonucleotides.

[0118] In some embodiments, the cyclic oligonucleotide may be the same as the capturing oligonucleotide, may include the capturing oligonucleotide, or may be included within the capturing oligonucleotide. In some embodiments, the cyclic oligonucleotide may comprise a separate molecule.

[0119] This disclosure provides a low-binding carrier with a coating, wherein the coating provides a low-nonspecific binding surface for proteins, carbohydrates, lipids, cell debris, or solution-loaded dye molecules. In some embodiments, tissue samples or cells or single cells can be placed on the surface of the carrier. Figure 3 (Left). In some embodiments, the low nonspecific binding surface includes multiple regions (e.g., feature portions) located at different predetermined locations on the carrier. Figure 3(Right). Different features on the carrier can be placed in non-overlapping or overlapping positions on the carrier. Features can be configured to have any shape, such as circular, oval, square, rectangular, or polygonal. Features can be arranged in a grid pattern of rows and columns, or can be arranged in rows or columns. In some embodiments, any given feature comprises a plurality of capturing oligonucleotides and a plurality of cyclized oligonucleotides fixed to the coating. The plurality of features includes at least first and second features.

[0120] In some embodiments, the first feature portion includes a plurality of first capture oligonucleotides having a first target capture region, a first spatial barcode sequence, a first sample barcode sequence, and a first cleavable region, and the first feature portion includes a plurality of first cyclic oligonucleotides having a first circularization anchoring binding sequence, a first amplification primer binding sequence, and a first sequencing primer binding sequence. In some embodiments, the first capture oligonucleotides further include a first amplification primer binding sequence and / or a first amplification primer binding sequence. In some embodiments, the first cyclic oligonucleotides further include sequences capable of binding / hybridizing the first spatial barcode sequence and / or sequences capable of binding the first sample barcode sequence.

[0121] In some embodiments, the second feature portion includes a plurality of second capture oligonucleotides having a second target capture region, a second spatial barcode sequence, a second sample barcode sequence, and a second cleavable region, and the second feature portion includes a plurality of second cyclic oligonucleotides having a second circularization anchoring binding sequence, a second amplification primer binding sequence, and a second sequencing primer binding sequence. In some embodiments, the second capture oligonucleotides further include a second amplification primer binding sequence and / or a second amplification primer binding sequence. In some embodiments, the second cyclic oligonucleotides further include sequences capable of binding / hybridizing the second spatial barcode sequence and / or sequences capable of binding the second sample barcode sequence.

[0122] In some embodiments, the sequence of the first target capture region in the first feature portion may be the same as or different from the sequence of the second target capture region in the second feature portion. In some embodiments, the first spatial barcode sequence in the first feature portion is different from the second spatial barcode sequence in the second feature portion. In some embodiments, the first sample barcode sequence in the first feature portion may be the same as or different from the second sample barcode sequence in the second feature portion. The first amplification primer binding sequence in the first feature portion may be the same as the second amplification primer binding sequence in the second feature portion. The first sequencing primer binding sequence in the first feature portion may be the same as the second sequencing primer binding sequence in the second feature portion. The first cleavable region in the first feature portion may be cleavable under the same or different conditions (e.g., the same enzyme, compound, light, or heat) as the second cleavable region in the second feature portion.

[0123] In some embodiments, the low nonspecific binding coating includes multiple regions (e.g., feature portions) in which the feature portions are attached to multiple capturing and cyclized oligonucleotides attached to the coating. In some embodiments, a first feature portion is attached to a first plurality of capturing oligonucleotides and a first plurality of cyclized oligonucleotides, and a second feature portion is attached to a second plurality of capturing oligonucleotides and a second plurality of cyclized oligonucleotides, wherein the first and second capturing oligonucleotides and the first and second cyclized oligonucleotides are in fluid communication with each other, such that the capturing oligonucleotides and cyclized oligonucleotides can react with reagents (e.g., enzymes including polymerases, polymer-nucleotide conjugates, nucleotides and / or divalent cations) in a large-scale parallel manner.

[0124] In some embodiments, the cleavable region of the captured oligonucleotide can be cleaved by an enzyme. In some embodiments, Figure 2 The cleavable region (250) shown contains at least one uracil base or polyuracil sequence, which can be cleaved by uracil DNA glycosylase (UDG) or DNA glycosylase-lyase endonuclease VIII (e.g., commercially available enzyme USER). TM In some embodiments, the cleavable site comprises at least one 8-oxoguanine (8-oxoG) that can be cleaved by DNA-formamidopyrimidine glycosylase (Fpg). In some embodiments, the cleavable region comprises a base-free site that can be cleaved by endonuclease IV or endonuclease VIII. In some embodiments, the enzyme-cleavable region comprises a nucleotide sequence that is recognized and cleaved by restriction endonucleases that cleave double-stranded or single-stranded nucleic acid chains (e.g., DNA). In some embodiments, the enzyme-cleavable region comprises a glycosidic bond that can be cleaved by amylase or a peptide bond that can be cleaved by protease.

[0125] like Figure 2 As shown, in some embodiments, the cleavable region (250) of the captured oligonucleotide can be cleaved by a compound containing unstable chemical bonds (e.g., including but not limited to ester bonds, thiol bonds, vicinal diol bonds, sulfone bonds, silyl ether bonds, and base-free or purine / pyrimidine-free (AP) sites). Ester bonds can be cleaved with acids, bases, or hydroxylamine. Thiol bonds can be disulfide bonds that can be cleaved by glutathione or a reducing agent. Vicinal diol bonds can be cleaved with sodium periodate. Sulfonate bonds can be cleaved with bases. Siliyl ether bonds can be cleaved with acids. Base-free or purine / pyrimidine-free (AP) sites can be cleaved with bases or AP endonucleases.

[0126] In some embodiments, the cleavable region (250) of the photocleavable captured oligonucleotide comprises a photocleavable portion that can be cleaved by exposure to light, UV light, or a laser. The photocleavable portion can be cleaved by exposure to light of any wavelength. The photocleavable portion includes 3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicumarol, 6-bromo-7-alkoxycoumarin-4-ylmethoxycarbonyl, benzoylmethyl ester derivatives, or 8-quinolinylbenzenesulfonate. The photocleavable portion includes a bimane-based linker, a bis(aryl)hydrazone-based linker, or an o-nitrobenzyl (ONB) linker. In some embodiments, the cleavable region (250) of the captured oligonucleotide is cleavable upon exposure to heat and comprises a Diels-Alder linker.

[0127] Vectors used for capturing and analyzing RNA. This article... Figure 4 The present invention provides a vector (700) comprising a plurality of immobilized oligonucleotides. The vector can be used to capture and analyze target nucleic acids, such as RNA molecules. In some embodiments, the vector includes a passivated surface (e.g., a coating or layer) disclosed elsewhere herein. Figure 1 This allows the surface to bind little or no to proteins, carbohydrates, lipids, cell debris, or solution-loaded dye molecules. In some embodiments, the surface has multiple oligonucleotides (e.g., capture oligonucleotides) bound thereto for capturing target nucleic acids. Figure 4 (700)). In some embodiments, each of the capturing oligonucleotides comprises a single-stranded oligonucleotide. The capturing oligonucleotides can be anchored to a passivated surface via their 5' ends, or the internal portion of the capturing oligonucleotide can be anchored to the passivated surface. Each capturing oligonucleotide may contain an extendable 3' end. Figure 4As shown, each capturing oligonucleotide may include a cleavable region (740) located near the end of the surface immobilized to a passivated surface. For example, each capturing oligonucleotide may include a cleavable region near the 5' end. The cleavable region may be cleaved by enzymes, compounds, light, or heat. In some embodiments, each capturing oligonucleotide includes a target capturing region (710) and a universal sequence region (720, 730). In some embodiments, the target capturing region of the capturing oligonucleotide includes a sequence that can hybridize with at least a portion of the target nucleic acid. The target capturing region may include, for example, a homopolymer sequence (e.g., poly-T or poly-dT), a random nucleotide sequence, or a target-specific sequence corresponding to a known sequence of the target nucleic acid. In some embodiments, the universal sequence region includes a sample barcode sequence (720) that can be used to distinguish target nucleic acids from different sample sources in multiplex assays. In some embodiments, the universal sequence region includes a spatial barcode sequence (730) that conveys location information of the capturing oligonucleotide on the vector, which in turn conveys location information of cells or single cells within a tissue sample. In some embodiments, the sample barcode sequence (720) may be upstream or downstream of the spatial barcode sequence (730). In some embodiments, the universal sequence region of the captured oligonucleotide includes at least one sequence that binds to / hybridizes with a universal primer sequence (e.g., a sequencing primer sequence and / or an amplification primer sequence). In some embodiments, the captured oligonucleotide includes a cleavable region (740) that can be cleaved by enzymes, compounds, light, or heat.

[0128] Still referencing Figure 4 In some embodiments, this document provides multiple type II oligonucleotides (e.g., cyclic oligonucleotides; 800) in soluble form or immobilized on a surface (e.g., a coating). Cyclic oligonucleotides can facilitate the cyclization of captured target nucleic acids. In some embodiments, each cyclic oligonucleotide comprises a single-stranded oligonucleotide. Cyclic oligonucleotides may be in soluble form, or they may be immobilized to a passivated surface via their 5' ends, or internal portions of the cyclic oligonucleotides may be immobilized to a passivated surface. Each cyclic oligonucleotide may contain an extendable 3' end. Each cyclic oligonucleotide contains an adaptor-binding region (810). In some embodiments, the adaptor-binding region includes a sequencing primer-binding region. In some embodiments, the adaptor-binding region includes an amplification primer-binding region. In some embodiments, each cyclic oligonucleotide contains a homopolymer region ( Figure 4 (830)). The homopolymer region may be selected from poly(T), poly(dT), poly(A), poly(dA), poly(C), poly(dC), poly(G), and poly(dG). In some embodiments, each cyclized oligonucleotide includes an anchoring region (830) and an anchoring moiety (840).

[0129] In some implementations, capture oligonucleotides (Figure 5, (700)) and cyclized oligonucleotides ( Figure 4(800)) can be immobilized on the passivated surface prior to contacting the passivated surface with the target nucleic acid molecule (e.g., RNA) for the target molecule capture step. In an alternative embodiment, the capture oligonucleotide is immobilized on the passivated surface prior to contacting the passivated surface with the target nucleic acid molecule for the target molecule capture step, and subsequently, multiple cyclic oligonucleotides (e.g., in soluble form) can be provided in solution and allowed to flow onto the passivated surface to immobilize the cyclic oligonucleotides.

[0130] In some embodiments, the cyclic oligonucleotide may be the same as the capturing oligonucleotide, may include the capturing oligonucleotide, or may be included within the capturing oligonucleotide. In some embodiments, the cyclic oligonucleotide may comprise a separate molecule.

[0131] In some implementations, the cleavable region of the oligonucleotide is captured. Figure 4 (740)) can be cleaved by an enzyme. In some embodiments, the cleavable region contains at least one uracil base or polyuracil sequence, which can be cleaved by uracil RNA glycosylase (UDG) or RNA glycosylase-lyase endonuclease VIII (e.g., commercially available enzyme USER). TM In some embodiments, the cleavable site comprises at least one 8-oxoguanine (8-oxoG) that can be cleaved by RNA-formamidopyrimidine glycosylase (Fpg). In some embodiments, the cleavable region comprises a base-free site that can be cleaved by endonuclease IV or endonuclease VIII.

[0132] In some embodiments, the enzyme-cleavable region comprises a nucleotide sequence that is recognized and cleaved by restriction endonucleases that cleave double-stranded or single-stranded nucleic acid chains (e.g., RNA). In some embodiments, the enzyme-cleavable region comprises a glycosidic bond that can be cleaved by amylase, or a peptide bond that can be cleaved by protease.

[0133] In some implementations, the cleavable region of the oligonucleotide is captured. Figure 4 (740) can be cleaved by compounds containing unstable chemical bonds (e.g., including but not limited to ester bonds, thiol bonds, vicinal diol bonds, sulfone bonds, silyl ether bonds, and no-base or no-purine / no-pyrimidine (AP) sites). Ester bonds can be cleaved with acids, bases, or hydroxylamine. Thiol bonds can be disulfide bonds that can be cleaved by glutathione or reducing agents. Visceral diol bonds can be cleaved with sodium periodate. Sulfonate bonds can be cleaved with bases. Silicyl ether bonds can be cleaved with acids. No-base or no-purine / no-pyrimidine (AP) sites can be cleaved with bases or AP endonucleases.

[0134] In some implementations, the cleavable region of the captured oligonucleotide that can be photocleaved ( Figure 4(740) contains an optically cleavable portion that can be cleaved by exposure to light, UV light, or a laser. The optically cleavable portion can be cleaved by exposure to light of any wavelength. The optically cleavable portion includes 3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicumarol, 6-bromo-7-alkoxycoumarin-4-ylmethoxycarbonyl, benzoylmethyl ester derivatives, or 8-quinolinylbenzenesulfonate. The optically cleavable portion includes a bimane-based linker, a bis(aryl)hydrazone-based linker, or an o-nitrobenzyl (ONB) linker. In some embodiments, the cleavable region of the oligonucleotide is captured ( Figure 4 (740) is cuttable when exposed to heat and includes a Diels-Alder connector.

[0135] Biological samples are immobilized onto a surface. This document provides solid carriers (e.g., low-nonspecific binding carriers) that also contain adjacent biological samples. In some embodiments, the biological sample includes single cells, multiple cells, tissues, organs, organisms, or slices of these biological samples. In some embodiments, the biological sample is derived from eukaryotes (e.g., animals, plants, fungi, protozoa), archaea, or eubacteria. The biological sample can be derived from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. The biological sample can be derived from primary or immortalized cell lines of rodents, pigs, cats, dogs, cattle, horses, primates, or humans.

[0136] Biological samples can be solid samples, such as tissue biopsies. Biological samples can also be fluid samples, such as blood or blood components (e.g., serum or plasma). In some embodiments, biological samples are taken from skin, heart, lungs, kidneys, respiratory fluid, bone marrow, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, pharyngeal swabs, biopsies, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive fluids, tears, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, nails, skin cells, plasma, nasal swabs or nasopharyngeal washes, cerebrospinal fluid, umbilical cord blood, emphatic fluid, and / or other excretions or body tissues. Biological samples can also be cell-free samples.

[0137] Biological samples may include cells. Cells described herein may be leukocytes, erythrocytes, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells derived from the heart, lungs, brain, liver, kidneys, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine. Cells may be normal or healthy cells. Alternatively or in combination, cells may be diseased cells, such as cancer cells, or pathogenic cells derived from an infected host. In some embodiments, the cells belong to a subpopulation of cells, such as immune cells (e.g., T cells, cytotoxic (killer) T cells, helper T cells, αβ T cells, γδ T cells, T cell progenitor cells, B cells, B cell progenitor cells, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells and / or macrophages, or any combination thereof), undifferentiated human stem cells, induced differentiated human stem cells, or rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblast cells). Other cells are considered and consistent with the disclosure herein.

[0138] Biological samples can be extracted from organisms (e.g., biopsies) or obtained from cell cultures grown in liquids or petri dishes. Biological samples include fresh, frozen, fresh-frozen, or archived samples (e.g., formalin-fixed paraffin-embedded; FFPE). Biological samples can be embedded in wax, resin, epoxy resin, or agar. Biological samples can be fixed, for example, in any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-triton, or glutaraldehyde. Biological samples may or may not be sectioned. Biological samples may be stained, destained, or unstained.

[0139] In some embodiments, biological samples may be permeabilized after being immobilized onto the surface described herein to allow nucleic acids within the sample, including target nucleic acid molecules, to migrate from one or more cells to multiple capturing oligonucleotides immobilized on the surface. Permeabilization allows reagents (e.g., phosphorus-selective antibodies, nucleic acid conjugated antibodies, nucleic acid probes, primers, etc.) to enter the cells and reach concentrations within the cells greater than concentrations that would normally permeate into the cells without such permeabilization. In some embodiments, cells may be permeabilized in the presence of at least about 60%, 70%, 80%, 90%, or higher methanol (or ethanol) and incubated on ice for a period of time. The incubation period may be at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, or more minutes.

[0140] Biological samples can be permeated by contacting them with one or more permeabilizing agents, said permeabilizing agents including organic solvents, detergents, cross-linking agents, and / or enzymes. In some embodiments, organic solvents include acetone, ethanol, and methanol. In some embodiments, detergents include saponins, Triton X-100, Tween-20, or sodium dodecyl sulfate (SDS) or N-lauroyl sarcosinate sodium salt solution. In some embodiments, cross-linking agents include paraformaldehyde. In some embodiments, enzymes include trypsin, pepsin, or proteases (e.g., prions K). In some embodiments, target nucleic acid molecules from the biological sample hybridize with (are captured by) capture oligonucleotides immobilized on a carrier in a manner that preserves the spatial location information of the target nucleic acid molecules in the biological sample.

[0141] Biological samples can be used to generate three-dimensional polymer matrices comprising cellular and subcellular components (e.g., nucleic acid molecules) of the biological sample. The three-dimensional polymer matrix can be covalently or non-covalently coupled to the surfaces described herein. In some embodiments, the three-dimensional polymer matrix is ​​porous and comprises polymerized or cross-linked subcellular components, including target nucleic acid molecules. A polymer matrix can be formed within a biological sample (e.g., cells or tissues) by infusing one or more polymer precursors (e.g., monomers, such as ethylene oxide for polyethylene glycol) into the biological sample and polymerizing or cross-linking the one or more polymer precursors. Before, during, or after the formation of the polymer matrix, immobilizers (e.g., formaldehyde) can be used to immobilize portions (e.g., DNA, RNA, proteins) within the biological sample. Porous matrices can be prepared according to various methods. For example, a polyacrylamide gel matrix can be polymerized with biotinylated DNA molecules and acrylamide-modified streptavidin monomers, wherein a suitable acrylamide:bisacrylamide ratio is used to control the crosslinking density. Further control over the size and density of the molecular sieve can be achieved by adding additional crosslinking agents (e.g., functionalized polyethylene glycol). PCT / US2019 / 055434 (which is incorporated herein by reference in its entirety) provides methods for immobilizing biological samples to a surface and generating a polymer matrix within the biological sample.

[0142] Biological samples include one or more target nucleic acid molecules, which are analyzed in some cases using the systems, methods, and compositions described herein. In some embodiments, the target nucleic acid includes naturally occurring nucleic acids, recombinant nucleic acids, and / or synthetic nucleic acids. The target nucleic acid includes linear and / or circular forms. In some embodiments, the target nucleic acid can be DNA. In some embodiments, the target nucleic acid can be genomic DNA. In some embodiments, the target nucleic acid can be viral DNA. In some embodiments, the target nucleic acid can be cell-free DNA (cfDNA). In some embodiments, the DNA is genomic DNA, methylated or unmethylated DNA, and / or organelle DNA. The DNA can be fragmented and / or unfragmented. In some embodiments, one or more target nucleic acid molecules include RNA, including poly(A) RNA and / or non-poly(A) RNA. The RNA includes coding and / or non-coding RNA. RNA includes tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), antisense RNA, non-coding RNA, and / or protein-coding RNA.

[0143] The target nucleic acid disclosed herein has a fixed three-dimensional relationship with the biological sample after surface conjugation. This fixed three-dimensional relationship enables, at least in part, the identification of spatial and cellular origin within the biological sample after nucleic acid identification using the systems and methods described herein.

[0144] Target nucleic acid capture and preparation. This article provides methods for hybridizing target nucleic acids with capture oligonucleotides coupled to a surface (e.g., a low-nonspecific binding surface) in the presence of a biological sample. In some cases, the described hybridization buffer formulations, combined with the disclosed low-binding vectors, provide improved hybridization rates, hybridization specificity (or stringency), and hybridization efficiency (or yield). As used herein, hybridization specificity is a measure of the ability of a tethered adaptor sequence, primer sequence, or oligonucleotide sequence to hybridize correctly only with its perfectly complementary sequence, while hybridization efficiency is a measure of the percentage of total available tethered adaptor sequences, primer sequences, or oligonucleotide sequences that typically hybridize with complementary sequences.

[0145] Improved hybridization specificity and / or efficiency can be achieved by optimizing hybridization buffer formulations for use with the disclosed low-binding surfaces, and will be discussed in more detail in the examples below. Examples of hybridization buffer components that can be adjusted to achieve improved performance include, but are not limited to, buffer type, organic solvent mixture, buffer pH, buffer viscosity, detergent and zwitterionic components, ionic strength (including adjustment of monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, other additives, etc.

[0146] As a non-limiting example, suitable buffers for preparing hybridization buffers may include, but are not limited to, phosphate-buffered saline (PBS), succinate, citrate, histidine, acetate, Tris, taps, mops, pyps, heptaes, hydroxypropyl methylisocyanate (HEPES), mesotherapy, and methylisocyanate (MES). The choice of a suitable buffer typically depends on the target pH of the hybridization buffer solution. Generally, the desired pH range for the buffer solution is from about pH 4 to about pH 8.4. In some embodiments, the buffer pH may be at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8.0, at least 8.2, or at least 8.4. In some embodiments, the buffer solution pH may be at most 8.4, at most 8.2, at most 8.0, at most 7.8, at most 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at most 6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at most 5.0, at most 4.5, or at most 4.0. Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the pH range is expected to be from about 6.4 to about 7.2. Those skilled in the art will recognize that the buffer solution pH may have any value within this range, such as about 7.25.

[0147] Detergents suitable for hybridization buffer formulations include, but are not limited to, zwitterionic detergents (e.g., 1-dodecanoyl-sn-glycerol-3-phosphocholine, 3-(4-tert-butyl-1-pyridyl)-1-propanesulfonate, 3-(N,N-dimethylmyristylammonium)propanesulfonate, 3-(N,N-dimethylmyristylammonium)propanesulfonate, ASB-C80, C7BzO, CHAPS, CHAPS hydrate, CHAPSO, DDMAB, dimethylethylammonium propanesulfonate, N,N-dimethyldodecylamine N-oxide, N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate or N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate) and anionic, cationic, and nonionic detergents. Examples of nonionic detergents include poly(oxyethylene) ethers and related polymers (e.g., TRITON X-100 and CA-630), bile salts and glycoside detergents.

[0148] The disclosed low nonspecific binding vector, used alone or in combination with optimized buffer formulations, can produce relative hybridization rates that are approximately 2 to approximately 20 times faster than conventional hybridization protocols. In some cases, the relative hybridization rate can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, or at least 40 times faster than conventional hybridization protocols.

[0149] The disclosed low nonspecific binding vector, used alone or in combination with optimized buffer formulations, can produce total hybridization reaction times of less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes for any of these completion metrics (i.e., the time required for the hybridization reaction to be 90%, 95%, 98%, or 99% complete).

[0150] Compared to conventional hybridization protocols, the disclosed low-nonspecificity binding vector, used alone or in combination with optimized buffer formulations, produces improved hybridization specificity. In some embodiments, achievable hybridization specificity is superior to that achieved in 10 hybridization events, 20 hybridization events, 30 hybridization events, 40 hybridization events, 50 hybridization events, 75 hybridization events, 100 hybridization events, 200 hybridization events, 300 hybridization events, 400 hybridization events, 500 hybridization events, 600 hybridization events, and 700 hybridization events. Base mismatch, 1 base mismatch in 800 hybridization events, 1 base mismatch in 900 hybridization events, 1 base mismatch in 1,000 hybridization events, 1 base mismatch in 2,000 hybridization events, 1 base mismatch in 3,000 hybridization events, 1 base mismatch in 4,000 hybridization events, 1 base mismatch in 5,000 hybridization events, 1 base mismatch in 6,000 hybridization events, 1 base mismatch in 7,000 hybridization events, 1 base mismatch in 8,000 hybridization events, 1 base mismatch in 9,000 hybridization events, or 1 base mismatch in 10,000 hybridization events.

[0151] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized buffer formulations, can produce improved hybridization efficiency (e.g., the fraction of available oligonucleotide primers that successfully hybridize with the target oligonucleotide sequence on the vector surface) compared to conventional hybridization protocols. In some cases, hybridization efficiencies better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% can be achieved for any input target oligonucleotide concentration specified below and any hybridization reaction time specified above. In some cases, for example where the hybridization efficiency is less than 100%, the resulting surface density of the target nucleic acid sequence hybridizing with the vector surface may be less than the surface density of the oligonucleotide adaptor or primer sequences on the surface.

[0152] In some cases, using the disclosed low-nonspecific binding vector for nucleic acid hybridization (or amplification) applications using conventional or optimized hybridization (or amplification) protocols can reduce the required input concentration of the target (or sample) nucleic acid molecules in contact with the vector surface. For example, in some cases, the target (or sample) nucleic acid molecules can be contacted with the vector surface at concentrations ranging from about 10 pM to about 1 μM (i.e., prior to annealing or amplification). In some cases, the target (or sample) nucleic acid molecules may be administered at the following concentrations: at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, or at least 1 μM. In some cases, the target (or sample) nucleic acid molecules can be administered at the following concentrations: up to 1 μM, up to 900 nM, up to 800 nM, up to 700 nM, up to 600 nM, up to 500 nM, up to 400 nM, up to 300 nM, up to 200 nM, up to 100 nM, up to 90 nM, up to 80 nM, up to 70 nM, up to 60 nM, up to 50 nM, up to 40 nM, up to 30 nM. The maximum values ​​are 20 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, target (or sample) nucleic acid molecules can be administered at concentrations ranging from about 90 pM to about 200 nM. Those skilled in the art will recognize that target (or sample) nucleic acid molecules can be administered at concentrations having any value within this range, such as about 855 nM.

[0153] In another example, the volume of biological sample that can contact the surface can be reduced relative to comparable biological samples analyzed using standard hybridization reagents on comparable surfaces. In some embodiments, the fluid sample containing the target (or sample) nucleic acid molecule can be in the range of about 5 μl to about 900 μl. In some cases, the sample volume range is about 5 μl to about 800 μl. In some cases, the sample volume range is about 5 μl to about 700 μl. In some cases, the sample volume range is about 5 μl to about 600 μl. In some cases, the sample volume range is about 5 μl to about 500 μl. In some cases, the sample volume range is about 5 μl to about 400 μl. In some cases, the sample volume range is about 5 μl to about 300 μl. In some cases, the sample volume range is about 5 μl to about 200 μl. In some cases, the sample volume range is about 5 μl to about 150 μl. In some cases, the sample volume range is about 5 μl to about 100 μl. In some cases, the sample volume ranges from about 5 μl to about 90 μl. In some cases, the sample volume ranges from about 5 μl to about 85 μl. In some cases, the sample volume ranges from about 5 μl to about 80 μl. In some cases, the sample volume ranges from about 5 μl to about 75 μl. In some cases, the sample volume ranges from about 5 μl to about 70 μl. In some cases, the sample volume ranges from about 5 μl to about 65 μl. In some cases, the sample volume ranges from about 5 μl to about 60 μl. In some cases, the sample volume ranges from about 5 μl to about 55 μl. In some cases, the sample volume ranges from about 5 μl to about 50 μl. In some cases, the sample volume ranges from about 15 μl to about 150 μl. In some cases, the sample volume ranges from about 15 μl to about 120 μl. In some cases, the sample volume ranges from 15 μl to about 100 μl. In some cases, the sample volume ranges from about 15 μl to about 90 μl. In some cases, the sample volume ranges from about 15 μl to about 85 μl. In some cases, the sample volume ranges from about 15 μl to about 80 μl. In some cases, the sample volume ranges from about 15 μl to about 75 μl. In some cases, the sample volume ranges from about 15 μl to about 70 μl. In some cases, the sample volume ranges from about 15 μl to about 65 μl. In some cases, the sample volume ranges from about 15 μl to about 60 μl. In some cases, the sample volume ranges from about 15 μl to about 55 μl. In some cases, the sample volume ranges from about 15 μl to about 50 μl.

[0154] In some cases, the surface density (i.e., before any subsequent solid-phase or clonal amplification reaction) of the disclosed low-nonspecific binding vector, used alone or in combination with optimized buffer formulations, that results in hybridization is in the range of approximately 0.0001 target oligonucleotide molecules / μm. 2 To approximately 1,000,000 target oligonucleotide molecules / μm 2 In some cases, the surface density of the target oligonucleotide molecule for hybridization can be at least 0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, or at least 600. At least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 2 00,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules / μm 2In some cases, the surface density of the target oligonucleotide molecules for hybridization can be up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000. 000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 2 5,000, up to 20,000, up to 15,000, up to 10,000, up to 9,500, up to 9,000, up to 8,500, up to 8,000, up to 7,500, up to 7,000, up to 6,500, up to 6,000, up to 5,500, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000 0, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, up to 10, up to 5, up to 1, up to 0.5, up to 0.1, up to 0.05, up to 0.01, up to 0.005, up to 0.001, up to 0.0005 or up to 0.0001 molecules / μm 2 Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the surface density of the target oligonucleotide molecules for hybridization may be around 3,000 molecules / μm. 2 Approximately 20,000 molecules / μm 2 Within this range. Those skilled in the art will recognize that the surface density of the target oligonucleotide molecules for hybridization can have any value within this range, for example, about 2,700 molecules / μm. 2 .

[0155] In other words, in some cases, the disclosed low-nonspecific binding vector, used alone or in combination with an optimized hybridization buffer formulation, can result in a surface density (i.e., prior to any subsequent solid-phase or clonal amplification reaction) of target (or sample) oligonucleotide molecules in the range of 100 hybridization target oligonucleotide molecules / mm². 2 Up to 1×10 7 oligonucleotide molecules / mm 2 Or approximately 100 target oligonucleotide molecules for hybridization per mm 2 To approximately 1×10 12 Target oligonucleotide molecules of hybridization / mm 2 In some cases, the surface density of the target oligonucleotide molecules for hybridization can be at least 100, at least 500, at least 1,000, at least 4,000, at least 5,000, at least 6,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000. 0, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 At least 5×10 7 At least 1×10 8 At least 5×10 8 At least 1×10 9 At least 5×10 9 At least 1×10 10 At least 5×10 10 At least 1×10 11 At least 5×10 11 Or at least 1×10 12 molecule / mm 2 In some cases, the surface density of the target oligonucleotide molecule for hybridization can be at most 1 × 10⁻⁶. 12 At most 5×10 11 At most 1×10 11At most 5×10 10 At most 1×10 10 At most 5×10 9 At most 1×10 9 At most 5×10 8 At most 1×10 8 At most 5×10 7 At most 1×10 7 Maximum 5,000,000, maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350,000, maximum 300,000, maximum 250,000, maximum 200,000, maximum 150,000, maximum 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 molecules / mm 2 Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the surface density of the target oligonucleotide molecules for hybridization may be around 5,000 molecules / mm². 2 Approximately 50,000 molecules / mm 2 Within this range. Those skilled in the art will recognize that the surface density of the target oligonucleotide molecules for hybridization can have any value within this range, for example, approximately 50,700 molecules / mm². 2 .

[0156] In some cases, the length of the target (or sample) oligonucleotide molecule (or nucleic acid molecule) that hybridizes with the oligonucleotide adapter or primer molecule attached to the surface of a low-binding carrier can range from about 0.02 kb to about 20 kb or from about 0.1 kb to about 20 kb. In some cases, the length of the target oligonucleotide molecule may be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, or at least 40 kb, or any intermediate value across the range described herein, such as a length of at least 0.85 kb.

[0157] In some cases, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded multimeric nucleic acid molecule, which further comprises repeats of regularly occurring monomeric units. In some cases, the length of the single-stranded or double-stranded multimeric nucleic acid molecule may be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb, at least 30 kb, or at least 40 kb, or any intermediate value across the range described herein, for example, a length of about 2.45 kb.

[0158] In some cases, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) may include a single-stranded or double-stranded multimeric nucleic acid molecule containing about 2 to about 100 copies of regularly repeating monomeric units. In some cases, the copy number of the regularly repeating monomeric units may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100. In some cases, the copy number of the regularly repeating monomeric unit can be up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, up to 5, up to 4, up to 3, or up to 2. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the copy number of the regularly repeating monomeric unit can be in the range of about 4 to about 60. Those skilled in the art will recognize that the copy number of the regularly repeating monomeric unit can have any value within this range, such as about 17. Therefore, in some cases, even if the hybridization efficiency is less than 100%, the surface density of the target sequence for hybridization can exceed the surface density of the oligonucleotide primers in terms of the copy number of the target sequence per unit area of ​​the vector surface.

[0159] As used herein, the phrase “nucleic acid surface amplification” (NASA) is used interchangeably with the phrase “solid-phase nucleic acid amplification” (or simply “solid-phase amplification”). In some aspects of this disclosure, nucleic acid amplification formulations, when combined with the disclosed low-binding vectors, provide improved amplification rates, amplification specificity, and amplification efficiency. As used herein, specific amplification refers to the amplification of template library oligonucleotide chains that are covalently or non-covalently tethered to a solid vector. As used herein, non-specific amplification is the amplification of guide dimers or other non-template nucleic acids. As used herein, amplification efficiency is a measure of the percentage of tethered oligonucleotides successfully amplified on the vector surface during a given amplification cycle or amplification reaction. Nucleic acid amplification performed on the surfaces disclosed herein can achieve amplification efficiencies of at least 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95% (e.g., 98% or 99%).

[0160] Any of the various thermal cycling or isothermal nucleic acid amplification protocols can be used with the disclosed low-binding vector. Examples of nucleic acid amplification methods that can be used with the disclosed low-nonspecific binding vector include, but are not limited to, polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridging amplification, isothermal bridging amplification, rolling circle amplification, loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification, or single-strand-binding (SSB) protein-dependent amplification.

[0161] In some embodiments, the rolling circle amplification reaction includes: (1) forming a capture nucleotide-polymerase complex by contacting a plurality of immobilized covalently closed circular nucleic acid molecules with: (i) a first plurality of polymerases having chain displacement activity; (ii) a plurality of nucleotides (e.g., a type of nucleotide or a mixture of dATP, dGTP, dCTP, and dTTP); (iii) a non-catalytic divalent cation (e.g., strontium or barium) that mediates nucleotide binding but not nucleotide incorporation; and optionally (iv) a plurality of amplification primers (if primers are lacking in the covalently closed circular molecules). The rolling circle amplification reaction also includes: (4) performing a nucleotide polymerization reaction by contacting the capture nucleotide-polymerase complex with: (i) at least one divalent cation (e.g., magnesium and / or manganese) that mediates both nucleotide binding and nucleotide incorporation and (ii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP) under conditions suitable for performing isothermal rolling circle amplification to produce a plurality of immobilized multiplyes: (i) at least one divalent cation (e.g., magnesium and / or manganese) that mediates both nucleotide binding and nucleotide incorporation and (ii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP).

[0162] In some embodiments, the rolling circle amplification reaction further comprises multiple compacting oligonucleotides that can hybridize with portions of the multiplex to collapse the multiplex into a more compacted shape and size. A compacting oligonucleotide is a single-stranded nucleic acid molecule having two identical sequences separated by short linker sequences, wherein the two identical sequences are anticomplementary to a portion of the multiplex. The compacting oligonucleotide can be of any length, for example, 20-100 nucleotides. The two identical sequence regions hybridize with the multiplex to pull the distal portions of the multiplex together, thereby compacting the multiplex. In some embodiments, the compacting oligonucleotide is resistant to 3' exonuclease degradation and / or single-stranded endonuclease degradation. In some embodiments, the compacting oligonucleotide comprises any combination of any two or more of the following: 3' phosphorylation; at least two 3' nucleotides having a phosphate thioester bond between them; at least one 3' nucleotide having a 2'-O-methyl moiety; and / or at least one 3' nucleotide having a 2' fluorobase.

[0163] In some embodiments, in the capture-nucleotide polymerase mixture of step (c), the first plurality of polymerases having strand substitution activity include phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase and Bca(exo-) DNA polymerase, a Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0164] In some embodiments, the primers used in amplification comprise single-stranded nucleic acid primers of about 5-25 nucleotides in length. In some embodiments, the amplification primers are resistant to 3' exonuclease degradation and / or single-stranded endonuclease degradation. In some embodiments, the amplification primers comprise any one or any combination of two or more of the following: 3' phosphorylation; at least two 3' nucleotides having a phosphate thioester bond between them; at least one 3' nucleotide having a 2'-O-methyl moiety; and / or at least one 3' nucleotide having a 2' fluorobase.

[0165] In some embodiments, the rolling circle amplification reaction further includes at least one auxiliary protein or enzyme, including helicase, single-strand binding (SSB) protein or recombinase (e.g., T4 uvsX) and / or recombinase cofactor (e.g., T4 uvsY or T4gp32).

[0166] In some implementations, the isothermal rolling ring amplification reaction can be carried out at temperatures of approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

[0167] In some implementations, the multiply may contain at least 2, 10, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more copies of repeating units.

[0168] Following rolling circle amplification, a multiple substitution amplification reaction using random sequence primers can be performed. The multiple substitution amplification reaction comprises: (1) forming a multiple substitution amplification (MDA) reaction mixture by contacting a plurality of fixed polymers with: (i) a second plurality of polymerases having strand substitution activity, and (ii) a plurality of soluble amplification primers, wherein a single amplification primer among the plurality of soluble amplification primers is exonuclease resistant and has a 3' extendable end and contains a random sequence that can partially hybridize with a single-stranded circular nucleic acid template, (iii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP), and (iv) at least one divalent cation (e.g., magnesium and / or manganese) that mediates nucleotide binding and nucleotide incorporation; and (2) performing an isothermal multiple substitution amplification (MDA) reaction to generate a plurality of fixed branched polymers.

[0169] In some embodiments, the second plurality of polymerases having strand displacement activity in the multiple substitution amplification (MDA) reaction mixture includes phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, a Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or the variant EquiPhi29 DNA polymerase (e.g., from ThermoFisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0170] In some embodiments, in the multiple substitution amplification (MDA) reaction mixture, the plurality of amplification primers comprise single-stranded nucleic acid primers of about 5-25 nucleotides in length. In some embodiments, the plurality of soluble amplification primers comprise unprotected single-stranded nucleic acid primers. In some embodiments, the plurality of soluble amplification primers comprise protected single-stranded nucleic acid primers resistant to 3' exonuclease degradation and / or single-stranded endonuclease degradation. In some embodiments, the plurality of soluble amplification primers comprise any combination of any two or more of the following: 3' phosphorylation; at least two 3' nucleotides having a phosphate thioester bond between them; at least one 3' nucleotide having a 2'-O-methyl moiety; and / or at least one 3' nucleotide having a 2' fluorobase. In some embodiments, the plurality of soluble amplification primers comprise a group of primers having the same length (e.g., 6 or 9 nucleotides). In some embodiments, the plurality of soluble amplification primers comprise a group of primers comprising mixtures of different lengths (e.g., a mixture comprising 6-mer and 9-mer primers). In some embodiments, the plurality of soluble amplification primers comprises a mixture of primers having a random sequence comprising up to 4 6 Different sequences (e.g., for 6-mers) or 4 9 Different sequence sequences (e.g., for 9-mer).

[0171] In some embodiments, the multiple substitution amplification (MDA) reaction mixture may further contain at least one auxiliary protein or enzyme, including helicase, single-strand binding (SSB) protein or recombinase (e.g., T4 uvsX) and / or recombinase cofactor (e.g., T4 uvsY or T4 gp32).

[0172] In some implementations, the isothermal multiple substitution amplification (MDA) reaction can be carried out at temperatures of approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C.

[0173] Following rolling circle amplification, a multiple substitution amplification reaction using a primase-polymerase can be performed. The multiple substitution amplification reaction comprises: (1) forming a multiple substitution amplification (MDA) reaction mixture by contacting a plurality of immobilized polymers with: (i) a second plurality of polymerases having strand substitution activity, (ii) a plurality of DNA primase-polymerases, (iii) a second plurality of nucleotides (e.g., a mixture of dATP, dGTP, dCTP, and dTTP), and (iv) at least one divalent cation (e.g., magnesium and / or manganese) that mediates nucleotide binding and nucleotide incorporation, and (2) performing an isothermal multiple substitution amplification (MDA) reaction to generate a plurality of immobilized branched polymers. In some embodiments, the multiple substitution amplification reaction is performed without the addition of amplification primers (e.g., primer-free reaction).

[0174] In some embodiments, the second plurality of polymerases having strand displacement activity in the multiple substitution amplification (MDA) reaction mixture includes phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, a Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or the variant EquiPhi29 DNA polymerase (e.g., from ThermoFisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0175] In some implementations, the multiple DNA primase-polymerases include enzymes from Thermus thermophilus HB27 (e.g., Tth PrimPol enzyme).

[0176] In some embodiments, the multiple substitution amplification (MDA) reaction mixture further comprises at least one auxiliary protein or enzyme, including helicase, single-strand binding (SSB) protein or recombinase (e.g., T4 uvsX) and / or recombinase cofactor (e.g., T4 uvsY or T4 gp32).

[0177] In some implementations, the isothermal multiple substitution amplification (MDA) reaction can be carried out at temperatures of approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C.

[0178] Another implementation of the two-stage amplification method involves exposing the polymers to a nucleic acid relaxant (first stage) followed by a bending amplification reaction during the second stage. Without being bound by theory, it is assumed that one or more nucleic acid relaxants can disrupt (e.g., denature) the hydrogen bonds in multiple immobilized nucleic acid polymers. This results in structural relaxation of the nucleic acid polymers and increases the number of new duplexes formed between the immobilized surface-capture primers and portions of the nucleic acid polymers, thereby increasing the chance of generating new polymers from the immobilized surface-capture primers. New polymers can be generated during the bending amplification reaction. Including relaxants can induce nucleic acid denaturation without the need for denaturing temperatures or denaturing chemicals.

[0179] In some embodiments, the amplification method includes: (1) performing rolling circle amplification on a vector to generate multiple single-stranded polymers, (2) forming a relaxation reaction mixture, (3) forming a bending amplification reaction mixture, (4) performing bending amplification on a vector (e.g., without the addition of soluble primers) to generate multiple double-stranded polymers, (5) washing, and (6) repeating steps (2)-(5) at least once.

[0180] In some embodiments, the relaxation reaction mixture of step (2) can be formed using at least one nucleic acid relaxant capable of disrupting hydrogen bonds in immobilized nucleic acid polysyntheses. Exemplary relaxants include nucleic acid denaturants, liquid-dissociating compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Liquid-dissociating compounds include urea, guanidine hydrochloride, or guanidine thiocyanate. Amide compounds include formamide, acetamide, or N,N-dimethylformamide (DMF). Aprotic compounds include acetonitrile, DMSO (dimethyl sulfoxide), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, or 2-methoxyethanol. Other relaxants include sodium iodide, potassium iodide, and polyamines.

[0181] In some embodiments, the relaxation reaction mixture comprises any one or a combination of two or more of the following: urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, N,N-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, sodium iodide, potassium iodide, and / or polyamines.

[0182] In some embodiments, the relaxation reaction mixture comprises formamide and SSC. In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, and SSC. In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, and MES (2-(4-morpholino)-ethanesulfonic acid). In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, guanidine hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). In some embodiments, the relaxation reaction mixture comprises acetonitrile, formamide, urea, and HEPES. In some embodiments, the SSC in the relaxation reaction mixture can be 1X, 2X, 3X, or 4X.

[0183] In some embodiments, during the formation of the relaxation reaction mixture in step (2), the temperature ramping condition can be applied from about 20°C to about 70°C, the relaxation incubation condition can be applied from about 40-70°C, and the temperature descent condition can be applied from about 70°C to about 20°C. Those skilled in the art will recognize that the temperature ramping condition, the relaxation incubation temperature, and the temperature descent condition can be modified.

[0184] In some embodiments, in the bent amplification reaction mixture of step (3), the second plurality of polymerases having strand displacement activity include a large fragment of Bst DNA polymerase (e.g., exonuclease-negative (minus)), phi29 DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, a Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0185] In some embodiments, the concentration (e.g., total concentration) of the third plurality of nucleotides in the bend amplification reaction mixture of step (2) can promote the nucleotide polymerization reaction. For example, the concentration (e.g., total concentration) of the third plurality of nucleotides is about 0.1-10 mM.

[0186] In some embodiments, the third plurality of nucleotides in the bending amplification reaction mixture of step (2) includes a mixture of two or more nucleotides selected from dATP, dGTP, dCTP and dTTP.

[0187] In some embodiments, in the bending amplification reaction mixture of step (2), at least one divalent cation mediating nucleotide binding and polymerization includes a catalytic divalent cation. In some embodiments, the catalytic divalent cation includes magnesium and / or manganese. The concentration of the catalytic divalent cation in the amplification reaction mixture may be about 1-20 mM.

[0188] In some embodiments, the bending amplification reaction mixture in step (2) may contain at least one accessory protein or enzyme, including helicase, single-strand binding (SSB) protein, or recombinase (e.g., T4 uvsX) and / or recombinase cofactor (e.g., T4 uvsY or T4 gp32). In some embodiments, these accessory proteins may be omitted.

[0189] In some embodiments, the temperature ramping conditions in the bending amplification reaction of step (4) can be from about 20°C to about 90°C. In some embodiments, the temperature ramping conditions in the bending amplification reaction of step (4) can be for about 5-15 seconds, or about 15-30 seconds, or about 30-45 seconds, or about 45-60 seconds, or longer. In some embodiments, the amplification incubation conditions in the bending amplification reaction of step (4) can be about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C or higher. In some embodiments, the amplification incubation conditions in the bending amplification reaction of step (4) can be for about 30-45 seconds, or about 45-60 seconds, or about 60-75 seconds, or about 75-90 seconds, or longer. In some implementations, the temperature gradient conditions in the bending amplification reaction of step (4) can be applied from about 90°C to about 20°C.

[0190] In some embodiments, during the bend amplification reaction in step (4), the temperature descent conditions can be performed for approximately 5-15 seconds, or approximately 15-30 seconds, or approximately 30-45 seconds, or approximately 45-60 seconds, or longer. In some embodiments, during the washing in step (5), the washing buffer comprises 1xSSC, or 1xSSC containing cobalt hexamine. In some embodiments, steps (2)-(5) can be repeated at least once, or up to 10 times, or up to 15 times, or up to 20 times, or up to 30 times or more.

[0191] Typically, improvements in amplification rate, amplification specificity, and amplification efficiency can be achieved by using the disclosed low-nonspecificity binding vector alone or in combination with formulations of amplification reaction components. In addition to containing nucleotides, one or more polymerases, helicases, single-stranded binding proteins, etc. (or any combination thereof), the amplification reaction mixture can be modified in various ways to achieve improved performance, including but not limited to the selection of buffer type, buffer pH, organic solvent mixture, buffer viscosity, detergents and zwitterionic components, ionic strength (including adjustment of monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, and other additives.

[0192] Compared to amplification rates obtained using conventional vectors and amplification protocols, the disclosed low-nonspecific binding vectors, used alone or in combination with optimized amplification reaction formulations, can produce increased amplification rates. In some cases, for any of the amplification methods described above, the achievable relative amplification rate can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times that of conventional vectors and amplification protocols.

[0193] In some cases, the disclosed low nonspecific binding vector, used alone or in combination with optimized buffer formulations, can produce amplification reaction times of less than 180 minutes, 120 minutes, 90 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, or 10 seconds (i.e., the time required for the amplification reaction to complete 90%, 95%, 98%, or 99%) for any of these completion metrics.

[0194] Some of the low-binding carriers disclosed herein exhibit specific to non-specific binding ratios of fluorophores (e.g., Cy3) at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value across the scope of this document. The ratio of specific fluorescence signal to non-specific fluorescence signal of some surfaces exhibiting fluorophores (e.g., Cy3) disclosed herein is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1 or greater than 100:1 or any intermediate value across the scope of this document.

[0195] In some cases, the disclosed low nonspecific binding vector, used alone or in combination with optimized amplification buffer formulations, can achieve faster amplification reaction times of no more than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes (i.e., the time required for the amplification reaction to complete 90%, 95%, 98%, or 99%). Similarly, the disclosed low nonspecific binding vector, used alone or in combination with optimized buffer formulations, can in some cases enable the amplification reaction to be completed in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or no more than 30 cycles.

[0196] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized amplification reaction formulations, may produce increased specific amplification and / or reduced nonspecific amplification compared to that obtained using conventional vectors and amplification protocols. In some cases, the achievable specific amplification to nonspecific amplification ratio is at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or 1,000:1.

[0197] In some cases, low-nonspecific binding vectors, used alone or in combination with optimized amplification reaction formulations, can produce increased amplification efficiency compared to those obtained using conventional vectors and amplification protocols. In some cases, amplification efficiencies better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% can be achieved at any of the amplification reaction times specified above.

[0198] In some cases, the length of the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) that hybridizes with the oligonucleotide adapter or primer molecule attached to the surface of the low-binding vector can range from about 0.02 kb to about 20 kb or from about 0.1 kb to about 20 kb. In some cases, the length of the cloned and amplified target oligonucleotide molecule may be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb, or any intermediate value across the range described herein, for example, a length of at least 0.85 kb.

[0199] In some cases, the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may include a single-stranded or double-stranded multimeric nucleic acid molecule, which also includes repeats of regularly occurring monomeric units. In some cases, the length of the cloned and amplified single-stranded or double-stranded multimeric nucleic acid molecule may be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb, or any intermediate value across the range described herein, such as a length of about 2.45 kb.

[0200] In some cases, the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may include a single-stranded or double-stranded multimeric nucleic acid molecule containing about 2 to about 100 copies of regularly repeating monomeric units. In some cases, the copy number of the regularly repeating monomeric units may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100. In some cases, the copy number of the regularly repeating monomeric unit can be up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, up to 5, up to 4, up to 3, or up to 2. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the copy number of the regularly repeating monomeric unit can be in the range of about 4 to about 60. Those skilled in the art will recognize that the copy number of the regularly repeating monomeric unit can have any value within this range, such as about 12. Therefore, in some cases, the surface density of the cloned and amplified target sequence can exceed the surface density of the oligonucleotide primers, even if the hybridization and / or amplification efficiency is less than 100%, in terms of the copy number of the target sequence per unit area of ​​the vector surface.

[0201] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized amplification reaction formulations, can produce increased clonal copy numbers compared to those obtained using conventional vectors and amplification protocols. In some cases, such as when the target (or sample) oligonucleotide molecule amplified by cloning contains a tandem multimeric repeat of a monomeric target sequence, the clonal copy number can be significantly lower than that obtained using conventional vectors and amplification protocols. Therefore, in some cases, the clonal copy number can be from about 1 molecule to about 100,000 molecules (e.g., target sequence molecules) per amplified community. In some cases, the clonal copy number can be at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 15,000, or at least 20,000 per amplified community. 0, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, or at least 100,000 molecules. In some cases, the clone copy number can be up to 100,000, 95,000, 90,000, 85,000, 80,000, 75,000, 70,000, 65,000, 60,000, 55,000, 50,000, 45,000, 40,000, or 35,000 per amplified community. The maximum number of clone copies may be 30,000, 25,000, 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 500, 100, 50, 10, 5, or 1 molecule. Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the clone copy number may be in the range of about 2,000 molecules to about 9,000 molecules. Those skilled in the art will recognize that the clone copy number may have any value within this range, such as about 2,220 molecules in some cases and about 2 molecules in others.

[0202] As described above, in some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain tandemly repeated monomeric target sequences. In some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain multiple molecules, each containing a single monomeric target sequence. Therefore, the disclosed low nonspecific binding vector, used alone or in combination with optimized amplification reaction formulations, can result in a surface density of target sequence copies ranging from approximately 100 target sequence copies / mm². 2 To approximately 1×10 12 Target sequence copies / mm 2 In some cases, the surface density of target sequence copies can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000. At least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 At least 5×10 7 At least 1×10 8 At least 5×10 8 At least 1×10 9 At least 5×10 9 At least 1×10 10 At least 5×10 10 At least 1×10 11 At least 5×10 11 Or at least 1×10 12 1 cloning and amplified target sequence molecule / mm 2 In some cases, the surface density of target sequence copies can be at most 1 × 10⁻⁶. 12 At most 5×10 11 At most 1×10 11 At most 5×10 10 At most 1×1010 At most 5×10 9 At most 1×10 9 At most 5×10 8 At most 1×10 8 At most 5×10 7 At most 1×10 7 Maximum 5,000,000, maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350,000, maximum 300,000, maximum 250,000, maximum 200,000, maximum 150,000, maximum 100, 000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 target sequence copies / mm 2 Any lower and upper limits described in this paragraph may be combined to form ranges included in this disclosure; for example, in some cases, the surface density of target sequence copies may be approximately 1,000 target sequence copies / mm². 2 Approximately 65,000 target sequence copies / mm 2 Within this range. Those skilled in the art will recognize that the surface density of target sequence copies can have any value within this range, for example, approximately 49,600 target sequence copies / mm². 2 .

[0203] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized amplification buffer formulations, can result in a surface density of approximately 100 molecules / mm² of target (or sample) oligonucleotide molecules (or clusters) amplified through cloning. 2 To approximately 1×10 12 Individual settlements / mm 2In some cases, the surface density of the cloned and amplified molecule can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000. 0, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 At least 5×10 7 At least 1×10 8 At least 5×10 8 At least 1×10 9 At least 5×10 9 At least 1×10 10 At least 5×10 10 At least 1×10 11 At least 5×10 11 Or at least 1×10 12 molecule / mm 2 In some cases, the surface density of cloned and amplified molecules can be as high as 1 × 10⁻⁶. 12 At most 5×10 11 At most 1×10 11 At most 5×10 10 At most 1×10 10 At most 5×10 9 At most 1×10 9 At most 5×10 8 At most 1×10 8 At most 5×10 7 At most 1×10 7Maximum 5,000,000, maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350,000, maximum 300,000, maximum 250,000, maximum 200,000, maximum 150,000, maximum 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 molecules / mm 2 Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the surface density range of cloned and amplified molecules may be approximately 5,000 molecules / mm². 2 Approximately 50,000 molecules / mm 2 Those skilled in the art will recognize that the surface density of a cloned and amplified community can have any value within this range, for example, approximately 48,800 molecules / mm². 2 .

[0204] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized amplification buffer formulations, can result in a surface density of approximately 100 molecules / mm² of target (or sample) oligonucleotide molecules (or clusters) amplified through cloning. 2 To approximately 1×10 12 Individual settlements / mm 2In some cases, the surface density of the cloned and amplified molecule may be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000. 0, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 At least 5×10 7 At least 1×10 8 At least 5×10 8 At least 1×10 9 At least 5×10 9 At least 1×10 10 At least 5×10 10 At least 1×10 11 At least 5×10 11 Or at least 1×10 12 molecule / mm 2 In some cases, the surface density of cloned and amplified molecules can be at most 1 × 10⁻⁶. 12 At most 5×10 11 At most 1×10 11 At most 5×10 10 At most 1×10 10 At most 5×10 9 At most 1×10 9 At most 5×10 8 At most 1×10 8 At most 5×10 7 At most 1×10 7Maximum 5,000,000, maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350,000, maximum 300,000, maximum 250,000, maximum 200,000, maximum 150,000, maximum 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 molecules / mm 2 Any lower and upper limits described in this paragraph may be combined to form the scope included in this disclosure; for example, in some cases, the surface density of cloned and amplified molecules may be around 5,000 molecules / mm². 2 Approximately 50,000 molecules / mm 2 Within this range. Those skilled in the art will recognize that the surface density of cloned and amplified molecules can have any value within this range, for example, approximately 48,800 molecules / mm². 2 .

[0205] In some cases, the disclosed low-nonspecific binding vector, used alone or in combination with optimized amplification buffer formulations, can result in surface densities of approximately 100 colonies / mm² of target (or sample) oligonucleotide colonies (or clusters) amplified by cloning. 2 To approximately 1×10 12 Individual settlements / mm 2In some cases, the surface density of the cloned amplified colonies can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000. 0, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 At least 5×10 7 At least 1×10 8 At least 5×10 8 At least 1×10 9 At least 5×10 9 At least 1×10 10 At least 5×10 10 At least 1×10 11 At least 5×10 11 Or at least 1×10 12 Individual settlements / mm 2 In some cases, the surface density of cloned amplified colonies can be at most 1 × 10⁻⁶. 12 At most 5×10 11 At most 1×10 11 At most 5×10 10 At most 1×10 10 At most 5×10 9 At most 1×10 9 At most 5×10 8 At most 1×10 8 At most 5×10 7 At most 1×10 7Maximum 5,000,000, maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350,000, maximum 300,000, maximum 250,000, maximum 200,000, maximum 150,000, maximum 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 settlements / mm 2 Any lower and upper limits described in this paragraph may be combined to form the range included in this disclosure; for example, in some cases, the surface density of cloned amplified colonies may be approximately 5,000 colonies / mm². 2 Approximately 50,000 clusters / mm 2 Within this range. Those skilled in the art will recognize that the surface density of cloned amplified colonies can have any value within this range, for example, approximately 48,800 colonies / mm². 2 .

[0206] In some cases, the disclosed low nonspecific binding vector, used alone or in combination with optimized amplification reaction formulations, can generate signals (e.g., fluorescence signals) from amplified and labeled nucleic acid populations with a variance coefficient not greater than 50%, such as 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less than 5%.

[0207] In some cases, the vector surfaces and methods disclosed herein allow amplification at elevated extension temperatures, such as 15°C, 20°C, 25°C, 30°C, 40°C or higher, or, for example, at about 21°C or 23°C.

[0208] In some cases, the vector surfaces and methods disclosed herein can simplify the amplification reaction. For example, in some cases, no more than 1, 2, 3, 4, or 5 discrete reagents can be used for the amplification reaction.

[0209] In some cases, the carrier surface and methods disclosed herein can be used to employ simplified temperature profiles during amplification, allowing the reaction to be carried out in a range from low temperatures of 15°C, 20°C, 25°C, 30°C, or 40°C to high temperatures of 40°C, 45°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, or above 80°C, for example, in the range of 20°C to 65°C.

[0210] The amplification reaction has also been improved, making it possible to generate a discernible signal on the surface with a smaller amount of template (e.g., target molecule or sample molecule), such as 1 pM, 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1,000 pM, 2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM, 9,000 pM, 10,000 pM, or samples greater than 10,000 pM, such as 500 nM. In an exemplary implementation, an input of approximately 100 pM is sufficient to generate a signal for reliable signal determination.

[0211] The disclosed solid-phase nucleic acid amplification reaction formulations and low-nonspecific binding vectors can be used in any of a variety of nucleic acid analysis applications, such as nucleic acid base identification, nucleic acid base classification, nucleic acid base determination, nucleic acid detection applications, nucleic acid sequencing applications, and nucleic acid-based (genetic and genomic) diagnostic applications. In many of these applications, fluorescence imaging techniques can be used to monitor hybridization, amplification, and / or sequencing reactions performed on the low-binding vectors.

[0212] Fluorescence imaging can be performed using any of a variety of fluorophores, fluorescence imaging techniques, and fluorescence imaging instruments known to those skilled in the art. Examples of suitable fluorescent dyes that can be used (e.g., by conjugation with nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, anthocyanins, and their derivatives, including anthocyanin derivatives such as anthocyanin dye-3 (Cy3), anthocyanin dye-5 (Cy5), and anthocyanin dye-7 (Cy7). Examples of fluorescence imaging techniques that can be used include, but are not limited to, fluorescence microscopy imaging, fluorescence confocal imaging, and two-photon fluorescence. Examples of fluorescence imaging instruments that can be used include, but are not limited to, fluorescence microscopes equipped with image sensors or cameras, confocal fluorescence microscopes, two-photon fluorescence microscopes, or custom instruments that include appropriately selected light sources, lenses, mirrors, prisms, dichroic reflectors, apertures, and image sensors or cameras. A non-limiting example of a fluorescence microscope equipped with images of the disclosed low-binding vector surface and cloned amplified colonies (or clusters) of target nucleic acid sequences hybridized thereon is the Olympus IX83 inverted fluorescence microscope, which is equipped with a 20x, 0.75NA, 532nm light source, a bandpass and dichroic mirror filter set (optimized for 532nm long-pass excitation) and a Cy3 fluorescence emission filter, a Semrock 532nm dichroic mirror, and a camera (Andors CMOS, Zyla 4.2), wherein the excitation light intensity is adjusted to avoid signal saturation. Typically, during image acquisition, the vector surface can be immersed in a buffer solution (e.g., 25mM ACES, pH 7.4 buffer).

[0213] In some cases, fluorescence imaging techniques can be used to evaluate nucleic acid hybridization and / or amplification reactions using disclosed reaction formulations and low-nonspecific binding vectors, where the contrast-to-noise ratio (CNR) of the image provides a key metric for assessing the specificity and nonspecific binding of the amplification on the vector. CNR is typically defined as: CNR = (Signal - Background) / Noise. The background term is generally considered to be the signal measured against the gap region surrounding a specific feature (diffraction-limited spot, DLS) within a designated region of interest (ROI). While signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it can be shown that an improved CNR relative to SNR can provide a significant advantage as a benchmark for signal quality in applications requiring rapid image capture (e.g., sequencing applications where cycle counts must be minimized), as illustrated in the following examples. The surface of this disclosure is also provided in co-examined International Application Serial No. PCT / US2019 / 061556 (which is incorporated herein by reference in its entirety).

[0214] In most ensemble-based sequencing methods, background terms are typically measured as signals associated with the "gap" region. Besides the "gap" background (B... interIn addition to the "intracellular" background (B) intra These two background signals exist within the regions occupied by amplified DNA colonies. The combination of these two background signals determines the achievable CNR, which in turn directly affects optical instrumentation requirements, architecture costs, reagent costs, runtime, cost / genome, and ultimately the accuracy and data quality of cyclic array-based sequencing applications. inter Background signals arise from a variety of sources; some examples include autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules (which produces spurious fluorescence signals that may mask signals from the ROI), and the presence of nonspecific DNA amplification products (e.g., those produced by primer dimers). In typical next-generation sequencing (NGS) applications, the background signal in the current field of view (FOV) is averaged and subtracted over time. The signal from a single DNA community (i.e., (S)-B in the FOV) is then removed. inter This produces classifiable and identifiable features. In some cases, intracellular background (B) intra This may produce mixed fluorescence signals that are not specific to the target but exist in the same ROI, making them more difficult to average and subtract.

[0215] As will be shown in the following examples, performing nucleic acid amplification on the low-binding substrate of this disclosure can reduce B by decreasing non-specific binding. inter Background signal enhancement can improve specific nucleic acid amplification and lead to a reduction in nonspecific amplification, which affects background signal from intercellular and intracellular regions. In some cases, the disclosed low-binding vector surface, optionally combined with the disclosed hybridization and / or amplification reaction formulations, can result in a CNR increase of 2, 5, 10, 100, or 1000-fold compared to those obtained using conventional vectors and hybridization, amplification, and / or sequencing protocols. Although described here in the context of using fluorescence imaging as a readout or detection mode, the same principles apply to the use of the disclosed low-nonspecific binding vector and nucleic acid hybridization and amplification formulations in other detection modes, including optical and non-optical detection modes.

[0216] The disclosed low-binding vector, optionally used in combination with the disclosed hybridization and / or amplification protocols, produces a solid-phase reaction exhibiting: (i) negligible nonspecific binding of proteins and other reaction components (thus minimizing substrate background), (ii) negligible nonspecific nucleic acid amplification products, and (iii) providing an adjustable nucleic acid amplification reaction.

[0217] Methods for capturing and analyzing DNA. This disclosure provides methods for analyzing nucleic acids in a cellular or spatially addressable manner, the methods comprising: (a) providing a carrier comprising a low nonspecific binding coating, wherein a plurality of capturing oligonucleotides and a plurality of cyclic oligonucleotides are immobilized onto the low nonspecific binding coating (e.g., Figure 2 The plurality of capturing oligonucleotides comprises (i) a target capturing region that hybridizes to at least a portion of a target nucleic acid molecule, (ii) a universal sequence region comprising a spatial barcode sequence, (iii) a circular anchoring sequence, and (iv) a cleavable region, wherein the plurality of circular oligonucleotides comprises (i) a homopolymer region, (ii) a universal sequence region comprising a sequencing primer binding sequence and (iii) a circular anchoring binding sequence, and wherein the low nonspecific binding coating comprises at least one hydrophilic polymer coating having a water contact angle of no more than 45 degrees.

[0218] In some embodiments, the low nonspecific binding coating in step (a) exhibits a low background fluorescence signal or a high contrast-to-noise (CNR) ratio relative to surfaces known in the art. In some embodiments, the low nonspecific binding coating exhibits less than about 0.25 molecules / μm. 2 The nonspecific absorption level of the Cy3 dye is such that no more than 5% of the target nucleic acids associate with the surface coating without hybridizing with the immobilized capture oligonucleotides. In some embodiments, when the fluorescence imaging system is used under non-signal saturation conditions, the fluorescence image of the surface coating having multiple cloned and amplified nucleic acid clusters exhibits a contrast-to-noise ratio (CNR) of at least 20 or at least 50 or higher.

[0219] In some implementations, the immobilized capture oligonucleotide in step (a) may comprise any combination of the following: (i) a target capture region that hybridizes to at least a portion of the target nucleic acid molecule, (ii) a universal sequence region containing a spatial barcode sequence, (iii) a cyclized anchoring sequence that binds to a portion of the cyclized oligonucleotide, and / or (iv) a cleavable region.

[0220] In some implementations, the target capture region of the fixed capture oligonucleotide in step (a) contains a target-specific sequence or a random sequence.

[0221] In some implementations, the immobilized cyclic oligonucleotide in step (a) may comprise any combination of the following: (i) a homopolymer region, (ii) a universal sequence region containing a sequencing primer binding sequence, and / or (iii) a cyclic anchoring binding sequence that binds to the cyclic anchoring sequence of the captured oligonucleotide.

[0222] The method for analyzing nucleic acids further includes the following steps: (b) contacting a low-nonspecific binding coating with a cellular biological sample in the presence of a high-efficiency hybridization buffer under conditions suitable for promoting the migration of target nucleic acid molecules from a cellular biological sample to one of an immobilized capture oligonucleotide, thereby forming an immobilized target nucleic acid duplex, wherein the target nucleic acid molecule is immobilized to the low-nonspecific binding coating in a manner that preserves the spatial location information of the target nucleic acid molecule in the cellular biological sample, wherein the target nucleic acid includes DNA or RNA (e.g., Figure 7 ).

[0223] In some implementations, the cellular biological sample in step (b) includes fresh, frozen, fresh-frozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples.

[0224] In some embodiments, the cellular biological sample in step (b) is permeabilized to facilitate the migration of one of the immobilized capture oligonucleotides from the cellular biological sample, including the target nucleic acid molecule (e.g., DNA and / or RNA).

[0225] In some embodiments, the high-efficiency hybridization buffer of step (b) comprises: (i) a first polar aprotic solvent having a dielectric constant of not more than 40 and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of not more than 115 and present in the high-efficiency hybridization buffer formulation in an amount that effectively denatures double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the high-efficiency hybridization buffer formulation in the range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding.

[0226] In some embodiments, the high-efficiency hybridization buffer in step (b) comprises: (i) a first polar aprotic solvent comprising 25%-50% acetonitrile by volume of the high-efficiency hybridization buffer; (ii) a second polar aprotic solvent comprising 5%-10% formamide by volume of the high-efficiency hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a congesting agent comprising 5%-35% polyethylene glycol (PEG) by volume of the high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer also comprises betaine.

[0227] In some implementations, the high-efficiency hybridization buffer in step (b) promotes a high degree of stringency (e.g., specificity), speed, and power of the nucleic acid hybridization reaction, and improves the efficiency of subsequent amplification and sequencing steps. In some implementations, the high-efficiency hybridization buffer significantly reduces nucleic acid hybridization time and lowers sample input requirements. Nucleic acid annealing can be performed under isothermal conditions, eliminating the need for a cooling step for annealing.

[0228] The method for analyzing nucleic acids further includes the step of: (c) using a hybridized target nucleic acid molecule as a template to perform a primer extension reaction on a fixed nucleic acid duplex, thereby forming a fixed target extension product. In some embodiments, the primer extension reaction includes contacting the fixed nucleic acid duplex with multiple nucleotides and a polymerase. In some embodiments, the polymerase includes E. coli DNA polymerase I, a Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase.

[0229] In some embodiments, the primer extension reaction in step (c) can be a reverse transcription reaction, comprising (i) a reverse transcriptase, (ii) multiple nucleotides, and (iii) multiple reverse transcriptase primers. In some embodiments, the reverse transcription reaction in step (a) comprises multiple nucleotides and an enzyme with reverse transcription activity, including reverse transcriptases from AMV (Avian Myeloblastemia Virus), M-MLV (Moloni Mule Leukemia Virus), or HIV (Human Immunodeficiency Virus). In some embodiments, the reverse transcriptase can be a commercially available enzyme, including MultiScribe. TM ThermoScript TM or ArrayScript TM In some embodiments, the reverse transcriptase includes Superscript I, II, III, or IV enzymes. In some embodiments, the reverse transcription reaction may include an RNase inhibitor.

[0230] The method for analyzing nucleic acids further includes the following steps: (d) performing a non-template tailing reaction on the fixed target extension product under conditions suitable for attaching homopolymer tails to a fixed target extension product, thereby forming a fixed tailed target extension product (e.g., Figure 27 In some embodiments, the non-template tailing reaction includes contacting the immobilized target extension product with multiple nucleotides and a polymerase, wherein the polymerase is Taq polymerase, Tfi DNA polymerase, 3' exonuclease-negative large (Klenow) fragment, or 3' exonuclease-negative T4 polymerase.

[0231] The method for analyzing nucleic acids further includes the step of: (e) cleaving the immobilized tailed target extension product to release the immobilized tailed target extension product from the low-binding coating, thereby forming a soluble tailed target extension product. In some embodiments, the cleavable region may be cleaved using enzymes, compounds, light, or heat.

[0232] The method for analyzing nucleic acids further includes the following steps: (f) binding a soluble tailed target extension product to one of a fixed cyclic oligonucleotide under certain conditions, said conditions being suitable for hybridizing the additional homopolymer tail of the soluble tailed target extension product to the homopolymer region of the fixed cyclic oligonucleotide, and suitable for hybridizing the cyclic anchoring sequence of the soluble tailed target extension product to the cyclic anchoring binding sequence of the fixed cyclic oligonucleotide, thereby forming an open-ring target extension product with a nick and / or break, such that the fixed cyclic oligonucleotide acts as a splint molecule to facilitate the cyclization of the soluble tailed target extension product (e.g., Figure 27 ).

[0233] The method for analyzing nucleic acids further includes the following steps: (g) closing the gap (if present) by performing a gap-filling primer extension reaction and closing the break (if present) by performing a ligation reaction on the open-loop target extension product, thereby forming a covalently closed circular target extension product that hybridizes with a fixed cyclic oligonucleotide, wherein the fixed cyclic oligonucleotide comprises a homopolymer region having a 3' extensionable end (e.g., Figure 27 ).

[0234] In some embodiments, step (g) of forming a covalently closed circular target extension product includes a polymerase-mediated gap-filling reaction, an enzymatic ligation reaction, or a polymerase-mediated gap-filling reaction and an enzymatic ligation reaction. In some embodiments, the polymerase-mediated gap-filling reaction includes contacting the open-loop target molecule with a DNA polymerase and multiple nucleotides, wherein the DNA polymerase includes *E. coli* DNA polymerase I, a Klenow fragment of *E. coli* DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the enzymatic ligation reaction includes using a ligase, including T3, T4, T7, or Taq DNA ligase. In some embodiments, forming a covalently closed circular target molecule includes contacting the open-loop target molecule with a CircLigase or CircLigase II enzyme.

[0235] The method for analyzing nucleic acids further includes the following steps: (h) performing rolling circle amplification using the 3' extensionable end of the homopolymer region of a fixed cyclic oligonucleotide under conditions suitable for forming a fixed nucleic acid polymolecule having a tandem repeat region (the tandem repeat region comprising a sequencing primer binding sequence, a target sequence, and a spatial barcode sequence). Figure 27 ).

[0236] In some embodiments, the rolling circle amplification reaction in step (h) includes contacting a covalently closed circularized lock probe (e.g., one or more circularized nucleic acid template molecules) with amplification primers, DNA polymerase, multiple nucleotides, and at least one catalytic divalent cation under conditions suitable for generating at least one nucleic acid polyp, wherein the at least one catalytic divalent cation includes magnesium or manganese.

[0237] In some embodiments, the rolling circle amplification reaction in step (h) includes: (1) contacting a covalently closed circularized lock probe (e.g., one or more circularized nucleic acid template molecules) with amplification primers, DNA polymerase, multiple nucleotides, and at least one non-catalytic divalent cation (which does not promote polymerase-catalyzed incorporation of nucleotides into the amplification primers), wherein the non-catalytic divalent cation includes strontium or barium; and (2) contacting the covalently closed circularized lock probe with at least one catalytic divalent cation under conditions suitable for generating at least one nucleic acid polymer, wherein the at least one catalytic divalent cation includes magnesium or manganese.

[0238] In some embodiments, the rolling ring amplification reaction in step (h) is carried out at a constant temperature (e.g., isothermal) of room temperature to about 50°C or room temperature to about 65°C.

[0239] In some embodiments, the rolling circle amplification reaction in step (h) can be carried out in the presence of a plurality of compacted oligonucleotides that compact the size and / or shape of the fixed multiply to form fixed compacted nanospheres.

[0240] In some embodiments, the rolling circle amplification reaction in step (h) includes a DNA polymerase with strand displacement activity selected from phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from ThermoFisher Scientific), and a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0241] In some embodiments, the rolling circle amplification reaction may be followed by a multiple substitution amplification (MDA) reaction. In some embodiments, the method further includes performing a multiple substitution amplification (MDA) reaction prior to step (f), wherein the MDA reaction comprises contacting at least one nucleic acid polyp with at least one amplification primer (which contains a random sequence), a DNA polymerase having strand substitution activity, multiple nucleotides, and a catalytic divalent cation (which includes magnesium or manganese).

[0242] In some embodiments, a multiple substitution amplification (MDA) reaction may follow the rolling circle amplification reaction. In some embodiments, the method further includes performing a multiple substitution amplification (MDA) reaction prior to step (f), wherein the MDA reaction comprises contacting at least one nucleic acid polyp with a DNA primase-polymerase, a DNA polymerase having strand substitution activity, multiple nucleotides, and a catalytic divalent cation (including magnesium or manganese). In some embodiments, the DNA primase-polymerase comprises an enzyme having both DNA polymerase and RNA primase activities. The DNA primase-polymerase can synthesize DNA primers on a single-stranded DNA template in a template sequence-dependent manner using deoxyribonucleotide triphosphates and can extend the primer chain by nucleotide polymerization (e.g., primer extension) in the presence of a catalytic divalent cation (e.g., magnesium and / or manganese). The DNA primase-polymerase comprises enzymes that are members of DnaG-like primases (e.g., bacteria) and AEP-like primases (archaea and eukaryotes). An exemplary DNA primase-polymerase is Tth PrimPol from thermophilic bacterium HB27.

[0243] In some embodiments, the rolling circle amplification reaction may be followed by a bending amplification reaction instead of a multiple substitution amplification (MDA) reaction. In some embodiments, the bending amplification reaction includes: (a) forming a nucleic acid relaxation reaction mixture by contacting nucleic acid polymers with a combination of one or more compounds selected from formamide, acetonitrile, ethanol, guanidine hydrochloride, urea, potassium iodide, and / or polyamines to produce relaxed nucleic acid polymers, wherein the formation of the nucleic acid relaxation reaction mixture is carried out under temperature ramping, relaxation incubation temperature, and temperature ramping; (b) washing the relaxed polymers; (c) forming a bending amplification reaction mixture by contacting the relaxed polymers with strand substitution DNA polymerase, multiple nucleotides, and catalytic divalent cations (in the absence of added amplification primers) to produce double-stranded polymers, wherein the formation of the bending amplification reaction mixture is carried out under temperature ramping, bending incubation temperature, and temperature ramping; (d) washing the double-stranded polymers; and (e) repeating steps (a)–(d) at least once.

[0244] Methods for capturing and analyzing RNA. This document provides methods for analyzing nucleic acids (e.g., RNA), the methods comprising: (a) providing a carrier comprising a low nonspecific binding coating, wherein a plurality of capturing oligonucleotides are immobilized onto the coating (e.g., Figure 4 and 28 The plurality of capturing oligonucleotides comprises (i) a target capturing region that hybridizes to at least a portion of the target nucleic acid molecule, (ii) a universal sequence region comprising a spatial barcode sequence and optionally a sample barcode sequence, and (iii) a cleavable region, wherein the low nonspecific binding coating comprises at least one hydrophilic polymer coating having a water contact angle of no more than 45 degrees. In some embodiments, the target capturing region comprises a homopolymer region having a poly-T sequence.

[0245] In some embodiments, the low nonspecific binding coating in step (a) exhibits a low background fluorescence signal or a high contrast-to-noise (CNR) ratio relative to surfaces known in the art. In some embodiments, the low nonspecific binding coating exhibits less than about 0.25 molecules / μm. 2 The nonspecific absorption level of the Cy3 dye is such that no more than 5% of the target nucleic acids associate with the surface coating without hybridizing with the immobilized capture oligonucleotides. In some embodiments, when the fluorescence imaging system is used under non-signal saturation conditions, the fluorescence image of the surface coating having multiple cloned and amplified nucleic acid clusters exhibits a contrast-to-noise ratio (CNR) of at least 20 or at least 50 or higher.

[0246] The method for analyzing nucleic acids further includes the following step: (b) contacting a low-nonspecific binding coating with a cell-biological sample in the presence of a highly efficient hybridization buffer under conditions suitable for promoting the migration of target nucleic acid molecules from a cellular biological sample to one of the immobilized capture oligonucleotides, thereby forming an immobilized target nucleic acid duplex, wherein the target nucleic acid molecule is immobilized to the low-nonspecific binding coating in a manner that preserves the spatial location information of the target nucleic acid molecule in the cellular biological sample, wherein the target nucleic acid comprises a polyA RNA molecule. In some embodiments, the target capture region having a polyT sequence can hybridize with the polyA RNA (e.g., Figure 28 ).

[0247] In some implementations, the cellular biological sample in step (b) includes fresh, frozen, fresh-frozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples.

[0248] In some embodiments, the cellular biological sample in step (b) is permeabilized to facilitate the migration of one of the immobilized capture oligonucleotides from the cellular biological sample, including the target nucleic acid molecule (e.g., DNA and / or RNA).

[0249] In some embodiments, the high-efficiency hybridization buffer of step (b) comprises: (i) a first polar aprotic solvent having a dielectric constant of not more than 40 and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of not more than 115 and present in the high-efficiency hybridization buffer formulation in an amount that effectively denatures double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the high-efficiency hybridization buffer formulation in the range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding.

[0250] In some embodiments, the high-efficiency hybridization buffer in step (b) comprises: (i) a first polar aprotic solvent comprising 25%-50% acetonitrile by volume of the high-efficiency hybridization buffer; (ii) a second polar aprotic solvent comprising 5%-10% formamide by volume of the high-efficiency hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a congesting agent comprising 5%-35% polyethylene glycol (PEG) by volume of the high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer also comprises betaine.

[0251] In some implementations, the high-efficiency hybridization buffer in step (b) promotes a high degree of stringency (e.g., specificity), speed, and power of the nucleic acid hybridization reaction, and improves the efficiency of subsequent amplification and sequencing steps. In some implementations, the high-efficiency hybridization buffer significantly reduces nucleic acid hybridization time and lowers sample input requirements. Nucleic acid annealing can be performed under isothermal conditions, eliminating the need for a cooling step for annealing.

[0252] The method for analyzing nucleic acids further includes the following steps: (c) using the hybridized target nucleic acid molecule as a template, performing a reverse transcription reaction on the fixed nucleic acid duplex to form a fixed target extension product (e.g., cDNA). Figure 28 ).

[0253] In some embodiments, the reverse transcription reaction in step (c) includes (i) a reverse transcriptase, (ii) multiple nucleotides, and (iii) multiple reverse transcriptase primers. In some embodiments, the reverse transcription reaction in step (a) includes multiple nucleotides and an enzyme with reverse transcription activity, including reverse transcriptases from AMV (Avian Myeloblastemia Virus), M-MLV (Moloni Mule Leukemia Virus), or HIV (Human Immunodeficiency Virus). In some embodiments, the reverse transcriptase may be a commercially available enzyme, including MultiScribe. TM ThermoScript TM or ArrayScript TMIn some embodiments, the reverse transcriptase includes Superscript I, II, III, or IV enzymes. In some embodiments, the reverse transcription reaction may include an RNase inhibitor.

[0254] In some embodiments, the method for analyzing nucleic acids (e.g., RNA) further includes: (d) attaching a nucleic acid adaptor to the non-fixed end of a fixed target extension product, thereby producing a fixed double-stranded target extension product with the adapted adaptor attached. Figure 28 Nucleic acid adaptors can be single-stranded or double-stranded. RNA ligase or DNA ligase can be used to attach nucleic acid adaptors. Single-stranded adaptors can be attached to the 3' end of one strand of a fixed target extension product using T4 RNA ligase, KOD ligase, Circligase, or SplintR ligase. Double-stranded adaptors can be attached to the non-fixed end of a fixed target extension product using T4 DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase, Ampligase, or SplintR ligase. The fixed double-stranded target extension product with the attached adaptor contains a fixed capture oligonucleotide that hybridizes to the target nucleic acid molecule (through reverse transcription elongation and adaptor attachment). In some embodiments, the fixed double-stranded target extension product with the attached adaptor is subjected to conditions that dissociate / remove or degrade the target nucleic acid molecule, thereby keeping the fixed single-stranded target extension product with the attached adaptor attached to the surface.

[0255] The method for analyzing nucleic acids may further include the following steps: (e) contacting a fixed single-stranded target extension product with an additional adaptor with a plurality of soluble cyclic oligonucleotides to form a target cyclic double strand, wherein each soluble cyclic oligonucleotide comprises (i) an adaptor-binding region, (ii) a homopolymer region, (iii) an anchoring region, and (iv) an anchoring moiety, wherein the homopolymer region comprises a poly-T sequence capable of hybridizing with a poly-A region of a target nucleic acid molecule, wherein the contact is performed under conditions suitable for immobilizing at least one soluble cyclic oligonucleotide in immediate proximity to the additional adaptor with a low nonspecific binding coating (e.g., Figure 28 ).

[0256] In some embodiments, the adaptor-binding region includes a sequencing primer-binding region. In some embodiments, the adaptor-binding region includes an amplification primer-binding region. In some embodiments, the homopolymer region contains a polynucleotide sequence selected from poly-T, poly-dT, poly-A, poly-dA, poly-C, poly-dC, poly-G, and poly-dG. In some embodiments, the homopolymer region contains a poly-T or poly-dT sequence. In some embodiments, the anchoring portion can be attached to a surface to generate a fixed cyclic oligonucleotide. The adaptor-binding region of the fixed cyclic oligonucleotide can hybridize with an additional adaptor sequence of a fixed single-stranded target extension product with an additional adaptor. The homopolymer region of the fixed cyclic oligonucleotide can hybridize with a homopolymer region (e.g., poly-A) of a fixed single-stranded target extension product with an additional adaptor.

[0257] Methods for analyzing nucleic acids may further include the following steps: (f) cleaving the cleavable region of a target cyclized duplex to release fixed ends from a low-nonspecific binding coating to produce a released target extension product, wherein an additional inode region of the released target extension product retains hybridization with an inode binding region of a fixed cyclized oligonucleotide, and a homopolymer region of the released target extension product can rehybridize with a homopolymer region of a fixed cyclized oligonucleotide to form an open-ring target cyclized duplex with a nick and / or break, such that the fixed cyclized oligonucleotide acts as a splint molecule to facilitate the cyclization of the released target extension product (e.g., Figure 8). In some embodiments, the cleavable region may be cleaved by an enzyme, a compound, light, or heat. In some embodiments, the additional inode region of the released target extension product retains hybridization with a fixed single-stranded target extension product with an additional inode. In some embodiments, the homopolymer region of the released target extension product can rehybridize with a homopolymer region of a fixed cyclized oligonucleotide to form an open-ring target extension product with a nick or break. Immobilized cyclized oligonucleotides can be used as splint molecules to facilitate the cyclization of released target extension products because the homopolymer region and the intransitive binding region of the immobilized cyclized oligonucleotide can hybridize with the ends of the released target extension products.

[0258] The method for analyzing nucleic acids may further include the following steps: (g) closing the gap (if present) by performing a gap-filling primer extension reaction and closing the break (if present) by performing a ligation reaction on the open-ring target cyclized duplex, thereby forming a covalently closed cyclic target extension product that hybridizes with a fixed cyclic oligonucleotide, wherein the fixed cyclic oligonucleotide includes an adaptor binding region containing a 3' extensionable end (e.g., Figure 28 ).

[0259] In some embodiments, step (g) of forming a covalently closed circular target extension product includes a polymerase-mediated gap-filling reaction, an enzymatic ligation reaction, or a polymerase-mediated gap-filling reaction and an enzymatic ligation reaction. In some embodiments, the polymerase-mediated gap-filling reaction includes contacting the open-loop target molecule with a DNA polymerase and multiple nucleotides, wherein the DNA polymerase includes *E. coli* DNA polymerase I, a Klenow fragment of *E. coli* DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the enzymatic ligation reaction includes using a ligase, including T3, T4, T7, or Taq DNA ligase. In some embodiments, forming a covalently closed circular target molecule includes contacting the open-loop target molecule with a CircLigase or CircLigase II enzyme.

[0260] Methods for analyzing nucleic acids may further include the following steps: (h) performing rolling circle amplification (e.g., by extending the 3' extendable end of the adaptor binding region of a fixed nucleic acid polymolecule having a tandem repeat region (which includes a sequencing primer binding sequence, a target sequence, and a spatial barcode sequence) under conditions suitable for forming a fixed nucleic acid polymolecule. Figure 28 ).

[0261] In some embodiments, the rolling circle amplification reaction in step (h) includes contacting a covalently closed circularized lock probe (e.g., one or more circularized nucleic acid template molecules) with amplification primers, DNA polymerase, multiple nucleotides, and at least one catalytic divalent cation under conditions suitable for generating at least one nucleic acid polyp, wherein the at least one catalytic divalent cation includes magnesium or manganese.

[0262] In some embodiments, the rolling circle amplification reaction in step (h) includes: (1) contacting a covalently closed circularized lock probe (e.g., one or more circularized nucleic acid template molecules) with amplification primers, DNA polymerase, multiple nucleotides, and at least one non-catalytic divalent cation (which does not promote polymerase-catalyzed incorporation of nucleotides into the amplification primers), wherein the non-catalytic divalent cation includes strontium or barium; and (2) contacting the covalently closed circularized lock probe with at least one catalytic divalent cation under conditions suitable for generating at least one nucleic acid polymer, wherein the at least one catalytic divalent cation includes magnesium or manganese.

[0263] In some embodiments, the rolling ring amplification reaction in step (h) is carried out at a constant temperature (e.g., isothermal) of room temperature to about 50°C or room temperature to about 65°C.

[0264] In some embodiments, the rolling circle amplification reaction in step (h) can be carried out in the presence of a plurality of compacted oligonucleotides that compact the size and / or shape of the fixed multiply to form fixed compacted nanospheres.

[0265] In some embodiments, the rolling circle amplification reaction in step (h) includes a DNA polymerase with strand displacement activity selected from phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from ThermoFisher Scientific), and a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0266] In some embodiments, the rolling circle amplification reaction may be followed by a multiple substitution amplification (MDA) reaction. In some embodiments, the method further includes performing a multiple substitution amplification (MDA) reaction prior to step (f), wherein the MDA reaction comprises contacting at least one nucleic acid polyp with at least one amplification primer (which contains a random sequence), a DNA polymerase having strand substitution activity, multiple nucleotides, and a catalytic divalent cation (which includes magnesium or manganese).

[0267] In some embodiments, a multiple substitution amplification (MDA) reaction may follow the rolling circle amplification reaction. In some embodiments, the method further includes performing a multiple substitution amplification (MDA) reaction prior to step (f), wherein the MDA reaction comprises contacting at least one nucleic acid polyp with a DNA primase-polymerase, a DNA polymerase having strand substitution activity, multiple nucleotides, and a catalytic divalent cation (including magnesium or manganese). In some embodiments, the DNA primase-polymerase comprises an enzyme having both DNA polymerase and RNA primase activities. The DNA primase-polymerase can synthesize DNA primers on a single-stranded DNA template in a template sequence-dependent manner using deoxyribonucleotide triphosphates and can extend the primer chain by nucleotide polymerization (e.g., primer extension) in the presence of a catalytic divalent cation (e.g., magnesium and / or manganese). The DNA primase-polymerase comprises enzymes that are members of DnaG-like primases (e.g., bacteria) and AEP-like primases (archaea and eukaryotes). An exemplary DNA primase-polymerase is Tth PrimPol from thermophilic bacterium HB27.

[0268] In some embodiments, the rolling circle amplification reaction may be followed by a bending amplification reaction instead of a multiple substitution amplification (MDA) reaction. In some embodiments, the bending amplification reaction includes: (a) forming a nucleic acid relaxation reaction mixture by contacting nucleic acid polymers with a combination of one or more compounds selected from formamide, acetonitrile, ethanol, guanidine hydrochloride, urea, potassium iodide, and / or polyamines to produce relaxed nucleic acid polymers, wherein the formation of the nucleic acid relaxation reaction mixture is carried out under temperature ramping, relaxation incubation temperature, and temperature ramping; (b) washing the relaxed polymers; (c) forming a bending amplification reaction mixture by contacting the relaxed polymers with strand substitution DNA polymerase, multiple nucleotides, and catalytic divalent cations (in the absence of added amplification primers) to produce double-stranded polymers, wherein the formation of the bending amplification reaction mixture is carried out under temperature ramping, bending incubation temperature, and temperature ramping; (d) washing the double-stranded polymers; and (e) repeating steps (a)–(d) at least once.

[0269] Methods and compositions for nucleic acid identification. This document provides methods for analyzing nucleic acids, said methods including determining the sequence of target nucleic acids (e.g., immobilized polynucleotides) mentioned herein. Sequencing can be targeted sequencing. Sequencing can be whole-genome sequencing. Whole-genome sequencing can include massively parallel sequencing (“next-generation sequencing” or “second-generation sequencing”). In some embodiments, sequencing is performed via ligation. In some embodiments, sequencing includes continuously monitoring the incorporation of labeled nucleotides into growing polynucleotide molecules. Sequencing can be performed by massively parallel array sequencing or single-molecule sequencing.

[0270] The method for analyzing nucleic acids further includes the following steps: (i) sequencing at least a portion of a fixed nucleic acid polyp, including sequencing the target sequence and the spatial barcode sequence, to determine the spatial location of the target nucleic acid in a cellular biological sample.

[0271] In some implementations, the sequencing in step (i) includes using a membrane containing a diameter greater than 1.0 mm. 2 An optical imaging system with a field of view (FOV) can sequence at least a portion of nucleic acid polysynthetic molecules.

[0272] In some embodiments, the sequencing in step (i) involves placing a cellular biological sample in a flow cell having walls (e.g., a top wall or first wall, and a bottom wall or second wall) and an intermediate gap, where the gap can be filled with fluid, thereby positioning the flow cell in a fluorescence optical imaging system. When using conventional imaging systems, the thickness of the cellular biological sample may require focusing the imaging system on the first and second surfaces of the flow cell, respectively. To improve imaging of the sequencing reaction of nucleic acids from the cellular biological sample, the flow cell can be placed in a high-performance fluorescence imaging system comprising two or more lens barrels designed to provide optimal imaging performance for the first and second surfaces of the flow cell at two or more fluorescence wavelengths. In some embodiments, the high-performance imaging system further includes a focusing mechanism configured to refocus the optics between acquiring images of the first and second surfaces of the flow cell. In some embodiments, the high-performance imaging system is configured to image two or more fields of view on at least one of the first or second flow cell surfaces.

[0273] In some implementations, step (i) of sequencing includes contacting a plurality of nucleic acid polymers with a plurality of sequencing primers, a plurality of polymerases and a plurality of multivalent molecules, wherein each multivalent molecule includes two or more repeats of a nucleotide portion connected to a core via a linker.

[0274] In some embodiments, the multivalent molecule includes multiple nucleotides bound to a particle (or core), said particle (or core) being, for example, a polymer, branched polymer, dendritic macromolecule, micelle, liposome, microparticle, nanoparticle, quantum dot or other suitable particles known in the art.

[0275] In some embodiments, the multivalent molecule includes: (a) a core; and (b) multiple nucleotide arms, comprising (i) a core attachment portion, (ii) a spacer containing a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the multiple nucleotide arms. In some embodiments, the spacer is attached to the linker. In some embodiments, the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group, and wherein the linker is attached to the nucleotide unit via a base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2-6 subunits and optionally the linker comprises an aromatic moiety.

[0276] In some implementations, the multivalent molecule includes a core attached to multiple nucleotide arms, wherein the multiple nucleotide arms have the same type of nucleotide units selected from dATP, dGTP, dCTP, dTTP, and dUTP.

[0277] In some embodiments, the multivalent molecule also includes a plurality of multivalent molecules, comprising a mixture of multivalent molecules having two or more different types of nucleotides selected from dATP, dGTP, dCTP, dTTP, and dUTP.

[0278] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein each nucleotide arm includes a nucleotide unit having a chain termination portion (e.g., a closing portion) at the 2' position of the sugar, the 3' position of the sugar, or at the 2' and 3' positions of the sugar.

[0279] In some embodiments, the chain-terminating portion comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion is selected from 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl, or derivatives thereof.

[0280] In some implementations, the chain termination portion may be cleaved / removed from the nucleotide unit.

[0281] In some embodiments, the chain termination portion is an azide, an azide group, or an azide-methyl group, which can be cleaved by the phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0282] In some embodiments, the multivalent molecule includes a core attached to multiple nucleotide arms, wherein the core is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore.

[0283] In some implementations, the core of the multivalent molecule includes an avidin-like portion and the core attachment portion includes biotin.

[0284] In some embodiments, the sequencing in step (i) comprises: (1) contacting a plurality of nucleic acid polymers with (i) a plurality of polymerases, (ii) at least one multivalent molecule comprising two or more repeats of a nucleotide portion connected to a core via a linker, and (iii) a plurality of sequencing primers hybridizing to a portion of the polymers, said conditions being adapted to bind at least one polymerase and at least one sequencing primer to a portion of one of the nucleic acid polymer molecules, and adapted to bind at least one nucleotide portion of the multivalent molecule to the 3' end of the sequencing primer at a position opposite to that of a complementary nucleotide in the multivalent molecule, wherein the bound nucleotide portion is not incorporated into the sequencing primer; (2) detecting and identifying the binding core of the multivalent molecule. (3) Optionally repeat steps (1) and (2) at least once; (4) contact the polyp molecule with (i) a plurality of polymerases and (ii) a plurality of nucleotides under conditions suitable for binding at least one polymerase to at least a portion of the polyp molecule and for binding at least one nucleotide of the plurality of nucleotides to the 3' end of a hybridization sequencing primer at a position opposite to that of a complementary nucleotide in the polyp molecule, wherein the bound nucleotide is incorporated into the hybridization sequencing primer; (5) Optionally detect the incorporated nucleotide; (6) Optionally identify the incorporated nucleotide to determine or confirm the sequence of the polyp; and (7) repeat steps (1)–(6) at least once.

[0285] In some embodiments, the sequencing in step (i) comprises: (1) contacting a plurality of fixed polymers with a plurality of sequencing primers, a plurality of polymerases, and a plurality of nucleotides hybridized to a sequence binding sequence of a sequencing primer under conditions suitable for binding at least one polymerase and at least one sequencing primer to a portion of the fixed polymers, and suitable for binding at least one of the nucleotides to the 3' end of the sequencing primer at a position opposite to that of a complementary nucleotide in the fixed polymers, wherein the bound nucleotide is incorporated into the 3' end of the sequencing primer; (2) detecting and identifying the incorporated nucleotide to determine the sequence of the fixed polymer molecule; and (3) optionally repeating steps (1) and (2) at least once. In some embodiments, at least one nucleotide of the plurality of nucleotides comprises a chain-terminating portion at the 2' or 3' position of a sugar. In some embodiments, the chain-terminating portion is an azide, an azide group, or an azide methyl group, which can be cleaved by a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0286] Sequencing methods may include contacting one or more target nucleic acids containing multiple linked or unlinked copies of a target sequence with a multivalent binding composition described herein. Contacting said one or more target nucleic acids containing multiple linked or unlinked copies of a target sequence with one or more polymer-nucleotide conjugates can provide a significantly increased local concentration of the correct nucleotide being queried in a given sequencing cycle, thus suppressing signals from improperly incorporated or phased nucleic acid chains (i.e., those elongated nucleic acid chains with one or more skipped cycles).

[0287] This document provides a method for obtaining nucleic acid sequence information, the method comprising contacting one or more target nucleic acids with one or more polymer-nucleotide conjugates. In some embodiments, the one or more target nucleic acids comprise multiple linked or unlinked copies of a target sequence. In some embodiments, the method results in a reduction in sequencing error rate, as indicated by a reduction in the identification of incorrect bases, the reporting of missing bases, or the failure to report correct bases. In some embodiments, the reduction in sequencing error rate may include a reduction of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the error rate observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides). In some embodiments, the method results in an average read length increase of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the average read length observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides). In some embodiments, the method results in an average read length increase of 10, 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or 500 nucleotides compared to the average read length observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides).

[0288] Using polymer-nucleotide conjugates for sequencing can shorten the total time of the sequencing reaction or sequencing run. Sequencing reaction cycles, including contact, detection, and incorporation steps, are performed in a total time range of about 5 minutes to about 60 minutes. In some embodiments, the sequencing reaction cycle is performed within a time range of at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the sequencing reaction cycle is performed within a time range of at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some embodiments, the sequencing reaction cycle can be performed in a total time range of about 10 minutes to about 30 minutes. Those skilled in the art will recognize that the sequencing cycle time can have any value within this range, such as about 16 minutes.

[0289] Using polymer-nucleotide conjugates for sequencing provides more accurate base readouts. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q score in the range of about 20 to about 50 for base determination accuracy during sequencing runs. In some embodiments, the average Q score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q score can have any value within this range, such as about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides.

[0290] This disclosure relates to polymer-nucleotide conjugates, each having multiple nucleotides conjugated to a particle or core (e.g., a polymer, branched polymer, dendritic macromolecule, or equivalent structure). Contacting the polymer-nucleotide conjugate with a polymerase and an initiated target nucleic acid can result in the formation of a detectable ternary complex, thereby enabling more accurate determination of the target nucleic acid bases.

[0291] When polymer-nucleotide conjugates are used instead of single unconjugated or untethered nucleotides to form complexes with polymerases and target nucleic acids, the local concentration of nucleotides increases many-fold, which in turn enhances signal strength, particularly compared to the correct signal from a mismatch. The polymer-nucleotide conjugates described herein may include at least one polymer-nucleotide conjugate for interaction with target nucleic acids. Multivalent compositions may also include two, three, or four different polymer-nucleotide conjugates, each having a different nucleotide conjugated to the particle.

[0292] In polymer-nucleotide conjugates having either a polymer-nucleotide conjugate form or a core-nucleotide conjugate form, multiple copies of the same nucleotide can be covalently or non-covalently bound to the particles. Examples of particles can include branched polymers; dendritic macromolecules; cross-linked polymer particles, such as agarose, polyacrylamide, acrylates, methacrylates, cyanoacrylates, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles; or any other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particles are branched polymers.

[0293] Nucleotides can be linked to particles or the core via linkers, and nucleotides can be attached to an end or site of the polymer. Nucleotides can be conjugated to particles via their bases or 5' ends. In some polymer-nucleotide conjugates, one nucleotide is attached to an end or site of the polymer. In some polymer-nucleotide conjugates, multiple nucleotides are attached to an end or site of the polymer. The conjugated nucleotides can spatially access one or more proteins, one or more enzymes, and nucleotide-binding moieties. In some embodiments, the nucleotides can be provided separately from the nucleotide-binding moieties, such as polymerases. In some embodiments, the linker does not include a light-emitting group or a light-absorbing group.

[0294] The particles or core may also have binding portions. In some embodiments, the particles or core can self-associate without using separate interacting portions. In some embodiments, the particles or core may self-associate due to buffering or salt conditions, for example, in the case of calcium-mediated interactions of hydroxyapatite particles, lipid or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

[0295] Polymer-nucleotide conjugates may have one or more labels (e.g., detectable reporting portions). Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radiolabels, or other such labels that enable the composition to be detected by methods known in the field of detecting macromolecules or molecular interactions. The label may be attached to a nucleotide (e.g., by attachment to a base or 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to a PEG subunit), or to a core (e.g., to a streptavidin core), to the end of the polymer, to a central portion, or to any other location within the polymer-nucleotide conjugate that will be recognized by those skilled in the art as sufficient to enable the composition, such as the particle, to be detected by methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or distinguish a particular polymer-nucleotide conjugate.

[0296] An example of a polymer-nucleotide conjugate (e.g., a polymer-nucleotide conjugate) is a polymer-nucleotide conjugate. Examples of branched polymers include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, dextran, or other such polymers. In one embodiment, the polymer is PEG. In another embodiment, the polymer may have PEG branches.

[0297] Suitable polymers may be characterized by repeating units having functional groups suitable for derivatization, such as amines, hydroxyl groups, carbonyl groups, or allyl groups. The polymer may also have one or more pre-derived substituents, such that one or more specific subunits include a derivatization site or branching site, regardless of whether other subunits include the same site, substituent, or moiety. Pre-derived substituents may include, or may further include, for example, nucleotides, nucleosides, nucleotide analogs, labels (e.g., fluorescent labels, radioactive labels, or spin labels), interacting moieties, additional polymer moieties, or any combination thereof.

[0298] In polymer-nucleotide conjugates (e.g., polymer-nucleotide conjugates), the polymer can have multiple branches. Branched polymers can have various configurations, including but not limited to star-shaped (“starburst”) forms, aggregated star-shaped (“helter skelter”) forms, bottle-brush-like, or dendritic macromolecules. Branched polymers can radiate from a central attachment point or a central portion, or may include multiple branch points, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each subunit of the polymer may optionally constitute a separate branch point.

[0299] In polymer-nucleotide conjugates, the length and size of the branches can vary depending on the type of polymer. In some branched polymers, the length of the branches can be 1 to 1,000 nm, 1 to 100 nm, 1 to 200 nm, 1 to 300 nm, 1 to 400 nm, 1 to 500 nm, 1 to 600 nm, 1 to 700 nm, 1 to 800 nm, or 1 to 900 nm, or greater, or have a length falling within or between any of the values ​​disclosed herein. In some branched polymers, the branches can have sizes corresponding to the following apparent molecular weights: 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K, 50K, 80K, 100K, or any value within the range defined by any two of the foregoing. The apparent molecular weight of the polymer can be calculated from the known molecular weights of a representative number of subunits, such as by size exclusion chromatography, by mass spectrometry, or by any other method known in the art. The polymer can have multiple branches. The number of branches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number falling within the range defined by any two of these values.

[0300] For polymer-nucleotide conjugates, branched polymers having 4, 8, 16, 32, or 64 branches can attach nucleotides to the ends of PEG branches, such that each end has 0, 1, 2, 3, 4, 5, 6, or more nucleotides attached. In a non-limiting example, a branched polymer having 3 to 128 PEG arms attaches one or more nucleotides to the ends of polymer branches, such that each end has 0, 1, 2, 3, 4, 5, 6, or more nucleotides or nucleotide analogs attached. In some embodiments, the branched polymer or dendritic macromolecule has an even number of arms. In some embodiments, the branched polymer or dendritic macromolecule has an odd number of arms.

[0301] In a polymer-nucleotide conjugate, each branch or subset of the polymer may be attached with a moiety comprising a nucleotide (e.g., adenine, thymine, uracil, cytosine, or guanine residues or derivatives or mimics), and this moiety is capable of binding polymerase, reverse transcriptase, or other nucleotide-binding domains. Optionally, the nucleotide moiety may be capable of binding to the polymerase-template-primer complex but not incorporating, or may be incorporated into the extended nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide moiety includes a chain termination portion that blocks the incorporation of subsequent nucleotides during a polymerase-mediated reaction. In some embodiments, the nucleotide moiety may be unblocked (reversibly blocked) such that subsequent nucleotides cannot be incorporated into the extended nucleic acid chain during a polymerase reaction until this blocking is removed, after which subsequent nucleotides can be incorporated into the extended nucleic acid chain during a polymerase reaction.

[0302] Polymer-nucleotide conjugates may also have a binding moiety in each branch or subset of branches. Some examples of binding moieties include, but are not limited to, biotin, avidin, streptavidin, polyhistidine domains, complementary pairing nucleic acid domains, G-tetramer-forming nucleic acid domains, calmodulin, maltose-binding protein, cellulase, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine ​​and its derivatives, antibodies, epitopes, protein A, and protein G. The binding moieties may be any interacting molecule or fragment thereof known in the art for binding or promoting interactions between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interacting domains or portions.

[0303] In some embodiments, the polymer-nucleotide conjugate may include one or more elements of a complementary interacting moiety. Exemplary complementary interacting moieties include, for example, biotin and avidin; SNAP-benzylguanosine; antibodies or FABs and epitopes; IgG FC and protein A, protein G, protein A / G, or protein L; maltose-binding proteins and maltose; lectins and homopolysaccharides; ion-chelating moieties; complementary nucleic acids; nucleic acids capable of forming triple or triple helical interactions; nucleic acids capable of forming G-quadriplexes, etc. Those skilled in the art will readily recognize that many paired moieties exist and are generally used for their properties (i.e., they interact strongly and specifically with each other); therefore, any such complementary pair or group is considered suitable for this purpose of constructing or conceiving the compositions of this disclosure. In some embodiments, the compositions disclosed herein may include compositions in which one element of the complementary interacting moiety is attached to a molecule or multivalent ligand, and another element of the complementary interacting moiety is attached to a separate molecule or multivalent ligand. In some embodiments, compositions as disclosed herein may include compositions in which two or all elements of the complementary interacting moiety are attached to a single molecule or multivalent ligand. In some embodiments, compositions disclosed herein may comprise compositions in which two or all elements of the complementary interacting portion are attached to a single arm or site of a single molecule or multivalent ligand. In some embodiments, compositions disclosed herein may comprise compositions in which two or all elements of the complementary interacting portion are attached to the same arm or site of a single molecule or multivalent ligand. In some embodiments, a composition comprising one element of the complementary interacting portion and a composition comprising another element of the complementary interacting portion may be mixed simultaneously or sequentially. In some embodiments, the interaction between molecules or particles disclosed herein allows for the association or aggregation of multiple molecules or particles, thereby, for example, increasing a detectable signal. In some embodiments, fluorescence, colorimetric, or radiometric signals are enhanced. In other embodiments, other interacting portions disclosed herein or known in the art are contemplated. In some embodiments, compositions as provided herein may be provided such that one or more molecules comprising a first interacting portion (e.g., one or more imidazole or pyridine portions) and one or more additional molecules comprising a second interacting portion (e.g., histidine residues) are mixed simultaneously or sequentially. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridine portions. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6 or more histidine residues. In such embodiments, the provided interactions between molecules or particles can be facilitated by the presence of divalent cations such as nickel, manganese, magnesium, calcium, strontium, etc. In some embodiments, for example, the (His)3 group can interact with the (His)3 group on another molecule or particle through coordination with nickel or manganese ions.

[0304] Polymer-nucleotide conjugates may include one or more buffers, salts, ions, or additives. In some embodiments, representative additives may include, but are not limited to, betaine, spermidine, detergents (e.g., Triton X-100, Tween 20, SDS, or NP-40), ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers (e.g., Pluronic® series polymers), arginine, histidine, imidazole, or any combination thereof, or any substance known in the art as a DNA “relaxant” (a compound that has the effect of altering the length of DNA, altering the number of linkers or crossovers within a polymer, or altering the conformational dynamics of a DNA molecule, thereby increasing the accessibility of intra-strand sites to the DNA binding site).

[0305] Polymer-nucleotide conjugates may include zwitterionic compounds as additives. Other representative additives can be found in Lorenz, TCJVis.Exp.(63),e3998,doi:10.3791 / 3998(2012) (which is incorporated herein by reference) regarding additives disclosed therein for promoting nucleic acid binding or kinetics, or for promoting processes involving the handling, use or storage of nucleic acids.

[0306] In some embodiments, the multivalent binding composition includes at least one cation, which may include, but is not limited to, sodium, magnesium, strontium, barium, potassium, manganese, calcium, lithium, nickel, cobalt, or other such cations known in the art, for promoting nucleic acid interactions, such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, etc.

[0307] When polymer-nucleotide conjugates are used instead of unconjugated or untethered nucleotides to form complexes with polymerase and target nucleic acids, the local concentration of nucleotides increases many times over, which in turn enhances the signal strength, particularly compared to the correct signal of a mismatch. This disclosure considers contacting polymer-nucleotide conjugates with polymerase and the initiated target nucleic acid to determine the formation of a ternary binding complex.

[0308] Due to the increased local concentration of nucleotides on the polymer-nucleotide conjugate, the binding between the polymerase, the initiated target strand, and the nucleotide becomes more favorable when the nucleotide is complementary to the next base of the target nucleic acid. The resulting binding complex has a longer duration, which in turn helps to shorten the imaging step. The high signal intensity generated using the polymer-nucleotide conjugate is maintained throughout the binding and imaging steps. The strong binding between the polymerase, the initiated target strand, and the nucleotide or nucleotide analogue also means that the resulting binding complex will remain stable during the washing step, and the signal will remain high as other reaction mixtures and unmatched nucleotide analogues are washed away. After the imaging step, the binding complex can be destabilized, and the initiated target nucleic acid can then be extended by one base. After extension, the binding and imaging steps can be repeated using the polymer-nucleotide conjugate to determine the identity of the next base.

[0309] The compositions and methods disclosed herein provide robust and controllable means for establishing and maintaining ternary enzyme complexes (e.g., during sequencing), and also provide significantly improved means for identifying and / or measuring the presence of said complexes, as well as means for controlling the persistence of said complexes. This provides an important solution to problems such as determining the identity of N+1 bases in nucleic acid sequencing applications.

[0310] Unbound by any particular theory, it has been observed that the multivalent binding compositions disclosed herein associate with polymerase nucleotide complexes at a time-dependent rate, but much slower than the known achievable association rate for nucleotides in free solution, to form ternary binding complexes. Thus, the association rate (Kon) is significantly and unexpectedly much slower than the association rate of mononucleotides or nucleotides not attached to the multivalent ligand complex. Importantly, however, the dissociation rate (Koff) of the multivalent ligand complex is much slower than the dissociation rate observed for nucleotides in free solution. Therefore, the multivalent ligand complexes of this disclosure provide a surprising and beneficial improvement to the persistence of ternary polymerase-polynucleotide-nucleotide complexes (particularly relative to such complexes formed with free nucleotides), for example, significantly improving imaging quality for nucleic acid sequencing applications, superior to currently available methods and reagents. Importantly, this property of the multivalent substrates disclosed herein makes the formation of visible ternary complexes controllable, allowing for subsequent visualization, modification, or processing steps with minimal regard to the dissociation of the complex—that is, the complex can be formed, imaged, modified, or otherwise used as needed and will remain stable until the user performs a confirmed dissociation step, such as exposing the complex to a dissociation buffer.

[0311] In various embodiments, the polymerase suitable for the binding interactions described herein (e.g., during sequencing) may include any polymerase known or potentially known in the art. Exemplary polymerases may include, but are not limited to: Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase; human α, δ, and ε DNA polymerases; bacteriophage polymerases, such as T4, RB69, and phi29 bacteriophage DNA polymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and Escherichia coli DNA polymerase III α and ε; 9°N polymerase, reverse transcriptases such as HIV M or O reverse transcriptase, avian myeloblastemia virus reverse transcriptase, or Moloney mouse leukemia virus (MMLV) reverse transcriptase, or telomerase. Other non-limiting examples of DNA polymerases may include those DNA polymerases or variants thereof from various archaea genera (e.g., *Aeropyrum*, *Archaeglobus*, *Desulfurococcus*, *Pyrobaculum*, *Pyrococcus*, *Pyrolobus*, *Pyrodictium*, *Staphylothermus*, *Stetteria*, *Sulfolobus*, *Thermococcus*, and *Vulcanisaeta*, etc.), including such polymerases known in the art, such as Vent. TM DeepVent TM ,Pfu,KOD,Pfx,Therminator TM And Tgo polymerase. In some implementations, the polymerase is Klenow polymerase.

[0312] The ternary complex has a longer duration of activity when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid compared to when they are not complementary. The ternary complex also has a longer duration of activity when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid compared to unconjugated or tethered complementary nucleotides. For example, in some embodiments, the duration of the ternary complex can be less than 1 s, more than 1 s, more than 2 s, more than 3 s, more than 5 s, more than 10 s, more than 15 s, more than 20 s, more than 30 s, more than 60 s, more than 120 s, more than 360 s, more than 3600 s, or longer, or within a range defined by any two or more of these values.

[0313] For example, the duration can be measured by observing the onset and / or duration of the binding complex, such as by observing signals from the labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in the binding complex, thereby allowing the detection of signals from the label during the duration of the binding complex.

[0314] It has been observed that different durations can be achieved using different salts or ions, suggesting that, for example, complexes formed in the presence of magnesium form faster than those formed in other ionic conditions. It has also been observed that complexes formed in the presence of, for example, strontium readily form and completely or substantially completely dissociate after ion withdrawal or washing with a buffer lacking one or more components of the compositions of the present invention (e.g., polymers and / or one or more nucleotides, and / or one or more interacting moieties) or a buffer containing, for example, a chelating agent (which can induce or accelerate the removal of divalent cations from complexes containing multivalent reagents). Therefore, in some embodiments, the compositions of this disclosure contain magnesium. In some embodiments, the compositions of this disclosure contain calcium. In some embodiments, the compositions of this disclosure contain strontium or barium. In some embodiments, the compositions of this disclosure contain cobalt. In some embodiments, the compositions of this disclosure contain MgCl2. In some embodiments, the compositions of this disclosure contain CaCl2. In some embodiments, the compositions of this disclosure contain SrCl2. In some embodiments, the compositions of this disclosure contain CoCl2. In some embodiments, the compositions do not contain or substantially do not contain magnesium. In some embodiments, the composition contains no or substantially no calcium. In some embodiments, the methods of this disclosure provide contacting one or more nucleic acids with one or more compositions disclosed herein, wherein said compositions are deficient in one of calcium or magnesium, or both calcium and magnesium.

[0315] The dissociation of the ternary complex can be controlled by altering the buffer conditions. After the imaging step, the ternary complex is dissociated using a buffer with increased salt content, thereby washing away the labeled polymer-nucleotide conjugate and providing a means to attenuate or terminate the signal, for example, during transitions between sequencing cycles. In some embodiments, this dissociation can be achieved by washing the complex with a buffer lacking the necessary metal or cofactor. In some embodiments, the wash buffer may comprise one or more compositions for maintaining pH control. In some embodiments, the wash buffer may comprise one or more monovalent cations, such as sodium. In some embodiments, the wash buffer lacks or substantially lacks divalent cations, such as strontium, calcium, magnesium, or manganese, which are absent or substantially absent. In some embodiments, the wash buffer also contains chelating agents such as EDTA, EGTA, hypozinotriacetic acid, polyhistidine, imidazole, etc. In some embodiments, the wash buffer may maintain the ambient pH at the same level as the binding complex. In some embodiments, the wash buffer may raise or lower the ambient pH relative to the level observed for the binding complex. In some implementations, the pH may be in the range of 2-4, 2-7, 5-8, 7-9, 7-10, or below 2, or above 10, or within the range defined by any two values ​​provided herein.

[0316] The addition of specific ions can affect the binding of polymerase to the initiated target nucleic acid, the formation of the ternary complex, the dissociation of the ternary complex, or, for example, the incorporation of one or more nucleotides into the extended nucleic acid during the polymerase reaction. In some embodiments, the relevant anions may include chloride, acetate, gluconate, sulfate, phosphate, etc. In some embodiments, ions may be included in the compositions of this disclosure by adding one or more acids, bases, or salts such as NiCl2, CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, BaCl2, etc. Representative salts, ions, solutions, and conditions can be found in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, AR, Ed. (2000) (which is incorporated herein by reference in its entirety), particularly concerning Chapter 17 and related disclosures on salts, ions, salt solutions, and ionic solutions.

[0317] This disclosure contemplates contacting a polymer-nucleotide conjugate with one or more polymerases. Contact may optionally be performed in the presence of one or more target nucleic acids. In some embodiments, the target nucleic acid is a single-stranded nucleic acid. In some embodiments, the target nucleic acid hybridizes with a nucleic acid primer. In some embodiments, the target nucleic acid is a double-stranded nucleic acid. In some embodiments, the contact comprises contacting the polymer-nucleotide conjugate with a polymerase. In some embodiments, the contact comprises contacting the composition containing one or more nucleotides with multiple polymerases. The polymerase can bind to a single nucleic acid molecule.

[0318] The binding between the target nucleic acid and the polymer-nucleotide conjugate can be provided in the presence of a polymerase that has become catalytically inactive. In one embodiment, the polymerase may have been mutated to become catalytically inactive. In another embodiment, the polymerase may have been chemically modified to become catalytically inactive. In some embodiments, the polymerase may have become catalytically inactive due to the lack of a necessary substrate, ion, or cofactor. In some embodiments, the polymerase may have become catalytically inactive due to the absence of magnesium ions.

[0319] The binding between the target nucleic acid and the polymer-nucleotide conjugate occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer lacks catalytic ions such as magnesium or manganese. Alternatively, the binding between the target nucleic acid and the polymer-nucleotide conjugate occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer contains non-catalytic ions such as strontium, barium, or calcium.

[0320] When a non-catalytically active polymerase is used to facilitate the interaction of nucleic acids with a multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex, enabling detection by fluorescence or by other methods disclosed herein or known in the art. Unbound polymer-nucleotide conjugates may optionally be washed away before detection of the ternary binding complex.

[0321] One or more nucleic acids are contacted with the polymer-nucleotide conjugate disclosed herein in a solution containing one or more of calcium or magnesium, or both of calcium and magnesium. Alternatively, one or more nucleic acids are contacted with the polymer-nucleotide conjugate disclosed herein in a solution lacking one or more of calcium or magnesium, or both of calcium and magnesium, and in a separate step (regardless of the order of the steps), by adding one or more of calcium or magnesium, or both of calcium and magnesium, to the solution. In some embodiments, one or more nucleic acids are contacted with the polymer-nucleotide conjugate disclosed herein in a solution lacking strontium or barium, and this includes adding strontium to the solution in a separate step (regardless of the order of the steps).

[0322] This document discloses polymer-nucleotide conjugates and their uses in the analysis of nucleic acids, including sequencing or other bioassay applications. Increasing the binding of nucleotides to enzymes (e.g., polymerases) or enzyme complexes can be achieved by increasing the effective concentration of the nucleotides. This increase can be achieved by increasing the concentration of nucleotides in free solution or by increasing the amount of nucleotides near the relevant binding site. This increase can also be achieved by physically confining some nucleotides to a limited volume, resulting in a localized increase in concentration, and thus the structure can bind to the binding site with a higher apparent affinity than observed with unconjugated, untethered, or otherwise unconfined single nucleotides. An exemplary means of achieving this confinement is by providing a polymer-nucleotide conjugate in which multiple nucleotides are bound to particles, such as polymers, branched polymers, dendritic macromolecules, micelles, liposomes, microparticles, nanoparticles, quantum dots, or other suitable particles known in the art.

[0323] The polymer-nucleotide conjugates disclosed herein may include multiple nucleotide motifs attached to a particle. In some embodiments, the multiple nucleotide motifs are composed of nucleotide motifs of the same type (e.g., having the same or similar base pairing characteristics). When the multiple nucleotide motifs are complementary to the next nucleotide in a target nucleic acid to be identified, the polymer-nucleotide conjugate forms a binding complex (multivalent binding complex) between at least two nucleotide motifs and the next nucleotide in at least two copies of the target nucleic acid sequence. In some embodiments, the multivalent binding complex comprises two or more polymerases that bind to the initiating template of the target nucleic acid molecule. The multivalent binding complexes described herein exhibit increased stability and longer duration of action compared to binding complexes formed using a single unconjugated or untethered nucleotide. When bound to polymerases, the multivalent binding complexes can withstand washing steps and thus maintain high signal intensity throughout the imaging and washing steps of the workflow, see, for example... Figure 7 The polymer core of a polymer-nucleotide conjugate can be labeled with two or more detectable tags, which at least partially contributes to enhancing the detectable signal.

[0324] In some embodiments, at least one polymer-nucleotide conjugate comprises two or more repeats of a nucleotide moiety linked to a core via a linker, for example... Figure 5A and Figure 5B In some embodiments, the polymer-nucleotide conjugate comprises: (a) a core, and (b) a plurality of nucleotide arms, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer containing a PEG portion, (iii) a linker, and (iv) a nucleotide unit, such as Figures 5A-5D and Figures 6A-6B As shown in the image.

[0325] In some embodiments, the spacer is attached to the linker, wherein the linker is attached to a nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group. In some embodiments, the linker is attached to the nucleotide unit via a base. In some embodiments, the linker comprises an aliphatic chain or an oligomeric glycol chain, wherein both linker chains have 2-6 subunits, and optionally, the linker comprises an aromatic moiety (…). Figure 6A and Figure 6B In some embodiments, the polymer-nucleotide conjugate comprises a core attached to a plurality of nucleotide arms, wherein the plurality of nucleotide arms have nucleotide units of the same type selected from dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the low-binding carrier further comprises a plurality of polymer-nucleotide conjugates comprising a mixture of polymer-nucleotide conjugates having two or more different types of nucleotides selected from dATP, dGTP, dCTP, dTTP, and dUTP.

[0326] In some embodiments, the polymer-nucleotide conjugate comprises a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide unit having a chain termination portion (e.g., a blocking portion) at the 2' position, 3' position, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain termination portion is selected from alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, sulfur groups, disulfide groups, carbonate groups, urea groups, or silyl groups.

[0327] In some embodiments, the chain termination portion includes a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the chain-terminating portion is selected from 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof. In some embodiments, the chain-terminating portion includes an azide, an azdo group, or an azdomethyl group.

[0328] In some embodiments, the chain-terminating portion is cleavable / removable from the nucleotide arm, for example, by a compound, light, or heat. In some embodiments, the chain-terminating portion includes an alkyl, alkenyl, alkynyl, or allyl group that can be cleaved by tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain-terminating portion includes an aryl or benzyl group that can be cleaved by Pd / C. In some embodiments, the chain-terminating portion includes an amine, amide, ketone, isocyanate, phosphate, sulfide, or disulfide group that can be cleaved by a phosphine or thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating portion includes a carbonate group that can be cleaved by potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating portion comprises a urea or silyl group, which may be cleaved by tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the chain-terminating portion is an azide, azide group, or azide-methyl group, which may be cleaved by a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0329] In some embodiments, the polymer-nucleotide conjugate includes a core attached to multiple nucleotide arms, wherein the core or nucleotide bases include a label. In some embodiments, the label is a detectable report portion. The polymer-nucleotide conjugate may have one or more labels. Examples of detectable report portions include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radiolabels, or other such labels that enable the composition to be detected by methods known in the field of macromolecular or molecular interactions. The detectable report portion may be attached to a nucleotide (e.g., by attachment to the 5' phosphate portion of the nucleotide), attached to the particle itself (e.g., attached to a PEG subunit), attached to the end of the polymer, attached to the central portion, or attached to any other location within the polymer-nucleotide conjugate that a person skilled in the art would recognize as sufficient to enable the composition (e.g., the particle) to be detected by methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or distinguish a particular polymer-nucleotide conjugate. The detectable report portion may be a fluorophore. In some embodiments, the core may be an avidin-like portion and the core attachment portion may be a biotinylate portion.

[0330] Exemplary polymer-nucleotide conjugates and methods of use are described in U.S. Application No. 16 / 579,794, filed September 23, 2019, the contents of which are hereby expressly incorporated herein by reference for all purposes.

[0331] Polymer-nucleotide conjugates (PNPs) can be used to localize detectable signals to active sites of biochemical interactions, such as protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions (e.g., polymerase reactions). For example, the PNPs described herein can be used to identify template-bound base sites or base sites incorporated into extended nucleic acid chains during polymerase reactions, providing base differentiation for sequencing and array-based applications. The increased binding between the target nucleic acid and the nucleotide in the multivalent binding composition when the nucleotide is complementary to the target nucleic acid provides an enhanced signal, thereby significantly improving base determination accuracy and reducing imaging time.

[0332] Furthermore, the use of polymer-nucleotide conjugates allows sequencing signals from a given sequence to originate from clusters containing multiple copies of the target sequence. Sequencing methods that include multiple copies of the target sequence (e.g., polymers) have the advantage that the signal can be amplified because multiple simultaneous sequencing reactions exist within a defined region, each providing its own signal. The presence of multiple signals within a defined region also reduces the impact of any single skipped cycle, as the signal from a large number of correct base determinations overwhelms the signal from a smaller number of skipped or incorrect base determinations, thus providing a method for reducing phasing errors and / or increasing read lengths in sequencing reactions.

[0333] The polymer-nucleotide conjugates disclosed herein and their uses result in one or more of the following: (i) stronger signals for higher base determination accuracy compared to conventional nucleic acid amplification and sequencing methods; (ii) better differentiation of sequence-specific signals from background signals; (iii) reduced requirements for the amount of starting material required; (iv) increased sequencing rates and shorter sequencing times; (v) reduced phasing errors; and (vi) increased read lengths in sequencing reactions.

[0334] Those skilled in the art will recognize that in a series of iterative sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides during a given cycle, resulting in one or more sites being out of sync with the extended nucleic acid strand body. These incorporation failures will produce individual errors in the output sequence when the sequencing signal originates from a reaction occurring on a single copy of the target nucleic acid. Sequencing using polymer-nucleotide conjugates can reduce this type of error in sequencing reactions. For example, by providing an increased rebinding probability after early dissociation of the ternary polymerase complex, the frequency of “skipped” cycles due to non-incorporation can be reduced by using multivalent substrates capable of binding to polymerase-template-primer complexes or incorporating into the extended strand. Therefore, in some embodiments, this disclosure contemplates the use of multivalent substrates as disclosed herein, comprising nucleotides having free or reversibly modified 5' phosphate, diphosphate, or triphosphate moieties, wherein said nucleotides are linked to particles or polymers as disclosed herein by unstable or cleavable links. In some embodiments, this disclosure contemplates the reduction of the inherent error rate due to skipped incorporation by using multivalent substrates disclosed herein.

[0335] This disclosure also considers sequencing reactions in which sequencing signals from or associated with a given sequence are derived from or originate from a definable region containing multiple copies of a target sequence. A sequencing method that binds multiple copies of a target sequence has the advantage that the signal can be amplified because multiple simultaneous sequencing reactions exist within the defined region, each providing its own signal. The presence of multiple signals within the defined region also reduces the impact of any single skipped cycle, as the signal from a large number of correct base decisions will overwhelm the signal from a smaller number of skipped or incorrect base decisions. This disclosure further considers including free, unlabeled nucleotides during the extension reaction or during a separate portion of the extension cycle to provide incorporation at sites that may have been skipped in previous cycles. For example, unlabeled blocking nucleotides can be added during or after an incorporation cycle so that they can be incorporated into skipped sites. The unlabeled blocking nucleotides may be of the same type as the nucleotides of the substrate attached to a multivalently bound substrate or present or previously present during a particular cycle, or may include a mixture of 1, 2, 3, 4, or more types of unlabeled blocking nucleotides.

[0336] When each sequencing cycle runs perfectly, each reaction within a defined region will provide the same signal. However, as described elsewhere herein, in a series of iterative sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides during a given cycle, resulting in one or more sites becoming out of sync with the elongated nucleic acid strand body. This problem, known as “phasing,” causes sequencing signal decay because the signal is contaminated by stray signals from sites that have skipped one or more cycles. This, in turn, increases the possibility of base identification errors. The gradual decay of the sequencing signal in each cycle, along with the gradual accumulation of skipped cycles over multiple cycles, also reduces the effective read length. Another object of this disclosure is to provide methods for reducing phasing errors and / or increasing read lengths in sequencing reactions.

[0337] Sequencing methods may include contacting one or more target nucleic acids comprising multiple linked or unlinked copies of a target sequence with a multivalent binding composition described herein. Contacting said one or more target nucleic acids comprising multiple linked or unlinked copies of a target sequence with one or more polymer-nucleotide conjugates can provide a significantly increased local concentration of the correct nucleotide being queried in a given sequencing cycle, thus suppressing signals from improperly incorporated or phased nucleic acid chains (i.e., those elongated nucleic acid chains with one or more skipped cycles).

[0338] Methods for obtaining nucleic acid sequence information may include contacting one or more target nucleic acids with one or more polymer-nucleotide conjugates, wherein the one or more target nucleic acids comprise multiple linked or unlinked copies of the target sequence. The methods result in a reduction in sequencing error rates, as indicated by the identification of incorrect bases, the reporting of missing bases, or a reduction in the reporting of correct bases. In some embodiments, the reduction in sequencing error rates may include a reduction of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more compared to error rates observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides).

[0339] Methods for obtaining nucleic acid sequence information may include contacting one or more target nucleic acids with one or more polymer-nucleotide conjugates, wherein the template nucleic acid or target nucleic acids comprises multiple linked or unlinked copies of the target sequence. The methods result in an increase in average read length of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the average read length observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides).

[0340] A method for obtaining nucleic acid sequence information, the method comprising contacting one or more target nucleic acids with one or more polymer-nucleotide conjugates, wherein the one or more target nucleic acids comprise multiple linked or unlinked copies of a target sequence. The method results in an increase in average read length of 10, 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or 500 nucleotides compared to the average read length observed using monovalent ligands (including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides).

[0341] Sequencing using polymer-nucleotide conjugates effectively reduces sequencing time. Sequencing reaction cycles, including contact, detection, and incorporation steps, are performed in a total time range of approximately 5 minutes to approximately 60 minutes. In some embodiments, sequencing reaction cycles are performed within a range of at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, sequencing reaction cycles are performed within a range of at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some embodiments, sequencing reaction cycles can be performed in a total time range of approximately 10 minutes to approximately 30 minutes. Those skilled in the art will recognize that sequencing cycle times can have any value within this range, such as approximately 16 minutes.

[0342] Using polymer-nucleotide conjugates for sequencing provides more accurate base readouts. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q score in the range of about 20 to about 50 for base determination accuracy during sequencing runs. In some embodiments, the average Q score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q score can have any value within this range, such as about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides.

[0343] This disclosure relates to polymer-nucleotide conjugates, each having multiple nucleotides conjugated to a particle or core (e.g., a polymer, branched polymer, dendritic macromolecule, or equivalent structure). Contacting the polymer-nucleotide conjugate with a polymerase and an initiated target nucleic acid can lead to the formation of a detectable ternary complex, thereby enabling more accurate determination of the target nucleic acid bases.

[0344] When polymer-nucleotide conjugates are used instead of single unconjugated or untethered nucleotides to form complexes with polymerases and target nucleic acids, the local concentration of nucleotides increases many-fold, which in turn enhances signal strength, particularly compared to the correct signal from a mismatch. The polymer-nucleotide conjugates described herein may include at least one polymer-nucleotide conjugate for interaction with target nucleic acids. Multivalent compositions may also include two, three, or four different polymer-nucleotide conjugates, each having a different nucleotide conjugated to the particle.

[0345] In polymer-nucleotide conjugates having either a polymer-nucleotide conjugate form or a core-nucleotide conjugate form, multiple copies of the same nucleotide can be covalently or non-covalently bound to the particles. Examples of particles can include branched polymers; dendritic macromolecules; cross-linked polymer particles, such as agarose, polyacrylamide, acrylates, methacrylates, cyanoacrylates, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles; or any other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particles are branched polymers.

[0346] Nucleotides can be linked to particles or the core via linkers, and nucleotides can be attached to an end or site of the polymer. Nucleotides can be conjugated to particles via their bases or 5' ends. In some polymer-nucleotide conjugates, one nucleotide is attached to an end or site of the polymer. In some polymer-nucleotide conjugates, multiple nucleotides are attached to an end or site of the polymer. The conjugated nucleotides can spatially access one or more proteins, one or more enzymes, and nucleotide-binding moieties. In some embodiments, the nucleotides can be provided separately from the nucleotide-binding moieties, such as polymerases. In some embodiments, the linker does not include a light-emitting group or a light-absorbing group.

[0347] The particles or core may also have binding portions. In some embodiments, the particles or core can self-associate without using separate interacting portions. In some embodiments, the particles or core may self-associate due to buffering or salt conditions, for example, in the case of calcium-mediated interactions of hydroxyapatite particles, lipid or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

[0348] Polymer-nucleotide conjugates may have one or more labels (e.g., detectable reporting portions). Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radiolabels, or other such labels that enable the composition to be detected by methods known in the field of macromolecular or molecular interactions. Labels may be attached to nucleotides (e.g., by attachment to a base or 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to a PEG subunit), or to the core (e.g., to a streptavidin core), to the ends of the polymer, to the central portion, or to any other location within the polymer-nucleotide conjugate that a person skilled in the art would recognize as sufficient to enable the composition, such as the particle, to be detected by methods known in the art or described elsewhere...

Claims

1. A method for in situ analysis of target nucleic acid sequences, the method comprising: (a) Contacting a cell or tissue with a detectable nucleotide conjugate under conditions suitable for forming a binding complex within the cell or tissue, wherein the cell or tissue comprises a plurality of nucleic acid molecules, the plurality of nucleic acid molecules comprising the target nucleic acid sequence, the target nucleic acid sequence being primer-hybridized, wherein the detectable nucleotide conjugate comprises a polymer core, at least two nucleotide portions connected to the polymer core via a linker, and a detectable portion, and wherein the binding complex is formed between (i) at least two nucleotide portions of the detectable nucleotide conjugate and (ii) at least one nucleotide of each of the target nucleic acid sequences of at least two of the plurality of nucleic acid molecules; (b) Detect the binding complex; and (c) Identifying the target nucleic acid sequence by performing (a) and (b) on at least two other nucleotides in the target nucleic acid sequence of the at least two of the plurality of nucleic acid molecules. The method described herein is for non-diagnostic purposes.

2. The method of claim 1, wherein the cells or the tissue are fixed to the inner surface of the flow cell.

3. The method of claim 2, wherein the inner surface of the flow cell comprises one or more hydrophilic polymer layers.

4. The method of claim 3, wherein the one or more hydrophilic polymer layers comprise a polymer of polyethylene glycol (PEG).

5. The method of claim 4, wherein the one or more hydrophilic polymer layers comprise a branched polymer.

6. The method of claim 2, wherein the inner surface has a water contact angle of less than or equal to 45 degrees.

7. The method of claim 1, further comprising permeating the tissue or the cells prior to the contact in (a).

8. The method of claim 2, wherein the image of the inner surface exhibits a CNR greater than or equal to 10 when the contrast-to-noise ratio (CNR) is measured in such a manner as follows: (a) Contacting the inner surface with fluorescently labeled nucleotide molecules, the fluorescently labeled nucleotide molecules comprising a nucleic acid sequence complementary to at least a portion of a capture oligonucleotide immobilized on the inner surface; and (b) Following (a), the inner surface is imaged using an inverted microscope and camera under non-signal saturation conditions while the inner surface is immersed in a buffer solution.

9. The method of claim 1, wherein the identification of the target nucleic acid sequence in (c) is performed with a base determination accuracy characterized by a Q score greater than 25 for at least 80% of the identified nucleotides.

10. The method of claim 1, further comprising determining the spatial location of the target nucleic acid sequence within the cell or the tissue.

11. The method of claim 1, further comprising determining the cell type of the cell based at least in part on the identification of the target nucleic acid sequence in (c).

12. The method of claim 1, further comprising determining the tissue type of the tissue based at least in part on the identification of the target nucleic acid sequence in (c).

13. The method of claim 1, wherein the detectable nucleotide conjugate comprises: (a) a common core; and (b) The at least two nucleotide moieties coupled to the common core, wherein the common core comprises a polymer, micelles, liposomes, microparticles, nanoparticles or quantum dots.

14. The method of claim 13, wherein the common core is spherical.

15. The method of claim 1, wherein the detectable portion is coupled to the polymer core.

16. The method of claim 1, wherein the detectable nucleotide conjugate in (a) is included in a mixture of a plurality of detectable nucleotide conjugates, wherein each of the plurality of detectable nucleotide conjugates comprises a nucleotide motif of a different type from each other.

17. The method of claim 16, wherein the different types of nucleotide moieties comprise at least three different types of nucleotide moieties.

18. The method of claim 1, wherein the at least two nucleotide moieties do not contain a blocking group coupled thereto.

19. The method of claim 1, wherein the plurality of nucleic acid molecules contain blocking groups sufficient to prevent the incorporation of the at least two nucleotide moieties into the at least two target nucleic acid sequences.

20. The method of claim 1, wherein the tissue is cancerous tissue.

21. The method of claim 1, wherein the cell is a cancer cell.

22. The method of claim 1, wherein detecting the binding complex comprises imaging the cell or the tissue using one or more image sensors.

23. The method of claim 22, wherein the one or more image sensors comprise a photodetector array.

24. The method of claim 1, further comprising washing the cells or tissue after (b) to remove the detectable nucleotide conjugate from the cells or tissue.

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