WO2024173380A2

WO2024173380A2 - Method for acquiring cellular spatial information using light-sensitive dna barcodes

Info

Publication number: WO2024173380A2
Application number: PCT/US2024/015575
Authority: WO
Inventors: Robert PISCOPIO; Alex CHIALASTRI; Max Wilson; Siddharth DEY
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-02-13
Filing date: 2024-02-13
Publication date: 2024-08-22
Anticipated expiration: 2025-08-13
Also published as: WO2024173380A3

Abstract

Many embodiments described herein employ photocleavable oligonucleotides that can be used to encode cells for spatial locality within a field, which can be assessed during downstream nucleic acid analysis (e.g., sequencing). Such oligonucleotides can be referred to as Photocleavable Hashtag Oligos (PHOs). Further embodiments attach these photocleavable oligonucleotides to a cell membrane. Such attachment can include permanent labelling of cells, such as through covalent bonding, strong antibody interactions or strong biotin-streptavidin linkage, or less permanent labelling through the use of cholesterol anchors.

Description

Method for Acquiring Cellular Spatial Information Using Light-Sensitive DNA Barcodes

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/484,701 , entitled “Method for Acquiring Cellular Spatial Information Using Light- Sensitive DNA Barcodes,” filed February 13, 2023, the disclosures of which is incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to assessing spatial and genetic information regarding a cell, and specifically, methods and systems using photocleavable nucleic acids for identifying spatial location when performing transcriptom ic, genomic, epigenomic and proteomic analysis.

BACKGROUND OF THE DISCLOSURE

[0003] The organization of cells within tissue plays a key role in tuning cellular function, and several methods have recently been developed to capture the transcriptome of single cells while retaining positional information. However, these methods typically utilize a spot or grid type arrangement to obtain spatial information, which fail to capture tissue information between individual spots or grids and lack single-cell resolution, as each spot or grid position tends to obtain nucleic acids from multiple cells. These grid-type arrangements are constrained by the size of predesigned arrays or devices and do not allow for dynamically controlling spatial resolution, which can assist with better analyzing complex tissue morphology. Additionally, these methodologies tend to capture mRNA molecules with low efficiency, making it difficult to capture unique mRNA molecules, as compared to established scRNA-seq methods. Further, these methods are limited to profiling within fixed cells, rather than live cells and therefore cannot capture tissue dynamics in real time and further lack the ability to quantify single cell epigenomic and transcriptom ic information, thus limiting the ability to understand spatial effects on epigenetic landscapes and gene regulation. [0004] Given the above limitations, a need exists for a methods and systems that can characterize cells, fixed or live, based on position, while maintaining and enabling analysis of mRNA, genomic DNA, the epigenome, and proteome.

SUMMARY OF THE DISCLOSURE

[0005] This summary is meant to provide examples and is not intended to be limiting of the scope of the invention in any way. For example, any feature included in an example of this summary is not required by the claims, unless the claims explicitly recite the feature.

[0006] In some implementations, a cellular labelling system comprises a first nucleic acid molecule comprising a first nucleic acid oligo having a first sequence, a second nucleic acid oligo having a second sequence, and a photocleavable moiety linking the first nucleic acid oligo and the second nucleic acid oligo.

[0007] In some implementations, a cellular labelling system comprises an anchoring system linked to the first nucleic acid molecule configured to anchor the first nucleic acid molecule to a cellular membrane.

[0008] In some implementations, a cellular labelling system comprises a second nucleic acid that links the anchoring system to the first nucleic acid molecule. A portion of the first nucleic acid molecule has a sequence that is complementary to a sequence of a portion of the second nucleic acid molecule. The first nucleic acid and the second nucleic acid are bound via the complementary sequences.

[0009] In some implementations, a cellular labelling system comprises a third nucleic acid molecule linked to the anchoring system. A portion of the third nucleic acid molecule comprises a sequence that is complementary to a sequence of a portion of the second nucleic acid molecule. The second nucleic acid and the second nucleic acid are bound via the complementary sequences.

[0010] In some implementations, the anchoring system comprises two cholesterol moieties capable of integrating within a membrane of a cell. The second nucleic acid is linked to a first cholesterol moiety and the third nucleic acid is linked to a second cholesterol moiety. [0011] In some implementations, the anchoring system comprises concanavalin A, biotin, and streptavidin, wherein the concanavalin A is configured to bind to a sugar moiety. A biotin molecule is linked to the concanavalin A. A biotin molecule is linked to the first nucleic acid molecule. Streptavidin is configured to bind to the concanavalin A and to the first nucleic acid molecule via biotin, resulting in an anchoring of the first nucleic acid molecule to a cellular membrane.

[0012] In some implementations, the anchoring system comprises biotin, streptavidin, and a biotinylation reagent. The biotinylation reagent is utilized to biotinylate cellular components. A biotin molecule is linked to the first nucleic acid molecule. Streptavidin is configured to bind to the first nucleic acid molecule and to biotinylated cellular components via biotin, resulting in an anchoring of the first nucleic acid molecule to a cellular membrane.

[0013] In some implementations, the biotinylation reagent comprises N- hydroxysuccinimide (NHS).

[0014] In some implementations, the anchoring system comprises an antigen-binding domain of an antibody, biotin, and streptavidin. The antigen-binding domain is configured to target and bind a cellular component. A biotin molecule is linked to the antigen-binding domain. A biotin molecule is linked to the first nucleic acid molecule. Streptavidin is configured to bind to the antigen-binding domain and to the first nucleic acid molecule via biotin, resulting in an anchoring of the first nucleic acid molecule to the cellular component.

[0015] In some implementations, the first nucleic acid oligo and the second nucleic acid oligo each comprise a barcoding sequence.

[0016] In some implementations, the barcoding sequence is between 4 and 10 nucleotides in length.

[0017] In some implementations, the first nucleic acid oligo and the second nucleic acid oligo each further comprise a unique molecular identifier sequence and a capture sequence.

[0018] In some implementations, the cellular labelling system further comprises a first fluorophore that is linked to either the first nucleic acid oligo or the second nucleic acid oligo. [0019] In some implementations, the cellular labelling system further comprises a second fluorophore. The first fluorophore is linked to the first nucleic acid oligo and the second fluorophore is linked the second nucleic acid oligo.

[0020] In some implementations, the first nucleic acid molecule further comprises a third nucleic acid oligo having a third sequence and a second photocleavable moiety linking the second nucleic acid oligo and the third nucleic acid oligo.

[0021] In some implementations, the cellular labelling system further comprises a blocking nucleic acid molecule bound to the first nucleic acid molecule. The blocking nucleic acid molecule comprising a first blocking nucleic acid oligo having a first blocking sequence, a second blocking nucleic acid oligo having a second blocking sequence and a flexible linker linking the first blocking nucleic acid oligo and the second blocking nucleic acid oligo. A portion of the first blocking sequence is complementary to a portion of the first sequence and a portion the second blocking sequence is complementary to a portion of the second sequence.

[0022] In some implementations, a method for labelling a field of cells comprises contacting a cellular labelling system to a field of cells such that cells within the field are labeled via the labelling system. The cellular labelling system comprises a first nucleic acid molecule comprising a first nucleic acid oligo having a first sequence, a second nucleic acid oligo having a second sequence, and a photocleavable moiety linking the first nucleic acid oligo and the second nucleic acid oligo. The cellular labelling system comprises an anchoring system linked to the first nucleic acid molecule, wherein the anchoring system is configured to anchor the first nucleic acid molecule to a cellular membrane.

[0023] In some implementations, a method for labelling a field of cells comprises illuminating a pattern of light onto the field of cells resulting in cleavage of the second nucleic acid oligo off of the first nucleic acid molecule via the photocleavable moiety from at least a portion of the first nucleic acid molecules.

[0024] In some implementations, a method for labelling a field of cells comprises identifying a spatial location of a plurality of cells within the field of cells based on: an amount of the first nucleic acid molecules comprising a first nucleic acid molecule and a second nucleic acid molecule, an amount of the first nucleic acid molecules comprising a first nucleic acid molecule and lacking a second nucleic acid molecule, and the pattern of light illuminated onto the field of cells.

[0025] In some implementations, identifying a spatial location of a plurality of cells within the field of cells comprises performing single-cell sequencing on the plurality of cells to determine the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and a second nucleic acid oligo and the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and lacking a second nucleic acid oligo.

[0026] In some implementations, the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and a second nucleic acid oligo and the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and lacking a second nucleic acid oligo is determined by a first barcoded sequence within the first sequence and a second barcoded sequence with the second sequence.

[0027] In some implementations, a method for labelling a field of cells further comprises the field of cells into single cells prior to performing single-cell sequencing.

[0028] In some implementations, performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell RNA sequencing on the plurality cells.

[0029] In some implementations, performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell DNA methylation sequencing on the plurality cells.

[0030] In some implementations, performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell DNA accessibility sequencing on the plurality cells.

[0031] In some implementations, the cellular labelling system further comprises a first fluorophore that is linked to the second nucleic acid oligo. Identifying a spatial location of a plurality of cells within the field of cells comprises identifying a presence of the first fluorophore via fluorescence.

[0032] In some implementations, the cellular labelling system further comprises a second fluorophore that is linked to the first nucleic acid oligo. Identifying a spatial location of a plurality of cells within the field of cells comprises identifying a ratio of an amount the first fluorophore to an amount of the second fluorophore via fluorescence. [0033] In some implementations, illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying light intensities onto the field of cells.

[0034] In some implementations, illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying duration of illumination onto the field of cells.

[0035] In some implementations, illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying wavelength bands onto the field of cells.

[0036] In some implementations, illuminating a pattern of light onto the field of cells comprises use of a digital micromirror device to generate the pattern of light.

[0037] In some implementations, the field of cells comprises an adherent cell culture, an organoid culture, tissue, or cell applied to an optical medium.

[0038] In some implementations, the steps of: contacting a cellular labelling system to a field of cells such that cells within the field are labeled via the labelling system and illuminating a pattern of light onto the field of cells are iteratively repeated at least once. Each time the steps are repeated, the steps are performed with a labelling system comprising unique barcoding sequences and with a unique pattern of light.

[0039] The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A and 1 B illustrate schematics of an example of a cellular labelling system in accordance with many embodiments.

[0041] Figures 2A-2D illustrate schematics of examples of cellular labelling systems using biotin-streptavidin interactions in accordance with many embodiments.

[0042] Figures 3A-3C illustrate schematics of examples and data showing the use of blocking oligos in accordance with many embodiments.

[0043] Figure 4 illustrates an example of Digital Light Processing (DLP) projection to illuminate cells to yield a particular pattern in accordance with many embodiments.

[0044] Figures 5A-5H provide example of illumination patterns and gradients on various fields of cells and tissues in accordance with many embodiments. [0045] Figures 6A-6C provide an example of an illumination pattern with single cell resolution in accordance with many embodiments.

[0046] Figures 7A-7F provide a schematic and results of an iterative labelling strategy with multiple unique Photocleavable Hashtag Oligos (PHOs) in accordance with many embodiments.

[0047] Figures 8A-8D provide schematics of examples of methods for distinguishing cells by position for assessing genetics, epigenetics, and/or transcriptom ics in accordance with many embodiments.

[0048] Figures 9A-9D provide violin plots showing that single-cell sequencing protocols utilizing PHO labeling yield similar results to standard single-cell sequencing protocols.

[0049] Figures 10A-10D provide violin plots showing the effect of light exposure on cleavage of PHOs based on ratios of PHO barcode reads computed utilizing sequencing results.

[0050] Figures 11A-11 B provides data showing no bias is created by ultraviolet illumination in accordance with many embodiments.

[0051] Figures 12A-12B provides data showing that different cell types can be classified based PHO cleavage in addition to gene transcription, DNA accessibility, and DNA methylation.

[0052] Figures 13A-13C provides data showing that multiplexing PHO labels improves the ability to identify the spatial position of cells.

[0053] Figures 14A-14C provides data showing that single-cell methyl/accessibility sequencing protocols utilizing PHO labels yield results similar to standard single-cell methyl/accessibility sequencing protocols.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0054] Local environment and cellular position within a tissue or other environment has significance for cellular function. However, spatially dissecting genetic, including epigenetic, and “omic” (e.g., transcriptom ics, genomics, genome organization, proteomics, etc.) data for analysis from live cells has proven to be very difficult. Many embodiments described herein overcome these hurdles utilizing photocleavable oligonucleotides that can be activated in spatial manner to encode a particular location, which can be assessed during downstream nucleic acid analysis (e.g., sequencing). Such oligonucleotides can be referred to as Photocleavable Hashtag Oligos (PHOs). In some embodiments, photocleavable oligonucleotides are attached to a cell membrane. Such attachment can include permanent labelling of cells, such as through covalent bonding, antibody interactions or streptavidin-biotin binding, or less permanent labelling through the use of cholesterol anchors.

[0055] Once cells of a tissue are labeled with photocleavable oligonucleotide, a subset of cells can be selectively illuminated with light, or a pattern of light, resulting in cleavage of the PHO. When sequencing is performed on the tissue, cells can be differentiated by whether they are encoded with a full-length oligonucleotide tag or partial-length (i.e., cleaved) oligonucleotide tag (or by ratios thereof). Light illumination can be two dimensional, such as by illuminating multiple patterns of light. Alternatively, illumination of a light pattern can be combined patterns of particular light wavelengths, particular intensities, and/or particular exposure times to yield a multiplex of cleavage reactions of PHOs, resulting in an ability to yield multiple labels to demarcate a plurality of different localities.

[0056] Figures 1A and 1 B illustrate schematics of an example of a cellular labelling system in accordance with many embodiments. Specifically, Figure 1A provides an example of a label 100 including a PHO 102 attached to a cholesterol modified oligonucleotide (CMO) anchor system 104 and Figure 1 B provides a detail of various sequences that can be included within PHO 102. In many embodiments, PHO 102 includes an inner sequence 106 and an outer sequence 108 that are joined by photocleavable moiety 110. Inner sequence 106 and outer sequence 108 can contain a sequence (e.g., a 20-nucleotide sequence) for amplification, including PCR, RCA, and/or any other applicable method of nucleic acid amplification. Additionally, inner sequence 106 and outer sequence 108 can contain a barcoding (BC) sequence, a unique molecular identifiers (UMI) sequences, and/or a capture sequence (e.g., poly-adenosine (pA) sequence). Photocleavable moiety 110 can be reactive to one or more bands of various wavelengths of light, including ultraviolet, infrared, and/or visible wavelengths.

[0057] Sequences for barcodes and/or unique identifiers can range in size depending on how many unique identifiers are needed or desired — for example 4-10 nucleotides in length, including 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or longer. A capture sequence can be of any size to allow for capture by a complementary sequence (e.g., 15-30 nucleotides in length). One example of a capture sequence is a string of poly-A, which can be captured by a string of poly-T.

[0058] By having two (or more) barcoding sequences that are each separated by photocleavable moiety 110, spatial locality of cells can be determined using light energy. Cells (or tissues) within a field can be labelled with PHO 102, in which the PHO extends away from the cell. Light energy can be impinged upon PHO 102 to cleave photocleavable moiety 110. When photocleavable moiety 110 is cleaved, inner sequence 106 remains attached to the cell and outer sequence 108 is released into solution, which can be washed away or otherwise removed. Cells that received light energy can be differentiated from cells that did receive light energy by the presence of the outer sequence (or a ratio of the amount of outer sequence present). Illuminating light in a particular spatial pattern can differentiate cells within one spatial location from another spatial location. Particular regions of interest can be differentially assessed in downstream applications (e.g., sequencing) based on presence of an outer sequence.

[0059] The example in Figure 1A includes a CMC anchor system that anchors the PHO to a membrane of the cell. In many embodiments, CMO anchor 104 comprises an oligonucleotide 112 linked to a cholesterol moiety 114. The cholesterol moiety 114 can be an amphiphilic cholesterol-TEG modification that is capable of integrating with the lipid bilayer of a cell’s membrane to label said cell. In some embodiments, a CMO anchor system comprises a second CMO co-anchor 116. CMO co-anchor 116 can be provided that is complimentary to CMO anchor 104 to allow for a more secure anchoring of a CMO and PHO to the cell membrane.

[0060] In various embodiments, alternative anchoring systems are utilized to perform PHO labelling, such as universal surface biotinylation (USB), NHS-biotin chemistry, biotin-conjugated concanavalin A-based sample barcoding (CASB), click chemistry, streptavidin and biotin-modified oligos (BMOs), or any other anchoring system for controlled attachment of nucleic acid molecules to cellular components such that the anchoring system anchors the PHO to a membrane of the cell. [0061] Figures 2A-2C illustrate examples of anchoring systems, where Figure 2A provides an example of universal surface biotinylation (USB) labelling, Figure 2B provides an example of CASB labelling, and Figure 2C provides an example of antibody labelling. One advantage of universal surface biotinylation (USB) labelling is that an irreversible amide bond is formed between free amines of cell surface proteins that and biotin, which can then be used to label the cell with a PHO conjugated to streptavidin (Figure 2A). Compared to a CMO anchor-based method, which relies on hydrophobic interactions of cholesterol to embed the molecule, the USB methodology yields a stronger linkage between a cell and PHO. One advantage of CASB labelling is that concanavalin A can bind to sugar molecules on a cell surface (e.g., glycoproteins) and click chemistry can be used to form similar, but stronger binding through covalent bond formation. Concanavalin A can be further conjugated with biotin, which can then be used to label the cell with a PHO bound to streptavidin (Figure 2B). One advantage of antibody labeling is that the antibody can specifically target and bind particular cellular constituents, thus particular cell-types and/or cells with a particular status can be labelled. An antibody can be further conjugated with biotin, which can then be used to label the cell with a PHO conjugated to streptavidin (Figure 2C). It should be understood that fragments of binding proteins can be utilized, assuming the fragment comprises the structure for providing the binding activity (e.g., variable regions of antigen-binding domain of an antibody).

[0062] Figure 2D provides an example of a method for performing universal surface biotinylation (USB) via a biotinylation reagent such as (for example) N- hydroxysuccinimide (NHS). In this example, NHS is contacted with a cell (which may require NHS to be dissolved in an organic solvent such as DMSO or DMF, and then added to a solution in contact with the cell). Free amines of proteins that come in contact with NHS form an amide bond with biotin, yielding a cell labelled with biotin species. The labelled cell can then be contacted with a PHO bound to streptavidin, labelling the cell with numerous PHO species.

[0063] In several embodiments, PHOs are utilized to label a field of cells. A field of cells is to be understood to be any collection of cells having a spatial area and/or volume. The cells can be in vitro or in vivo, alive or fixed, 2-dimensional, or 3-dimensional. Examples include, but are not limited to adherent cell cultures, organoid cultures, tissues, and cells applied to an optical medium (e.g., microscope slide).

[0064] It should be noted that the labeling strategies illustrated in Figure 1 A and Figures 2A-2D are merely examples. Further examples include a PHO comprising more than two barcode sequences — e.g., inner, middle, and outer sequences; first, second, third, and fourth sequences; and up to any number of sequences. Each barcoded sequences can be separated with additional photocleavable moieties or any other cleavable moiety. In some implementations, when multiple photocleavable moieties are utilized, cleavage of each photocleavable moiety can be independently controlled using different wavelengths of light, different intensities, and/or different exposure times.

[0065] A PHO can comprise one or more photocleavable moieties. Generally, a photocleavable moiety is chemical structure that disassociates upon exposure to light energy. Several various photocleavable moieties are known and can be utilized in accordance with the various embodiments of PHO molecules. A particular photocleavable moiety generally is cleaved by one or more particular bands of wavelengths, and thus the choice of photocleavable moiety should be paired with light energy that is capable to cleave a PHO. In various embodiments, a photocleavable moiety is cleaved via ultraviolet light (e.g., less than 405 nm), a photocleavable moiety is cleaved via green light (e.g., more than 400 nm and less than 530 nm), or a photocleavable moiety is cleaved via red light (e.g., more than 500 nm and less than 620 nm). Particular wavelengths bands can be determined by the particular photocleavable moiety used. For more on photocleavable moieties and examples of moieties that can be utilized, see, e.g., P. J. LeValley, et al., Am Chem Soc. 2020 Mar 11 ;142(10):4671 -4679; T. L. Rapp and C. A. DeForest, Nat Commun. 2023 Aug 29; 14(1 ):5250; and M. J. Hansen, hem Soc Rev. 2015 Jun 7;44(11 ):3358-77; the disclosures of which are incorporated herein by reference).

[0066] In various embodiments, multiple dimensions of information and greater spatial resolution can be obtained through iterative spatial barcode labeling and subsequent addition of blocking oligos (BO). As illustrated in Figure 3A, BOs can include a complementary sequences to the inner and outer portions of a PHO that are linked together via a linker (e.g., a triethylene glycol (TEG) spacer). In some implementations, the linker is flexible and has a length such that it can configure to the dimensions of the photocleavable moiety on a PHO to allow for adequate complementation between the BO and the PHO. The BOs anneal to PHOs to lock an outer sequence to inner sequence, preventing dissociation of the outer sequence after photocleavage. By controlling the number of BOs supplied, the dissociation ratio of the outer sequence can be controlled. Figure 3B illustrates exemplary data showing that stepwise increases of BO concentrations yield a stepwise prevention of outer sequence release. After labelling a field cells with PHOs, multiple concentrations of BOs can be added in a spatially controlled manner to specifically label regions, which can be determined by the dissociation ratio of the outer sequence. Several rounds of spatial labelling can be done following this strategy. For example, as illustrated in Figure 3C, eight rounds of sequential labelling with PHOs and BOs and three discrete illumination levels provides a spatial resolution of 81x81 pixels within a field.

[0067] Additionally, the complementary binding interaction of BOs can be made more efficient and robust using nucleosides that form covalent bonds with its complementary nucleoside when illuminated with light. For example, 3-cyanovinylcarbazole nucleoside (CNVK) forms a covalent bond with its complement after illumination with UV light. In another example, the photocleavable moiety can be chemically modified to reduce and/or prevent cleavage. In some embodiments, an enzyme is utilized to modify a photocleavable moiety. For example, nitroreductase (NTR) enzymes can perform a reduction reaction upon the photocleavable moiety resulting in a chemical change in the moiety that prevents cleavage when illuminated by light illumination.

[0068] Once labelled, subsets of cells within a field can be controllably illuminated. Illumination can be performed via any source for providing light energy (e.g., lasers, diodes, tungsten, halogen, xenon, etc.). Projection can be controlled with any relevant process, such as DLP technologies using mirrors, filters, lenses, prisms, and/or any other means for directing and controlling light. In some embodiments, a digital micromirror device (DMD) is utilized to project patterns of light. Figure 4 illustrates an example of using DLP projection to controllably illuminate cells with a pattern, while Figures 5B-5H provide example of illuminated various fields of cells to yield various patterns and results. [0069] Visualization of labelling of a field of cells spatially controlled cleavage can be visualized by fluorescently labelling the inner and/or outer sequences of a PHO. Provided in Figure 5A is an example of a PHO that comprises an inner sequence with an inner fluorophore and an outer sequence with an outer fluorophore. A cell that is labelled with the PHO can emit light provided by the inner fluorophore and outer fluorophore. A cell that labelled with the PHO and then illuminated with light energy to cleave the outer sequence can only emit light provided by the inner fluorophore, as the outer fluorophore is cleaved and removed.

[0070] Figures 5B to 5D provide an example of spatially controlling light energy to selectively control cleavage PHO that has been labelled onto cells. Figure 5B provides a pattern that is utilized to selective control micromirrors of an array of micromirrors. Figure 5C and 5D provide results of specifically cleaving PHO in accordance with the pattern of shown in Figure 5B. In particular, Figure 5C shows a field of HeLa cells that have been labelled with a PHO as shown in Figure 5A, having an inner fluorophore (Cy3) and an outer fluorophore (Cy5). Upon selective illumination of the pattern, HeLa cells that received the illuminated light released the outer fluorophore (Cy3 only, depicted in blue) whereas the rest of the HeLa cells remained labelled with the outer fluorophore (Cy3 and Cy5, depicted in red). Figure 5D is a low-melting point agarose embedded mouse tissue that was labelled with the PHO shown in Figure 5A and illuminated with the pattern of 5B. Again, cells of the tissue that received the illuminated light released the outer fluorophore (Cy3 only, depicted in blue) whereas the rest of the tissue remained labelled with the outer fluorophore (Cy3 and Cy5, depicted in red). Any type of tissue preparation can be utilized, such as (for example) flash frozen with OCT, flash frozen with low melting point agarose, 4% PFA with paraffin.

[0071] As noted previously, controlling of light intensity can be used to demarcate subsets of cells of cells in a field by varying the amount of light intensity within the field. Figures 5E-5F provide an example of using a light gradient to controllably cleave a relative amount of a PHO. Figure 5E provides an image of the light pattern utilized to illuminate a field of labelled HeLa cells. As can be appreciated, the light projected as a stripe in which a gradient of light intensity was provided, increasing from left to right along the stripe. Figure 5F depicts the results of illumination of the graded on the labelled HeLa cells, which were labelled with a PHO as shown in Figure 5A, having an inner fluorophore (Cy3) and an outer fluorophore (Cy5). Upon illumination of the gradient, HeLa cells that received the greatest amount of illuminated light released the outer fluorophore (Cy3 only, depicted in blue) and decreased in intensity along with the gradient with cells having the least amount of illuminated light retaining the outer fluorophore (Cy3 and Cy5, depicted in red). Figure 5G depicts a data graph of controlling and varying both the amount of light intensity provided and the duration of light provided. As can be readily interpreted from the graph, the ratio of outer fluorophore (Cy5) to inner fluorophore (Cy3) decreases as the intensity of light increases and as the duration of light illumination increases. Although Figures 5E- 5F provide an example of a gradient of illumination projected along the X-axis, any gradient pattern can be provided. And in addition to light intensity and duration of exposure, wavelength band of light can be controlled and varied to yield patterns and/or gradients.

[0072] Provided in Figure 5H is an example of labelling multiple cell types, including HeLA cells, HEK293T cells, and U2OS cells, showing that the methodology can be applied to a variety of cells types.

[0073] With the use of DMDs and other devices for precise patterning, light patterns can have single cell precision. Provided in Figure 6A is another example of PHO in which an outer fluorophore (Cy5) is provided on the outer sequence but the inner sequence does not include an inner fluorophore. Figure 6B provides data results of an experiment in which a light pattern was projected with single cells precision to yield a pattern of PHO cleavage with single cell resolution. In this experiment, a co-culture of U2OS and HeLa cells was created to yield a field of cells. The HeLa cells expressed an mCherry transgene. As can be seen in the left panel, both the U2OS and HeLa cells are labeled with the outer fluorophore (Cy5) prior to illumination. The spatial expression of mCherry expression was used to set a pattern of illumination via a DMD. As can be seen in the right panel and in the data graph of Fig. 6C, the cells that were illuminated (i.e., the mCherry expressing HeLa cells), have low signal for outer fluorophore (Cy5), indicating that the outer sequence was cleaved from the PHOs attached to those cells.

[0074] Once labelled and illuminated, the position of individual cells, such as through single-cell sequencing, can be identified. In various embodiments, multiple PHOs are utilized for multiplex labelling, including multiple rounds of labelling with PHOs and/or by using PHOs with multiple different photocleavable moieties separating multiple oligonucleotide strings. Figures 7A-7F illustrate an example of how to label a tissue with multiple unique PHOs via multiple rounds. Specifically, Figure 7A provides a schematic for iteratively illuminating quadrants of a field to generate a pattern of labels and Figure 7E shows images of photomasks of the field in accordance with this example of patterning method. In this example, a collection of cells is separated into four quadrants (i.e., Q1 , Q2, Q3, and Q4). A first PHO conjugated with an outer fluorophore of Cy5 (i.e., PHO1 ) is used label all four quadrants and Q1 remains unilluminated while Q2, Q3, and Q4 are illuminated, resulting in cleavage of PHO1 in those quadrants. A second PHO conjugated with an outer fluorophore of FAM (i.e., PHO2) is used label all four quadrants and Q1 and Q2 remain unilluminated while Q3 and Q4 are illuminated, resulting in cleavage of PHO2 in those quadrants. A third PHO conjugated with an outer fluorophore of Cy3 (i.e., PHO3) is used label all four quadrants and 01 , Q2, and 3 remain unilluminated while 04 is illuminated, resulting in cleavage of PHOs in that quadrant. Figure 7B provides a summary of the readout, based on the selective cleavage of the three PHOs within the four quadrants. Figures 7C and Figure 7E provide the resulting fluorescence pattern for each of the quadrants after performing the sequential method as described. Figure 7D provides a 3D plot of sequencing results of outer-to-inner barcode read counts yield from the labelling and illumination strategy. Figure 7F provides a 3D plot of fluorescence intensity values results of the outer fluorophore from the labelling and illumination strategy. As can inferred from the results, sequential use of PHOs and patterned illumination can specifically label subsets of cells that are able to be differentiated via fluorescence imaging and downstream sequencing analysis.

[0075] Utilizing PHO labelling on cells and tissues, the underlying genetics, epigenetics, transcriptom ics, proteomics, and genomics of the labelled cells can be analyzed, maintaining an identification of spatial origin. Figures 8A to 8D provide schematics of examples of method to spatially distinguish cells by position when assessing genetics and transcriptomics.

[0076] In Figure 8A, Method 800 begins by labelling cells 802 with PHO labels 100. Cells 802 are illuminated with a pattern 804 in which a subset of cells are illuminated and a subset of cells remain unilluminated. Because of the photocleavable moiety, unilluminated cells 803 will possess both inner and outer sequences of label 100, while illuminated cells 805 will possess just an inner sequence of label 100. It should be noted that light intensity, duration of illumination, and/or wavelength band can be controlled and varied to yield a ratio of cleaved and non-cleaved labels 100. Upon labelling two or more subsets of cells based on an illumination pattern, the cells can be dissociated and isolated into single cells and assessed for single-cell transcription assessment by preparing a sequencing library comprising cDNA molecules of mRNA and PHOs. The sequencing library can be optionally separated by beads into an mRNA library and PHO library, and then sequenced via a next-generation sequencing technique.

[0077] Figure 8B provides an example of a method to generate a library of PHO molecules for a next-generation sequencing platform. First, uncleaved and cleaved PHO are reverse transcribed using a capture primer (e.g., poly-T primer) that contains a promoter (e.g., T7 promoter), a cell specific barcode, a UMI, adapter sequences (e.g., RA5 handle), and a complementary capture sequence (e.g., poly-T sequence). Second strand synthesis is subsequently performed, and PHO library fragments are optionally separated from endogenous transcript cDNA fragments using SPRI beads. Next, adapter sequences with sequencing primers are added to amplify and extend PHO molecules to generate sequencing libraries.

[0078] Figure 8C provides an example of a method to generate a library of PHO molecules on a droplet-based 10X platform. First, uncleaved and cleaved PHO are reverse transcribed using a capture primer (e.g., poly-T primer) that contains an Illumina TruSeq read 1 handle, a cell specific 10X barcode, a UMI, and a capture sequence (e.g., poly-T sequence). Next, a template switching oligo (TSO) is added and transcripts are extended. Prior to cDNA amplification, PHO library fragments are optionally separated from endogenous transcript cDNA fragments using SPRI beads. Next, adapter sequences with sequencing primers are added to amplify and extend PHO molecules to generate sequencing libraries.

[0079] Figure 8D provides an example of a method for concurrent transcriptom ic, epigenomic, and spatial library processing using PHO labelling of cells. First, cells are incubated with PHOs and illuminated with light patterns. Cells of the field are then dissociated and isolated into single cells, sorted into 384-well plates, and lysed. The following two steps are then performed simultaneously - (1) mRNA is reverse transcribed and the ‘inner’ and ’outer’ DNA sequences of PHOs are copied using a capture primer with an overhang containing a cell- and mRNA/PHO-specific barcode, a UMI, a sequencing adapter, and a T7 promoter; and (2) a methyltransferase (e.g., M.CviPI) is used to methylate cytosines in a GpC context within open chromatin. Next, after second strand synthesis (i), gDNA chromatin is stripped using protease (ii), 5hmC sites in the genome are glucosylated to block downstream detection by the restriction enzyme MspJI (iii), and MspJI is subsequently added that recognizes methylated cytosines in the genome and creates double-stranded (ds)-DNA breaks (iv). The excised gDNA molecules are ligated to ds-adapters containing a cell and genomic DNA (gDNA)-specific barcode, a UMI, a sequencing adapter and a T7 promoter (v). Following this, as all molecules are tagged with cell- and molecule-of-origin specific barcodes, individual wells of the 384-well plate are pooled (vi), and the PHO molecules are optionally separated from barcoded endogenous mRNA- and gDNA-derived molecules using SPRI beads (vii). PHO-derived molecules are then amplified by PCR to generate spatial Illumina libraries (viii). The mRNA- and gDNA-derived molecules are amplified by IVT (ix), followed by a further enrichment using pA biotin/streptavidin coated beads to separate mRNA and gDNA molecules (x). Finally, the remaining mRNA and gDNA molecules are amplified (xi) to generate endogenous transcriptome and epigenome sequencing libraries.

[0080] Based on a resultant ratio of cleaved (e.g., inner sequences) and non-cleaved (e.g., inner and outer) sequences, position of a cell, within a confidence interval, can be determined for a cell. Using a poly-T capture sequence, mRNA and PHO sequences can be captured simultaneously. Reverse transcription can be used to create cDNAs from the endogenous mRNA. After separation of cDNAs and PHOs, PCR or other amplification be used to amplify PHO sequences (e.g., inner and outer sequences). Quantitative PCR can be used to identify and/or quantify PHO sequences and/or sequencing can be used to simultaneously generate read counts for PHO sequences and endogenous nucleic acid sequences (e.g., RNA, DNA, etc.). Numerous sequencing platforms and library construction methods are compatible with the methods described herein, including 10X Genomics, Illumina, PacBio, and/or any other sequencing platform of interest.

[0081] Cell extrinsic signaling cues, tissue morphogenesis, and epigenetic landscapes play a key role in regulating cell type-specific gene expression programs in mammalian tissues. This process involves direct chemical modification to DNA, DNA binding to histone proteins, and chemical modifications to histones. Therefore, some embodiments enable spatially-resolved joint profiling of the proteome, epigenome, genome, and transcriptome from the same cell. The methodologies described herein provide downstream modularity allowing for genomic, epigenomic, and/or proteomic measurements and can be coupled with 10X Genomics droplet-based technologies enabling high-throughput cell partitioning and measurements.

Experimental Results

[0082] Utilizing the various systems and methods of the disclosure, experiments were performed to test the ability these systems and methods to spatially label and analyze cells.

[0083] Provided in Figures 9A to 9D are sequencing results from performing sequencing protocols utilizing PHOs as indicators of spatial labelling of cells. Provided in Figures 9A- 9B are violin plots of the number of genes detected per cell, number of transcripts detected per cell, and number of PHO detected per cell using a plate-based sequencing protocol (Figure 9A) and droplet-based cell capture protocol (Figure 9B). These results suggest that robust quantification of the transcriptome and PHOs can be determined using either sequencing protocol. Figures 9C and 9D provide violin plots assessing the number genes detected per cell and the number of UMI detected per cell, comparing standard single cell RNA sequencing (scRNA-seq) protocols using PHO labelling of cells (scSTAMP-seq), and protocols using PHO labelling of cells combined with methylation sequencing analysis (scSTAMP-MAT-seq). As can be inferred from the resultant data, PHO labeling and adding methylation analysis to sequencing protocols had no significant effect on detection of genes and UM Is.

[0084] Provided in Figures 10A-10D are violin plots depicting results of experiments assessing the effect of illumination exposure time on cleavage of PHO labels. Figures 10A-10B exposed UV light on cells via floodlight, and PHO cleavage was determined by sequencing using a plate-based sequencing protocol (Figure 10A) and droplet-based cell capture protocol (Figure 10B). Figure 10C exposed UV light on cells via DLP, and PHO cleavage was determined by sequencing using a plate-based sequencing protocol. In each experiment, greater exposure time resulted in a lower ratio of outer-to-inner PHO barcode ratios, suggesting that these ratios can be controlled by illumination exposure time to differentiate cells in accordance with a spatial pattern. The results also suggest that other mechanisms to control PHO cleavage, such as illumination intensity and illumination wavelength band can be utilized to differentiate cells based on outer-to-inner PHO barcode ratios. Figure 10D shows that even when six PHOs are sequentially multiplexed, each PHO has a similar level of cleavage in response to light exposure time. [0085] Provided in Figures 11 A-11 B are data plots assessing UV bias on the endogenous transcriptome of illuminated cells, as determined by sequencing using a plate-based sequencing protocol (Figure 11 A) and droplet-based cell capture protocol (Figure 11 B). Cells receiving full saturation of UV exposure to cleave PHOs (Max UV) were compared with cells receiving no UV exposure (<s> UV). Pearson’s correlation analysis and principal component analysis show that there is no differentiation of expression between the population of cells.

[0086] Provided in Figures 12A-12B are data plots assessing PHO cleavage as determined by sequencing. In Figure 12A, Hela and U2OS cells were labeled with PHOs and multiplex oligos (MPOs), which are similar to inner oligos of PHOs but lack a photocleavable moiety and an outer oligo. The principal component analysis shows that cells can be accurately classified based on their transcriptome, MPOs, or the fraction of PHOs cleaved. In Figure 12B, Hela and U2OS cells were labeled with PHOs and then assessed for gene transcription, DNA accessibility, and DNA methylation using a dropletbased cell capture protocol. The principal component analysis shows that cells can be accurately classified based on their gene expression, DNA accessibility, and DNA methylation, and further by the fraction of PHOs cleaved.

[0087] Provided in Figures 13A-13C are data plots showing that multiplexing of PHOs can be achieved and improves spatial assignment accuracy. Figure 13A provides violin plots demonstrating the error correction feature when multiple unique PHOs are applied simultaneously. Figure 13B shows that spatial accuracy improves the greater number of unique PHOs are added. Figure 13B provides a heatmap depicting the amount of PHO1 to PHO6 cleaved for each cell. [0088] Provided in Figures 14A-14C are data plots showing DNA accessibility and DNA methylation results of protocols utilizing PHOs to label cells. Figure 14A provides averaged single-cell DNA accessibility and DNA methylation profiles at DNase I hypersensitivity sites in Hela cells. Figures 14B-14C show that number of unique accessibility sites and number of unique methylation as a function of sequencing depth detected via single-cell methyl/accessibility sequencing protocols utilizing PHOs to label cells (scSTAMP-MAT-seq) was similar to standard single-cell methyl/accessibility sequencing protocols (sc-MAT-seq).

DOCTRINE OF EQUIVALENTS

[0089] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, several well-known processes and elements have not been described to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

[0090] Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the components or steps of the present invention may be made within the spirit and scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described herein, but, rather, is defined by the scope of the appended claims.

Claims

CLAIMS:

1 . A cellular labelling system, comprising: a first nucleic acid molecule comprising a first nucleic acid oligo having a first sequence, a second nucleic acid oligo having a second sequence, and a photocleavable moiety linking the first nucleic acid oligo and the second nucleic acid oligo; and an anchoring system linked to the first nucleic acid molecule configured to anchor the first nucleic acid molecule to a cellular membrane.

2. The cellular labelling system of claim 1 further comprising a second nucleic acid that links the anchoring system to the first nucleic acid molecule, wherein a portion of the first nucleic acid molecule has a sequence that is complementary to a sequence of a portion of the second nucleic acid molecule, wherein the first nucleic acid and the second nucleic acid are bound via the complementary sequences.

3. The cellular labelling system of claim 2 further comprising a third nucleic acid molecule linked to the anchoring system, wherein a portion of the third nucleic acid molecule comprises a sequence that is complementary to a sequence of a portion of the second nucleic acid molecule, wherein the second nucleic acid and the second nucleic acid are bound via the complementary sequences.

4. The cellular labelling system of claim 3, wherein the anchoring system comprises two cholesterol moieties capable of integrating within a membrane of a cell, wherein the second nucleic acid is linked to a first cholesterol moiety and the third nucleic acid is linked to a second cholesterol moiety.

5. The cellular labelling system of claim 1 , wherein the anchoring system comprises concanavalin A, biotin, and streptavidin, wherein the concanavalin A is configured to bind to a sugar moiety, wherein a biotin molecule is linked to the concanavalin A, wherein a biotin molecule is linked to the first nucleic acid molecule, and wherein streptavidin is configured to bind to the concanavalin A and to the first nucleic acid molecule via biotin, resulting in an anchoring of the first nucleic acid molecule to a cellular membrane.

6. The cellular labelling system of claim 1 , wherein the anchoring system comprises biotin, streptavidin, and a biotinylation reagent, wherein the biotinylation reagent is utilized to biotinylate cellular components, wherein a biotin molecule is linked to the first nucleic acid molecule, wherein streptavidin is configured to bind to the first nucleic acid molecule and to biotinylated cellular components via biotin, resulting in an anchoring of the first nucleic acid molecule to a cellular membrane.

7. The cellular labelling system of claim 6, wherein the biotinylation reagent comprises N-hydroxysuccinimide (NHS).

8. The cellular labelling system of claim 1 , wherein the anchoring system comprises an antigen-binding domain of an antibody, biotin, and streptavidin, wherein the antigenbinding domain is configured to target and bind a cellular component, wherein a biotin molecule is linked to the antigen-binding domain, wherein a biotin molecule is linked to the first nucleic acid molecule, and wherein streptavidin is configured to bind to the antigen-binding domain and to the first nucleic acid molecule via biotin, resulting in an anchoring of the first nucleic acid molecule to the cellular component.

9. The cellular labelling system of claim 1 , wherein the first nucleic acid oligo and the second nucleic acid oligo each comprise a barcoding sequence.

10. The cellular labelling system of claim 9, wherein the barcoding sequence is between 4 and 10 nucleotides in length.

11 . The cellular labelling system of claim 9, wherein the first nucleic acid oligo and the second nucleic acid oligo each further comprise a unique molecular identifier sequence and a capture sequence.

12. The cellular labelling system of claim 1 further comprising a first fluorophore that is linked to either the first nucleic acid oligo or the second nucleic acid oligo.

13. The cellular labelling system of claim 12 further comprising a second fluorophore, wherein the first fluorophore is linked to the first nucleic acid oligo and the second fluorophore is linked the second nucleic acid oligo.

14. The cellular labelling system of claim 1 , wherein the first nucleic acid molecule further comprises a third nucleic acid oligo having a third sequence and a second photocleavable moiety linking the second nucleic acid oligo and the third nucleic acid oligo.

15. The cellular labelling system of claim 1 further comprising a blocking nucleic acid molecule bound to the first nucleic acid molecule, wherein the blocking nucleic acid molecule comprising a first blocking nucleic acid oligo having a first blocking sequence, a second blocking nucleic acid oligo having a second blocking sequence and a flexible linker linking the first blocking nucleic acid oligo and the second blocking nucleic acid oligo, wherein a portion of the first blocking sequence is complementary to a portion of the first sequence and a portion the second blocking sequence is complementary to a portion of the second sequence.

16. A method for labelling a field of cells, comprising: contacting a cellular labelling system to a field of cells such that cells within the field are labeled via the labelling system, wherein the cellular labelling system comprises: a first nucleic acid molecule comprising a first nucleic acid oligo having a first sequence, a second nucleic acid oligo having a second sequence, and a photocleavable moiety linking the first nucleic acid oligo and the second nucleic acid oligo; and an anchoring system linked to the first nucleic acid molecule, wherein the anchoring system is configured to anchor the first nucleic acid molecule to a cellular membrane; illuminating a pattern of light onto the field of cells resulting in cleavage of the second nucleic acid oligo off of the first nucleic acid molecule via the photocleavable moiety from at least a portion of the first nucleic acid molecules; and identifying a spatial location of a plurality of cells within the field of cells based on: an amount of the first nucleic acid molecules comprising a first nucleic acid molecule and a second nucleic acid molecule, an amount of the first nucleic acid molecules comprising a first nucleic acid molecule and lacking a second nucleic acid molecule, and the pattern of light illuminated onto the field of cells.

17. The method of claim 16, wherein identifying a spatial location of a plurality of cells within the field of cells comprises performing single-cell sequencing on the plurality of cells to determine the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and a second nucleic acid oligo and the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and lacking a second nucleic acid oligo.

18. The method of claim 17, wherein the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and a second nucleic acid oligo and the amount of the first nucleic acid molecules comprising a first nucleic acid oligo and lacking a second nucleic acid oligo is determined by a first barcoded sequence within the first sequence and a second barcoded sequence with the second sequence.

19. The method of claim 17 further comprising: dissociating the field of cells into single cells prior to performing single-cell sequencing.

20. The method of claim 17, wherein performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell RNA sequencing on the plurality cells.

21 . The method of claim 17, wherein performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell DNA methylation sequencing on the plurality cells.

22. The method of claim 17, wherein performing single-cell sequencing on the plurality of cells further comprises concurrently performing single-cell DNA accessibility sequencing on the plurality cells.

23. The method of claim 16, wherein the cellular labelling system further comprises a first fluorophore that is linked to the second nucleic acid oligo, wherein identifying a spatial location of a plurality of cells within the field of cells comprises identifying a presence of the first fluorophore via fluorescence.

24. The method of claim 23, wherein the cellular labelling system further comprises a second fluorophore that is linked to the first nucleic acid oligo, wherein identifying a spatial location of a plurality of cells within the field of cells comprises identifying a ratio of an amount the first fluorophore to an amount of the second fluorophore via fluorescence.

25. The method of claim 16, wherein illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying light intensities onto the field of cells.

26. The method of claim 16, wherein illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying duration of illumination onto the field of cells.

27. The method of claim 16, wherein illuminating a pattern of light onto the field of cells comprises illuminating a pattern of varying wavelength bands onto the field of cells.

28. The method of claim 16, wherein illuminating a pattern of light onto the field of cells comprises use of a digital micromirror device to generate the pattern of light.

29 . The method of claim 16, wherein the field of cells comprises an adherent cell culture, an organoid culture, tissue, or cell applied to an optical medium.

30. The method of claim 16, wherein the steps of: contacting a cellular labelling system to a field of cells such that cells within the field are labeled via the labelling system and illuminating a pattern of light onto the field of cells are iteratively repeated at least once, wherein each time the steps are repeated, the steps are performed with a labelling system comprising unique barcoding sequences and with a unique pattern of light.