Targeted cell profiling via in SITU hybridization with mass spectrometry detection

Abstract

Disclosed herein is a high throughput workflow that uses in-situ hybridization base pairing and a cleavable mass tag to visualize gene expression using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry imaging (MSI). After hybridizing the DNA sequence to the RNA gene sequence, the complementary sequences can as an example be exposed to ultraviolet light. The photolysis reaction releases the mass tag that is ionizable during MALDI MSI and can be detected by the mass spectrometer when being used in an imaging context. This allows one to visualize hundreds of genes within a single imaging experiment without the need to cycle. This can also be performed at < 10 μm spatial resolution for cm2 pieces of tissue. This is significantly higher in spatial resolution and imaging area than current in situ or spatial transcriptomics measurements.

Description

TARGETED CELL PROFILING VIA IN SITU HYBRIDIZATION WITH MASS SPECTROMETRY DETECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 542,893, filed October s, 2023, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “320317-2110 Sequence Listing” created on August 23, 2024, and having 1 ,779 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] The ability to profile cells within a biological tissue is a powerful tool for understanding fundamental biology and its transition towards a diseased state. Cell profiling / naming provides context to the role of expressed genes within each cell, providing information on the potential functional state of the cell. Methods exist that harness the capability to sequence genes entirely, but these methods are expensive and require lengthy workflows.SUMMARY OF THE INVENTION

[0004] Disclosed herein is a high throughput workflow that uses in-situ hybridization base pairing and a cleavable mass tag to visualize gene expression using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry imaging (MSI). After hybridizing the DNA sequence to the RNA gene sequence, the complementary sequences can as an example be exposed to ultraviolet light. The photolysis reaction releases the mass tag that is ionizable during MALDI MSI and can be detected by the mass spectrometer when being used in an imaging context. This allows one to visualize hundreds of genes within a single imaging experiment without the need to cycle. This can also be performed at < 10 pm spatial resolution for cm2pieces of tissue. This is significantly higher in spatial resolution and imaging area than current in situ or spatial transcriptomics measurements.

[0005] Therefore, disclosed herein is a method of generating a high resolution two- dimensional image of a tissue sample, the method involving: (a) administering to the sample a plurality of probes that each bind distinct tissue targets, wherein each probe is conjugated to a unique mass tag by a cleavable linker, and wherein each of the unique mass tags are ionizable with a UV laser, charged, and have similar ionization efficiencies; (b) washing the sample under conditions suitable to remove unbound ISH probes; (c) treating the sample to cleave the linker and release the mass tag; (d) subjecting the sample to a mass spectrometry imaging method to generate a data set that comprises the identity and spatially-addressable abundance of each of the released mass tags across an area of saidsample; and (e) generating an image from the data set with each unique mass tag being assigned a unique color.

[0006] In some embodiments the cleavable linker is a photo-labile linker, and wherein step (c) comprises treating the sample with UV light. For example, in some embodiments the photo-labile linker is an o-nitrobenzyl-based linker.

[0007] In some embodiments the cleavable linker is an acid-labile linker, and wherein step (c) comprises treating the sample with an acidic solution, e.g., a solution with a pH less than 7.0, less than 6.5, less than 6.0, less than 5.5, less than 5.0, less than 4.5, less than 4.0 and the like. For example, the pH of the MALDI matrix is such that it provides a pH sufficient to cleave the acid-labile linker.

[0008] In some embodiments each of the unique mass tags is a peptide having a molecular weight from 500 Da to 3,000 Da, wherein the peptide comprises at least one charged residue selected from the group consisting of Arginine, Lysine, Histidine, Aspartate, Glutamate. In some cases only one permanently charged residue is needed. In some embodiments at least or about 5 to 10% of the amino acids in the peptide are charged.

[0009] In some embodiments each of the unique mass tags is a lipid between 400 Da and 1200 Da.

[0010] In some embodiments each of the unique mass tags is a small molecule greater than 250 Da. In some embodiments each of the unique mass tags has a molecular weight at least 0.1 Da higher or lower than any other mass tag.

[0011] In some embodiments the sample is a formalin-fixed, paraffin-embedded (FFPE) section, fresh frozen section, or fixed section.

[0012] In some embodiments the image has at least a 1 ppm accuracy.

[0013] In some embodiments the probe is an in situ hybridization (ISH) probe comprising a single stranded DNA or RNA oligonucleotide having a first portion that is complementary to a target single stranded nucleic acid.

[0014] In some embodiments the oligonucleotide further comprises a second portion that is complementary to a fluorescently labeled detection probe, wherein the method further comprises administering to the sample the detection probe and imaging the sample for fluorescence. For example, in some embodiments the detection probe has the formula: (a) 5 -RNA Binding Sequence-Modified Nucleobase-Cleavable Linker-Ionizable Mass Tag - 3’; (b) 5 -RNA Binding Sequence-Modified Nucleobase-Cleavable Linker-Ionizable Mass Tag -Cleavable Linker-RNA Binding Sequence-Modified Nucleobase- 3’; (c) 5 -RNA Binding Sequence-Modified Nucleobase-Cleavable Linker-Ionizable Mass Tag -Cleavable Linker-5’-RNA Binding Sequence- 3’; (d) 5 -RNA Binding Sequence-Cleavable Linker- Ionizable Mass Tag -Cleavable Linker-5’-RNA Binding Sequence-Modified Nucleobase-3’;or (e) 5’-lonizable Mass Tag -Cleavable Linker-5’-RNA Binding Sequence-Modified Nucleobase- 3’.

[0015] In some embodiments the target is endogenous RNA or an administered therapeutic. For example, in some embodiments the target is endogenous mRNA. In some embodiments, the target is an mRNA vaccine.

[0016] In some embodiments the plurality of ISH probes such that the plurality of probes can bind at least 10, 100, 1 ,000, or 10,000 distinct RNA targets.

[0017] In some embodiments the probe is a probe comprising an antibody, antibody fragment, or aptamer having a first portion that is capable of binding to a target peptide or polypeptide. For example, in some embodiments the detection probe has the formula: (a) Antibody-Cleavable Linker-Ionizable Mass Tag - 3’; (b) Antibody Fragment-Cleavable Linker-Ionizable Mass Tag - 3’; or (c) Aptamer-Cleavable Linker-Ionizable Mass Tag - 3’.

[0018] In some embodiments the antibody, antibody fragment, or aptamer having a first portion that is capable of binding to a target peptide or polypeptide further comprises an fluorescently labelled amino thereon, wherein the method further comprises administering to the sample the detection probe and imaging the sample for fluorescence. For example, in some embodiments the detection probe has the formula: (a) Antibody(Fluorescently-labelled Amino Acid)-Cleavable Linker-Ionizable Mass Tag - 3’; (b) Antibody Fragment(Fluorescently-labelled Amino Acid)-Cleavable Linker-Ionizable Mass Tag - 3’; or (c) Aptamer(Fluorescently-labelled Amino Acid)-Cleavable Linker-Ionizable Mass Tag - 3’.

[0019] In some embodiments a plurality of probes comprising an antibody, an antibody fragment or aptamer such that the plurality of probes can bind at least 10, 100, 1 ,000, or 10,000 distinct peptide or polypeptide targets.

[0020] In some embodiments the plurality of probes comprises a mixture of (a) a plurality of ISH probes such that the plurality of probes can bind at least 10, 100, 1 ,000, or 10,000 distinct RNA targets; and (b) a plurality of probes comprising an antibody, an antibody fragment or aptamer such that the plurality of probes can bind at least 10, 100, 1 ,000, or 10,000 distinct peptide or polypeptide targets.

[0021] In some embodiments the mass spectrometry imaging method is carried out using an ionization source selected from the group consisting of matrix-assisted laser desorption ionization (MALDI), nanoparticle initiator mass spectrometry (NIMS), secondary ion mass spectrometry (SIMS), laser desorption, desorption electrospray ionization (DESI), probe electrospray ionization (PESI), laser spray, and laser ablation electrospray ionization (LAESI).

[0022] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0023] FIG. 1 shows a scheme illustrating MALDI MSI probes disclosed herein. The designed contiguous nucleotide sequence can be modified with a modified nucleobase, which can undergo a covalent conjugation reaction to attach a unique ionizable mass tag to correlate the detectable mass to the respective contiguous nucleotide sequence.

[0024] FIG. 2 shows a scheme for producing a MALDI MSI photocleavable probe disclosed herein. In this depiction, the chemically synthesized oligonucleotide is modified to include a 5-ethynyl thymidine base at the 3’ end of the contiguous nucleotide sequence. The alkyne is then subjected to the appropriate chemical conditions to attach a unique mass tag. In this case, copper click chemistry is used to make the respective triazole. Finally, the appropriate cleavage conditions are applied, such as UV light, and the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.

[0025] FIG. 3 shows a sagittal sectioned mouse brain depicting an example MSI three color image of wild type mouse brain at 30 pm spatial resolution. The cleaved ionizable mass tags detected in the image indicate the distribution of nucleic acid probe sequences for Sox10, HpcaH, and Foxp2.

[0026] FIG. 4 shows general MALDI ISH MSI workflow. Synthesized photocleavable peptide mass tags are conjugated to alkyne-tailed RNA binding probes and applied to fresh frozen tissue in a mass spectrometry-compatible in-situ hybridization assay. After photolytically cleaving the peptide mass tags from their RNA binding sequences, the tissue is coated in a MALDI matrix and put through a MALDI MSI experiment on a Bruker timsTOF FleX.

[0027] FIG. 5 shows detection of Foxp2 MALDI ISH at 10 pm spatial resolution. Ultimately, the power of MS can be used to detect hundreds of mRNA within a single tissue section at cellular resolution for larger areas of tissue. Top is the positive signal and bottom is a negative control.

[0028] FIG. 6 shows probes designed to target 20 mRNA relevant to neuroscience. Each of these probes were tested within sagittal sections and display high definition and signal intensity. In total 6 mass tags were tested across these 20 genes, showing probes and sequences can be exchanged. Additionally, probes were tested in sets of three, demonstrating that multiplexing is possible.

[0029] FIG. 7 shows MALDI IHC used to detect neurofilament light chain (top) and NeuN proteins within fresh frozen murine brain using MALDI MSI.

[0030] FIGs. 8A and 8B show both spatial lipidomics and gene expression were obtained from the same sagittal section using the MALDI ISH workflow, demonstrating that multiomic measurements are possible. FIG. 8A is an MSI image of Pl(38:5) (green), Pl(36:4) (blue), and Gpr17 (mRNA expressed in oligodendrocytes) detected within the same section. FIG. 8B shows the ISH profile (top) and lipid profile (bottom) of an individual oligodendrocyte is shown, demonstrating the information that can be gained from combining spatial metabolomics with gene expression.

[0031] FIG. 9 shows general MALDI ISH MSI workflow. Fresh frozen tissue is sectioned to 10 pm and mounted to an indium-tin oxide (ITO) conductive glass slide. The native tissue is coated in 2,5 dihydroxy acetophenone (DHA) for untargeted lipid analysis using MALDI MSI. The same tissue section has the matrix removed and undergoes MALDI ISH analysis using photocleavable mass tagged probes to detect respective mRNA targets. After photolytically separating the mass tags from their attached oligonucleotide, the slide is once again coated in DHA and run on the BrukertimsTOF FleX.

[0032] FIG. 10 shows a MALDI ISH probe synthesis scheme (Scheme 1). TdT tailing with 5-EdUTP, CuAAC addition of previously synthesized azide PC mass tags and photolysis reaction products using UV-A light to yield ionizable mass tag. XXXYRVDXAR (SEQ ID NO:1).

[0033] FIG. 11 shows 50 ng / pL was determined to be the amount of probe necessary for detectable MS signal at 50 pm. Panel A shows a probe targeting mRNA associated with Foxp2 was titrated at 0 ng (blank), 1 ng / pL, 10 ng / pL, and 50 ng / pL. The mass tag had a peptide sequence of DALYRVDAAR and was detected with an m / z value of 1312.6377. Panel B shows the ion intensity box plot between the probe concentration used in hybridization and detected signal from the sections in Panel A). Panels C, D, and E) 50 ng / pL was sufficient for other tested probes, Foxp2, Proxl and Gpr17, within a different serial sections of mouse brain, demonstrating that this concentration was consistent across animals and across mRNA targets.

[0034] FIG. 12 shows 20 mRNA probes were designed using unique combinations of probe sequences and mass tags were multiplexed in sets of two or three on serial sections of mouse brain (Rows 1-8). New RNA binding sequences were paired with previously tested mass tags (Columns A / B) and new mass tags were paired with known RNA binding sequences (Column C). MALDI MSI was run at 50 pm to yield a time efficient screening demonstrating a controlled method to visualize correctly designed probe sequences and high preforming mass tags.

[0035] FIG. 13 shows both spatial lipidomics and gene expression were obtained from the same sagittal section using the MALDI ISH workflow, demonstrating that multiomic measurements are possible. Panels 13A to 13D are MSI images of Pl(38:5) (green), Pl(36:4)(blue), and Gpr17 (mRNA expressed in oligodendrocytes) detected within the same section. Also shown are ISH profiles (left) and lipid profiles (right) of an individual, demonstrating the information that can be gained from combining spatial metabolomics with gene expression.

[0036] FIGs. 14A-14B show exemplary depictions of a MALDI MSI cleavable probe that can bind a protein target. FIG. 14A shows a depiction in which a chemically synthesized oligonucleotide is linked on either side of a peptide mass tag using methods as generally disclosed herein. Finally, the appropriate cleavage conditions are applied, such as UV light, and the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI. FIG. 14B shows a depiction in which a chemically synthesized oligonucleotide is linked on either side of a peptide mass tag using methods as generally disclosed herein. Finally, the appropriate cleavage conditions are applied, such as UV light or suitably acidic conditions, and the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.

[0037] FIG. 15 shows an exemplary depiction of a MALDI MSI cleavable probe disclosed herein. In this depiction, the chemically synthesized oligonucleotide is modified to comprise a suitable alkyl linker further linked to a trityl moiety that is linked to the peptide mass tag. Finally, the appropriate cleavage conditions are applied, such as UV light or suitably acidic conditions, and the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.

[0038] FIG. 16 shows an alternate exemplary depiction of a MALDI MSI cleavable probe disclosed herein in which the probe structure is related to that shown in FIG. 15, but in which the relative orientation and of the oligonucleotide and peptide mass tag are reversed as shown. As shown, under the appropriate cleavage conditions are applied, such as UV light or suitably acidic conditions, the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.

[0039] FIG. 17 shows an exemplary depiction of a MALDI MSI cleavable probe disclosed herein. In this depiction, a protein binding portion, e.g., an antibody, an antibody fragment, or an aptamer, is covalently linked, via an amino acid on the antibody, antibody fragment, or a suitable moiety on the aptamer, via linker further covalently linked to a cleavable moiety such as the trityl moiety depicted. As shown, under the appropriate cleavage conditions are applied, such as UV light or suitably acidic conditions, the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.

[0040] FIG. 18 shows an alternate exemplary depiction of a MALDI MSI cleavable probe disclosed herein in which the probe structure is related to that shown in FIG. 17, but in which the relative orientation and of the protein binding portion and peptide mass tag are reversed as shown. As shown, under the appropriate cleavage conditions are applied, suchas UV light or suitably acidic conditions, the modified peptide mass tag from the contiguous nucleotide sequence is detected using MALDI MSI.DETAILED DESCRIPTION

[0041] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0044] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0045] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0046] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0047] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0048] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0049] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0050] The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solidphase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide disclosed herein is man-made, e.g., chemically synthesized. The oligonucleotide disclosed herein may also comprise one or more modified nucleosides or nucleotides.

[0051] The term “contiguous nucleotide sequence” refers to the region of the oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”. In some embodiments all the nucleotides of the oligonucleotide are present in the contiguous nucleotide sequence. In some embodiments the oligonucleotide comprises the contiguous nucleotide sequence and may, optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used toattach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0052] Nucleotides are the building blocks of oligonucleotides and polynucleotides, and can include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

[0053] The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In some embodiments, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers".

[0054] The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages or natural phosphate linkages that covalently couples two nucleosides together. Nucleotides with modified internucleoside linkage are also termed “modified nucleotides”. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide disclosed herein, for example, within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides.

[0055] In some embodiments, the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester to a linkage that is, for example, more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay [e.g., snake venom phosphodiesterase (SVPD)], both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages.

[0056] In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof,are modified. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are modified.

[0057] It will be recognized that, in some embodiments, the internucleoside linkages which link the oligonucleotide to a non-nucleotide functional group, such as a conjugate, may be phosphodiester. In some embodiments, the internucleoside linkages which link the oligonucleotide to a non-nucleotide functional group are modified.

[0058] In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.

[0059] Modified internucleoside linkages may, for example, be selected from the group comprising phosphorothioate, phosphoramidate, diphosphorothioate, and boranophosphate. In some embodiments, the modified internucleoside linkages are compatible with the RNase H recruitment of the oligonucleotide disclosed herein, for example, phosphorothioate, diphosphorothioate, or boranophosphate.

[0060] In some embodiments the internucleoside linkage comprises sulphur (S), such as a phosphorothioate internucleoside linkage.

[0061] A phosphorothioate internucleoside linkage is particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In preferred embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80%, or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

[0062] In some embodiments, the oligonucleotide comprises one or more neutral internucleoside linkage, particularly a internucleoside linkage selected from phosphotriester, methylphosphonate, MMI, amide-3, formacetal or thioformacetal. Further internucleoside linkages are disclosed in WO2009 / 124238 (incorporated herein by reference). In an embodiment the internucleoside linkage is selected from linkers disclosed in W02007 / 031091 (incorporated herein by reference).

[0063] Nuclease resistant linkages, such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers, or the non-modified nucleoside region of headmers and tailmers. Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and / or affinity enhancing regions such as regions F and F' for gapmers, or the modified nucleoside region of headmers and tailmers.

[0064] Each of the design regions may however comprise internucleoside linkages other than phosphorothioate, such as phosphodiester linkages, in particularly in regions where modified nucleosides, such as LNA, protect the linkage against nuclease degradation. Inclusion of phosphodiester linkages, such as one or two linkages, particularly between or adjacent to modified nucleoside units (typically in the non-nuclease recruiting regions) can modify the bioavailability and / or bio-distribution of an oligonucleotide. W02008 / 113832 is incorporated herein by reference for the teaching of oligonucleotides having phosphodiester linkages.

[0065] In some embodiments, all the internucleoside linkages in the oligonucleotide are phosphorothioate and / or boranophosphate linkages. In some embodiments, all the internucleoside linkages in the oligonucleotide are phosphorothioate linkages.

[0066] The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. The term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants.

[0067] In some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl-cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0068] The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine (5mC). Combinations of these modifications may also be used. For example, 5mC LNA nucleosides may be used. Likewise, 2 '-hydroxymethyl (2'-OMe) 5mC may be used.

[0069] The term “complementarity” describes the capacity for Watson-Crick basepairing of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example, 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases.

[0070] The term “% complementary” as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., oligonucleotide) which, at a given position, are complementary to (i.e., form Watson Crick base pairs with) a contiguous nucleotide sequence, at a given position of a separate nucleic acid molecule (e.g., the target nucleic acid). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences, dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase / nucleotide which does not align (form a base pair) is termed a mismatch.

[0071] The term “hybridizing” or “hybridizes” as used herein is to be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (Tm) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions, Tm is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy AG° is a more accurate representation of binding affinity and is related to the dissociation constant (Kd) of the reaction by AG°=-RTIn(Kd), where R is the gas constant and T is the absolute temperature. Therefore, a very low AG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and target nucleic acid. AG° is the energy associated with a reaction where aqueous concentrations are 1 M, the pH is 7, and the temperature is 37° C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements. AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides disclosed herein hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°. The oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or -16 to -27 kcal such as -18 to -25 kcal.

[0072] In some embodiments, the disclosed oligonucleotide comprises a contiguous nucleotide sequence of at least 8 nucleotides which is complementary to or hybridizes to a target sequence present in the target nucleic acid molecule. The contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 8 contiguous nucleotides, such as 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.

[0073] The disclosed oligonucleotide may comprise one or more nucleosides which have a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA. Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance. Such modifications include those where the ribose ring structure is modified, e.g., by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleosides (WO2011 / 017521) or tricyclic nucleosides (WO2013 / 154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0074] Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example, be introduced at the 2', 3', 4' or 5' positions. Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.

[0075] A 2' sugar modified nucleoside is a nucleoside which has a substituent other than H or — OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradical, and includes 2’ substituted nucleosides and LNA (2'-4' biradical bridged) nucleosides. For example, the 2' modified sugar may provide enhanced binding affinity and / or increased nuclease resistance to the oligonucleotide. Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA (O-Me), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'-fluoro-ANA (F-ANA). For further examples, please see Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213; and Deleavey and Damha, Chemistry and Biology 2012, 19, 937.

[0076] Locked Nucleic Acid (LNA) nucleosides are modified nucleosides which comprise a linker group (referred to as a biradical or a bridge) between C2' and C4' of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.

[0077] Conjugation of the disclosed oligonucleotide to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g., by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue, or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue, or cell type. At the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g., off target activity or activity in non-target cell types, tissues or organs. WO 93 / 07883 and WO 2013 / 033230 provides suitable conjugate moieties, which are hereby incorporated by reference. WO 2012 / 143379 provides a method of delivering a drug across the blood-brain-barrier by conjugation to an antibody fragment with affinity to the transferrin receptor, which are hereby incorporated by reference.

[0078] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids) or combinations thereof. In some embodiments the non-nucleotide moiety an antibody or antibody fragment, such as an antibody or antibody fragment that facilitates delivery across the blood-brain-barrier, in particular an antibody or antibody fragment targeting the transferrin receptor.

[0079] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.Disclosed Probes: ISH Probes

[0080] Turning now to FIG. 1 , MALDI MSI probes include a first single stranded portion that is complementary to a target single stranded nucleic acid and an ionizable mass tag conjugated to the probe by a cleavable linker. The single stranded target nucleic acidcan be a natural RNA molecule, e.g. in a tissue of a subject. In other embodiments, the single stranded nucleic acid target is a synthetic DNA or RNA oligonucleotide, e.g. attached to a molecule of interest as a tag.

[0081] As shown in FIG. 1 , the mass tag is too large to be detected by MSI when conjugated to the MALDI MSI probe and bound to a target. Once the tissue has been sufficiently washed to remove unbound probes, the linker can be cleaved, e.g. by UV light for a photocleavable linker. Once cleaved, the ionizable mass tag can be detected by MSI.

[0082] The MALDI MSI probes can optionally also include a second single stranded portion that is complementary to a secondary sequence as part of the detection probe, where a fluorescently labeled oligonucleotide with a single stranded portion that is complementary to the second single stranded portion of the MALDI MSI probe is capable of hybridizing.

[0083] ISH probes can be designed in silica, using tools available in the art. For example, Stellaris Probe Designer Version 4.2 can be used to generate probe sequences from respective genes of interest. Likewise, Thermo Fischer Scientific’s PCR Primer Dimerization tool can be used to cross-reference and eliminate probes that are not involved in primer dimerization. In some embodiments the probes have a GC content ranging from 25% GC to 65% GC. In some embodiments candidate probes are screened in silico for potential secondary structure interactions, e.g. using VECTORBUILDER.

[0084] The ISH probe can be from 15 to 45 bp in length, including 20bp. In the case of using shorter probe lengths (<20bp) locked nucleic acids (LNAs) can be used to increase the melting temperature. Aptamers and nonconical bases can also be used.

[0085] Turning to FIG. 14A, a further embodiment of an ISH probe is depicted in which the peptide mass tag is flanked by cleavable linkers, each of which is covalently linked to an ISH probe such as a DNA probe. Upon cleavage, e.g., photocleavage when a photolabile cleavable linker is used, the peptide mass tag is released and can be detected as described herein. In some instances, the flanking cleavable linker comprises a structure represented by the following chemical formula:Turning FIG. 14B, a further embodiment of an ISH probe is depicted in which the peptide mass tag is flanked by cleavable linkers, each of which is covalently linked to an ISH probe such as a DNA probe. Upon cleavage, e.g., photocleavage as discussed above or in thepresence of a suitably acidic pH, such as may be present in the MALDI matrix, the peptide mass tag released. In some embodiments, the exemplary linker, including the photo- or acid- labile portion (on the right side below) is a structure represented by the following chemical formula:

[0086] Turning to FIG. 15, a further embodiment of an ISH probe is depicted in which the probe comprises an alkyl linker to a DNA probe complementary to a nucleotide or polynucleotide target of interest which is covalently linked to a trityl linker moiety further covalently linked to a peptide mass tag. Upon cleavage, e.g., photocleavage as discussed above or in the presence of a suitably acidic pH, such as may be present in the MALDI matrix, the peptide mass tag released. In some embodiments, the exemplary linker, including the photo- or acid-labile portion (on the right side below) is a structure represented by the following chemical formula:As depicted in FIG. 16, the relative orientation of the DNA probe portion and the peptide mass tag can be reversed.Synthesize mass tags (<3,000 Da)

[0087] A plurality of different ISH probes can be used with distinct mass tags. The mass tags need to carry charge and the ionization efficiency needs to be roughly the same. The mass tags also need to carry charge in either positive or negative mode. They also need to be ionizable with a UV laser for MALDI MS. They also need to be robust and non- degradable in a vacuum or upon laser ablation.

[0088] In some embodiments, the probe has the amino acid sequence XXXYRVDXAR (SEQ ID NO:1). Other arrangements of the static peptide sequence in SEQ ID NO:1 are also contemplated. In some embodiments, the charged amino acids are 2-4 amino acids apart. In some embodiments, the static sequence can vary in length as discussed above. Variable amino acids can also be put on either end of the sequence.

[0089] Classes of mass tags include: peptides, small molecules, lipids; and carbohydrates Mass tags can be virtually any peptide sequence. The key is that they can reliably charge residues (Arginine, Lysine, Histidine, Aspartate, Glutamate).

[0090] In some embodiments the amino acids are modified amino acids. This could serve to ionize the sequence better, incorporate different isotopic labels, deuterium, or affect hydrophobicity.Cleavable Linkers

[0091] Options for cleavable linkers include enzymatically cleaved linkers (Trypsin (amino acid linker) or Restriction Enzyme (DNA / RNA linker) or Endo / Exonuclease); pH- sensitive linkers (acid or base); photolabile linkers (UV or near IR); thermolabile linkers; and disulfide reduction / Redox active linkers. In some embodiments the linker is a MALDI laser- cleavable linker.Applications photocleaveable mass tags to ISH probes

[0092] ISH probes with photocleavable mass tags can be used in a variety of different applications and to detect a variety of different targets, including antibodies, proteins, beads (Polystyrene, PEGA, Tantagel or polyacrylamide), nanobodies, CRIPSR- cas9 editing products, mRNA therapies (siRNA / mRNA vaccines), Adeno-associated Viral Vectors, Lipid nanoparticles, and oligonucleotide microarrays. The ISH probes can also be used as a drug delivery vehicle (e.g. Antibody Drug Conjugates). For example, the ISH probe can be specific for a nucleic acid sequence attached to an antibody.Established Molecular Biology Techniques to Synthesize Probes

[0093] Molecular biology techniques to synthesize probes include terminal transferase extension, Rolling Circular Amplification / Polymerization (RCA / RCP), Primer Exchange Reaction (PER), Tyramide Signal Amplification (TSA), Reverse Transcription PCR (RT-PCR), Polymerase Chain Reaction (PCR), Avidin-biotin complexation, CARD-ISH, SABER-ISH, and hybridization chain reaction.Hybridization of the ISH probe

[0094] The buffer used for ISH probe hybridization can in some embodiments include a denaturation Agent (e.g. anywhere from 0-50% of final hybridization buffer). Examples include formamide and ethylene carbonate.

[0095] The buffer used for ISH probe hybridization can in some embodiments include cations (e.g. from 50mM to 500mM). Examples include Na+, K+, Mg+2, Ca+2, Li+, and NH4+.

[0096] The buffer used for ISH probe hybridization can in some embodiments include a crowding Agent (e.g. 0-25% of final hybridization buffer). Examples include Dextran Sulfate, Ficoll, Poly(Ethylene Glycol), Carrageenan, Alginate, Agarose, Cellulose (and modified derivatives), chitin and Acrylamide.

[0097] Hybridization can in some embodiments be conducted from 4 hours to overnight at 37°C. Other suitable temperatures and hybridization times can be adapted as needed.Tissues

[0098] The ISH probes can be used on any tissue of a human or animal. Examples include brain, spinal cord, spinal column, liver, kidney, spleen, lung, breast, tonsils, lymph nodes, eyes, teeth, hair, liquid biopsy, femur, and heart. The ISH probes can be used on plants, bacteria, viruses, protists, monera, and fungi. In some embodiments the target is an organelle in a cell, such as mitochondria.

[0099] The ISH probes can be used to evaluate a wide variety of disease states, including cancer, Alzheimer’s Disease (AD), Parkinson’s, autism, diabetes, inflammation, chronic pain, etc.

[0100] The target tissue can be formalin-fixed, paraffin-embedded (FFPE), fresh frozen, fixed, embedded, not embedded, cell dissociates, cell culture, or FACS sorted cells. The tissue can be sectioned at from 5-25 m.

[0101] The ISH probes can be used to evaluate a variety of animal developmental models including Aplysia californica, nematodes, zebra fish, and fruit flies.Disclosed Probes: Protein Probes Comprising an Antibody, Antibody Fragment, and / or Aptamer

[0102] Turning now to FIGs. 17-18, MALDI MSI probes can comprise a first protein binding portion, e.g., an antibody, an antibody fragment, and / or an aptamer, that is capable of binding to a target peptide or polypeptide and an ionizable mass tag conjugated to the probe by a cleavable linker. The target peptide or polypeptide can be a natural peptide or polypeptide molecule, e.g. in a tissue of a subject. In other embodiments, the target peptide or polypeptide can be peptide or polypeptide, e.g. covalently attached to a molecule of interest as a tag. Suitable exemplary cleavable linkers and covalent linkages are as shown in FIGs. 17-18, using the methods as described herein throughout.

[0103] As shown in FIGs. 17-18, the mass tag is too large to be detected by MSI when conjugated to the MALDI MSI probe and bound to a target. Once the tissue has been sufficiently washed to remove unbound probes, the linker can be cleaved, e.g. by UV light fora photocleavable linker or under suitable pH conditions for an acid-labile linker. Once cleaved, the ionizable mass tag can be detected by MSI.

[0104] The MALDI MSI probes can optionally also include a second portion comprising an antibody, an antibody fragment, or an aptamer, where a fluorescently labeled moiety is covalently linked to the antibody, antibody fragment, or aptamer that can bind a target peptide or polypeptide to the second portion of the MALDI MSI probe.

[0105] Suitable antibody or an antibody fragment probes can be prepared using antigens designed in silica, using tools available in the art.Synthesize mass tags (<3,000 Da)

[0106] A plurality of different protein binding probes can be used with distinct mass tags. The mass tags need to carry charge and the ionization efficiency needs to be roughly the same. The mass tags also need to carry charge in either positive or negative mode. They also need to be ionizable with a UV laser for MALDI MS. They also need to be robust and non-degradable in a vacuum or upon laser ablation.

[0107] In some embodiments, the probe has the amino acid sequence XXXYRVDXAR (SEQ ID NO:1). Other arrangements of the static peptide sequence in SEQ ID NO:1 are also contemplated. In some embodiments, the charged amino acids are 2-4 amino acids apart. In some embodiments, the static sequence can vary in length as discussed above. Variable amino acids can also be put on either end of the sequence.

[0108] Classes of mass tags include: peptides, small molecules, lipids; and carbohydrates Mass tags can be virtually any peptide sequence. The key is that they can reliably charge residues (Arginine, Lysine, Histidine, Aspartate, Glutamate).

[0109] In some embodiments the amino acids are modified amino acids. This could serve to ionize the sequence better, incorporate different isotopic labels, deuterium, or affect hydrophobicity.Cleavable Linkers

[0110] Options for cleavable linkers include enzymatically cleaved linkers (Trypsin (amino acid linker) or Restriction Enzyme (DNA / RNA linker) or Endo / Exonuclease); pH- sensitive linkers (acid or base); photolabile linkers (UV or near IR); thermolabile linkers; and disulfide reduction / Redox active linkers. In some embodiments the linker is a MALDI laser- cleavable linker, such as the examples shown in FIGs. 17-18.Applications photocleaveable mass tags to protein probes

[0111] Protein probes with photocleavable or acid-labile mass tags can be used in a variety of different applications and to detect a variety of different targets, including antibodies, proteins, beads (Polystyrene, PEGA, Tantagel or polyacrylamide), nanobodies, CRIPSR-cas9 editing products, mRNA therapies (siRNA / mRNA vaccines), Adeno- associated Viral Vectors, Lipid nanoparticles, and oligonucleotide microarrays. The proteinprobes can also be used as a drug delivery vehicle (e.g. Antibody Drug Conjugates). For example, the protein probe can be specific for a target peptide or polypeptide attached to an antibody.Established Molecular Biology Techniques to Synthesize Probes

[0112] Molecular biology techniques to synthesize probes include terminal transferase extension, Rolling Circular Amplification / Polymerization (RCA / RCP), Primer Exchange Reaction (PER), Tyramide Signal Amplification (TSA), Reverse Transcription PCR (RT-PCR), Polymerase Chain Reaction (PCR), Avidin-biotin complexation, CARD-ISH, SABER-ISH, and hybridization chain reaction.Binding of the Protein Probe

[0113] The buffers and binding conditions for the use of antibodies, antibody fragments, and aptamers to peptide or polypeptides in tissue sections, as contemplated herein, are known to the skilled artisan.

[0114] Bind can in some embodiments be conducted from 4 hours to overnight at 20- 37°C. Other suitable temperatures and hybridization times can be adapted as needed.Tissues

[0115] The protein probes can be used on any tissue of a human or animal. Examples include brain, spinal cord, spinal column, liver, kidney, spleen, lung, breast, tonsils, lymph nodes, eyes, teeth, hair, liquid biopsy, femur, and heart. The protein probes can be used on plants, bacteria, viruses, protists, monera, and fungi. In some embodiments the target is an organelle in a cell, such as mitochondria.

[0116] The protein probes can be used to evaluate a wide variety of disease states, including cancer, Alzheimer’s Disease (AD), Parkinson’s, autism, diabetes, inflammation, chronic pain, etc.

[0117] The target tissue can be formalin-fixed, paraffin-embedded (FFPE), fresh frozen, fixed, embedded, not embedded, cell dissociates, cell culture, or FACS sorted cells. The tissue can be sectioned at from 5-25 pm.

[0118] The protein probes can be used to evaluate a variety of animal developmental models including Aplysia californica, nematodes, zebra fish, and fruit flies.Mass Spectrometry Imaging

[0119] The target tissue can be sprayed with any suitable MALDI matrix. For example, the MALDI marix can be CHCA, DAN, DHB, CLCCA, DPH, DHA, CMBT, norharmane, 9AA, CNDA, CNAA, curcumin, MAPS, THAP, DANHCL, PNA, NEDC, GAQ, HZN, metal nanoparticles, or ionic liquids and can be applied using pneumatic sprayers, airbrushes, sublimation, dried droplet, electrospray deposition, etc.

[0120] Mass Spectrometry Imaging (MSI) can then be performed. This can be done at a spatial resolution of from 1 pm to 500 pm, including from 10 to 50 pm, such as 10 pm. This can be done in positive or negative ion mode.

[0121] The disclosed methods can involve mapping and / or displaying the spatial measurements of cleaved mass tags in the sample. Such mapping or display thus providing a spatial topology or distribution of the localizations of the targets (e.g. RNA transcripts) in the sample.

[0122] In some embodiments, where the sample is a clinical sample, the mapping or display of the spatial measurements may be performed in situ in living tissues to provide topographic and localization images of the intact tissue sample upon which an informed clinical decision, diagnosis or prognosis can be made.

[0123] Sample collection can include, for example, taking a surgical biopsy; surgical removal of a tissue or portion of a tissue; performing a percutaneous, endoscopic, transvascular, radiographic-guided or other non-surgical biopsy; euthanizing an experimental animal and removing tissue; collecting ex vivo experimental preparations; removing tissue at post-mortem examination; or other methods of collecting tissues.

[0124] Methods for preparing histopathologic slides from cell or tissue sample(s) by standard (non)-fixing methods known in the art, including cryopreservation, ethanol dehydration, OCL preservation, or other methods known in the art.

[0125] In some embodiments, the histopathology slide is exposed to a spatially- organized volatilization of molecules and ions wherein a focused energy source, such as a laser beam, or desorption system is rastered across the tissue sample to create a series of discrete packets or a continuous flow of volatilized molecules in a spatially-organized manner. The molecules are charged (ions) or neutrals subjected to subsequent ionization. Energy-induced volatilization processes include, for example, matrix-assisted laser desorption ionization (MALDI), nanoparticle initiator mass spectrometry (NIMS), secondary ion mass spectrometry (SIMS), laser desorption, desorption electrospray ionization (DESI), probe electrospray ionization (PESI), laser spray, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), and laser ablation electrospray ionization (LAESI).

[0126] In some embodiments, the volatilized molecules are directed into a mass analyzer using instrument modalities that include, for example, time-of-flight (TOF), Orbitrap, Fourier-transform ion cyclotron (FTIR), magnetic sector, quadrupole, or other mass spectrometers. In a preferred embodiment, tandem mass spectrometers (MS / MS) are used, such as TOF-TOF or Quadrupole-TOF, wherein the second MS collects fragmentation spectra for molecular characterization of ions analyzed by the first mass spectrometer.

[0127] In some embodiments, the method involves visually displaying, on a spatially defined basis, locations and abundance of the mass tags / targets wherein said displays are in the form of heat maps, contour maps or other spatial representations.

[0128] Analysis of serial sections from a tissue specimen allows assembly of three- dimensional images of a tissue. Also provided herein is also an output generated according to any one of the methods described above.

[0129] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1: MALDI MSI photocleavable probesProbe SynthesisPeptide Mass Tag Synthesis

[0130] A CEM Liberty Blue 2.0 microwave peptide synthesizer was used to synthesize 0.1 mmol variable static sequences prior to combinatorial manual synthesis. The first seven residues of the peptide were made on Fmoc-Arg(Pbf)-Wang resin (0.3 mmol / g). The resin was swollen in anhydrous N,N-dimethylformamide (DMF) for five minutes before deprotecting with 4-methylpiperidine (10% in DMF). Amino acid reagents (0.2 M) were coupled using 0.1 M diisopropylcarbodiimide (DIC) and 0.1 M OxymaPure for 2 min under microwave radiation at 90°C. After synthesizing the static region of the peptide, the resin was removed from the synthesizer and combinatorically exposed to manual solid phase synthesis using Thermo Scientific™ Pierce™ 5 mL disposable columns. Briefly, peptides were swollen in DMF for 2 hours and washed with DMF, followed by deprotection of the Fmoc protecting groups by addition of 4-methylpiperidine (25% in DMF) for 10 mins at room temperature. Amino acid coupling was performed by adding 5 molar equivalents of amino acid and 4.9 molar equivalents of DIC / OxymaPure to the column and heating at 95°C for 20 min. The 4-azidomethyl-3-nitrobenzoic acid photoclevable linker is then coupled, allowing for coupling to the modified oligonucleotide. Final deprotection and cleavage of the peptide was carried out by using trifluoroacetic acid (TFA), phenol, triisopropylsilane (TIPS), 88:5:5:2 for 2 hours at room temperature. The peptide was then precipitated in cold diethyl ether and spun down to remove excess deprotection cocktail. After washing the solid pellet 3 times, the ether was evaporated and the peptide dried under vacuum. The peptide was solubilized to 5mM concentration in 50% acetonitrile and stored for nucleic acid probe coupling.4-Azidomethyl-3-Nitrobenzoic Acid Synthesis

[0131] 4-Bromomethyl-3-nitrobenzoic acid (2.5g, 9.61 mmol) was dissolved in 50mL of methanol and stirred on ice. Sodium azide (0.75g, 11 .5mmol) was added as a 0.38Mmethanol solution over 10 minutes. The mixture turned a dark yellow upon addition and after stirring on ice for 1 hour was removed to stir at room temperature overnight. The resulting mixture was evaporated dry and resuspended in a minimal amount of dichloromethane (DCM) (~300ml_). The organic layer was washed with 1M HCI (50mL), dH2O (50ml_) and a brine solution (50ml_) before being dried over sodium sulfate. The DCM was filtered from the sodium sulfate, evaporated on a rotary evaporator, and dried under high vacuum overnight. The resulting solid, a light-yellow powder, (1.86g, 87%) was weighed and examined using ESI-MS in negative mode (Exact Mass = 222.04, Exp Mass = 222.02).Terminal Transferase Labeling with Modified deoxynucleotide triphosphates (dNTPs)

[0132] For a 50pL reaction volume, 3’ OH oligonucleotide sequences (1 uM) were incubated with terminal transferase (1 U / pL) and its provided buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate), cobalt chloride (1 mM), and 5- ethynyl deoxyuracil triphosphate (50uM) for 90 min at 37°C. Following the incubation, the enzyme was heat inactivated at 75°C for 20 min. The reaction was cooled to 4°C and purified using a spin column.Copper Click Coupling of Photocleavable Peptide Mass Tag

[0133] The alkyne-modified oligonucleotide was mixed with the photocleavable peptide mass tag and sodium ascorbate (75uM). After degassing with N2, a pre-chelated mixture of CuSO4(47uM) and Tris(benzyltriazolylmethyl)amine (THPTA) (94uM) to a final volume of 50uL. The mixture was degassed under N2, before allowing to incubate at room temperature overnight. The resulting mixture was purified and concentrated using a spin column.Matrix Assisted Laser Desorption / lonization In-Situ Hybridization Assay

[0134] Fresh frozen tissue was sectioned to 10pm and mounted on an Indium Tin Oxide (ITO) coated glass slide. The mounted tissue was dried in a vacuum desiccator for 30 minutes prior to being subjected to fixation. 10% cold neutral-buffered formalin was used to fix the tissue for 15 minutes, followed by thorough washing with 1X phosphate buffered saline (PBS). The tissue was then gradually dehydrated through a series of ethanol (70%, 95% 100%) and chloroform washes. After drying the tissue, the mass-tagged oligonucleotide probes were hybridized (10% dextran sulfate, 10% formamide, 2X saline sodium citrate (SSC)) at 37°C overnight. Following the incubation, the sample was washed three times with the post-hybridization wash buffer (2X SSC, 10% formamide) for 5 minutes each at 37°C. A cation exchange was then carried out using 300mM ammonium bicarbonate, using an orbital shaker for gentle agitation. The buffer exchange was performed for twice, each for 5 min. The sample was then air dried for one hour under darkness. Photocleavage was preformed using the Phrozen Tech UV illuminator, exposing the UV-A light for 20minutes at full power.Mass Spectrometry Imaging Parameters / lmage Processing

[0135] The sample is then coated in a-Cyano-4-hydroxycinnamic acid (5 mg / mL in 70% methanol) on an M3+ automated aerosolized sprayer (HTX Instruments). The sample was sprayed over 8 passes using N2 heated to 80°C, using a flow rate of 120 pL / min. a nozzle velocity 1200mm / min and drying time of 4s. The sample was then run on the Bruker timsTOF fleX, using a the microgrid-assisted laser footprint of 5pm. The mass spectrometer was run in positive ion mode, using the quadrupole as a mass filter for ions less than 1 ,000 Da and higher than 3,000 Da.

[0136] Results

[0137] Figure 3 shows afresh frozen sagittal section of a wild type mouse brain.Example 2: Expand the number of MALDI MSI Probes Available for Use

[0138] MALDI ISH was developed for highly multiplexed analysis of gene expression (Figure 4). MALDI ISH MSI is a technique that replaces a fluorescent label with a photocleavable mass tag to enable spatial visualization of nucleic acids using a mass spectrometer. This platform has primarily been used to analyze mRNA through an smFISH- like approach, where a hybridization probe directly interacts with the mRNA target. Terminal transferase (TdT) was have previously used to create a modified tail to which specific mass tags can attach, effectively amplifying the detectable signal for the mass spectrometer. This approach allows for the simultaneous detection of multiple RNA targets and has the capability of detecting other endogenous metabolites, such as lipids, small molecules, glycans, and more. By using a mass spectrometry system, it is possible to plex higher than is achievable with fluorescence microscopy as well as higher sensitivity and selectivity that is afforded by mass spectrometry. In brief, a copper-click cycloaddition reaction can be used to attach a photocleavable linker to complementary RNA probe for genes of interest. This enables indiscriminate coupling of a unique mass tag (e.g. peptide, lipid, small molecule, etc.) to hundreds of different probes that can be detected using MALDI MSI (Figure 5). This enables mapping of thousands of cell types / states and transcripts using modern MSI on a single instrument. To date, a total of 20 neuroscience-related MALDI ISH probes have been curated (Figure 6) that have high sensitivity and specificity by MALDI MSI. This demonstrates that the technology works and that a single ISH probe can be used to detect designated mRNA features, rather than traditional FISH, which often requires several probes targeting one mRNA for sufficient detection. MALDI ISH can therefore be expanded to incorporate at least an additional 50 neuroscience-related genes (e.g., reactive astrocytes, activated neurons, oligodendrocytes, microglia, etc.) and at least an additional 50 immune system-related genes (e.g., T-cells, B-cells, macrophages, monocytes, etc.).Example 3: Methods for Amplification of MALDI ISH Signals

[0139] The ability to amplify nucleic acids has been leveraged in various ways to address issues associated with instrumental sensitivity and detection. The polymerase chain reaction revolutionized the use of DNA polymerases to analyze and utilize the biochemical properties of nucleic acids. One particularly interesting method of amplification is rolling circular amplification (RCA), where a circular padlock probe containing two complementary arm sequences hybridizes to a target (RNA / DNA). The padlock probe is specific to the target, aligning a 5’ phosphate group with the 3’ hydroxyl end of the circular probe. The proximity of the two ends allows T4 DNA ligase to close the loop, and the double-stranded region between the probe and the target serves as a primer site for the polymerase to begin amplification. This process forms a DNA nanoball from the circular template being copied repeatedly, creating a repeating sequence to which a detection probe can be applied.

[0140] RCA has been recently used in popular techniques like Xenium and Stereo- seq to enable amplification of designed probe sequences. RCA allows for simultaneous amplification of padlock probes, creating potential for high-plexity applications. While TdT tailing amplification has been successful, RCA offers the opportunity to further amplify the signal and enhance the spatial detection of lowly expressed genes. In this approach, the DNA nanoball is created just as in fluorescent detection, however, a photocleavable mass- tagged probe is used instead. From a synthetic standpoint, RCA allows for the opportunity to singly label a probe because of the increased number of hybridization sites within the DNA nanoball. Singly labeling a probe is more efficient and robust compared to labeling the tail due to the increased entropy of the hundreds of bases in the tail. From a mass spectrometry perspective, the amplification is compatible with simultaneous untargeted metabolomic analysis and enriches the relative abundance compared to highly abundant lipids. Because mass spectrometry has a large dynamic range, amplifying the signal is a helpful way to visualize ions / molecules that are a small part of complex mixtures. While still detectable, the intensity of detectable ions is an important consideration in design because of the amount of ionization energy applied and the instrument’s ability to accurately measure the complex mixture. As such, RCA can be used in the disclosed methods to increase the number of mass tags associated with each mRNA sequence for enhanced detection and sensitivity. This can increase the sensitivity of MALDI ISH by over an order of magnitude. The sequences included those previously curated (Figure 6) as well as those developed in Example 2.Example 4: Develop an integrated MSI approach for measuring metabolomics, cell type, and gene expression

[0141] The breadth of spatial multiomic capabilities can be further expaned by integrating a similar photocleavable mass strategy into an immunohistochemistry workflow,known as MALDI IHC. This approach involves directly tagging antibodies with photocleavable mass tags and applying these custom probes to a standard IHC assay. However, the current version of the MALDI IHC that is commercialized faces sensitivity issues, because there is no amplification and only a limited and unknown number of mass tags can be added. Ultimately, this not an issue for highly expressed proteins, but often lowly expressed proteins are critical for cell typing or disease states. Thus, it can be difficult to characterize proteins that are in low abundance when multiplexing other more abundant molecules, including other proteins, mRNA, or lipids, because of instrumental dynamic range. Additionally, directly conjugating a mass tag to an antibody may affect the anybody’s binding efficiency and specificity due to the steric bulk involved when indiscriminately labeling amines with a bulky peptide mass tag. These changes and alterations are often observed in the MSI images when compared to fluorescence tags and images using the same antibodies. Strategies like co-detection by indexing (CODEX) have demonstrated the multiplexing capability of IHC by conjugating a DNA oligonucleotide barcode that can then be probed with a labeled detection probe (in our case a mass tag). CODEX has been shown to be compatible with standard MALDI MSI workflows and can be combined with immunorolling circular amplification, which can be simultaneously used with standard RCA for RNA detection, significantly simplifying the assay and amplifying signal, while maintaining the antibody binding site. As disclosed herein, oligonucleotide barcodes and RCA can be used to increase the number of mass tags associated with each antibody for enhanced detection and sensitivity. This can increase the sensitivity of MALDI ICH by over an order of magnitude.

[0142] The future of understanding biological systems will come from truly integrated multiomics. Modern MS systems are highly sensitive (<ppb), capable of imaging entire tissue regions at cellular resolutions, and have sufficient mass resolution for distinguishing tens of thousands of features in a single pixel. Because of this, adding MS mass tags to antibodies for immunohistochemistry (IHC, Figure 7) and in situ hybridization (ISH, Figures 5 and 6) enable their detection with a mass spectrometer, along with their metabolomic counterparts within a single experiment (Figure 8). Through this, and validation from the orthogonal experiments, this all-in-one omics methodology enables true comparison of transcriptomics, proteomics, and metabolomics. With this data one can calculate metabolomic profiles of discrete cell types or cell states, such as single oligodendrocytes (Figure 8B). One can also use this data to understand molecular neighborhoods within a variety of organ systems. In brief, a mixture of open-source software can be used for cell segmentation, neighborhood analysis, and image registration. Machine learning methods will be used to process the “big data” that is produced.

[0143] In brief, murine or human tissue (bought from vendors or provided by collaborations) are frozen over an isopentane slurry, embeded in carboxylmethylcellulose, cryosection to 10 pm, thaw mounted onto conductive slides, and stored at -80 °C until analysis. Prior to analysis, slides are thawed inside a desiccator to prevent metabolite delocalization. MALDI IMS analysis is performed on a Bruker timsTOF fleX MS system at <10 pm spatial resolution in positive and negative ion mode to obtain metabolomic profiles of the tissue. Detected molecules are putatively identified via accurate mass measurements (<1 ppm error) and database searching. Based on prior experimentation, one can identify >100 molecular features. MALDI matrix is then be removed, and tissue is concurrently fixed. Tissues then undergo MALDI ISH (Figure 8) and then MALDI IHC (Figure 7) to obtain gene expression and protein data using the same instrument. CODEX MxlF processing or spatial transcriptomics using traditional methods (e.g. DSP spatial profiler or Visium) can be performed to validate the data from the all-in-one method as well as provide an alternative if the all-in-one is not applicable to all tissues.

[0144] This has extensive applicability to study a variety of disease states, but may have the largest impact on understanding lipid metabolism, signaling, and impact on human health and disease. In summation, this approach stands to develop workflows for spatially assaying gene expression, protein abundance, and metabolomics using the same imaging system on the same sample.Example 5: Spatial Multiomics Combining Lipids and Gene Expression using MALDI ISH MSIResults

[0145] This Example describes a method of performing in-situ hybridization using MALDI MSI, enabling single-cell resolution, multiplexed, multiomic assessment of gene expression and metabolomic signatures within a single, fresh frozen tissue section (Figure 9). This approach involves the incorporation of peptide-encoded mass tags to the nucleic acid hybridization moieties capable of being analyzed by MALDI MSI. By incorporating a photocleavable linker between the nucleic acid sequence and the uniquely massed peptide tags, mass tags can be temporally released after being delivered to their appropriate spatial location. In brief, our method for mass labeling an mRNA probe is constructed in a flexible and modular format (tags and probes can be mixed and matched) for affordable and scalable synthesis and application. An o-nitrobenzylic azide photocleavable linker was synthesized that is compatible with standard Fmoc protected peptide synthesis. With the goal of having a flexible, modular, and easily expandable design, an azide-modified linker allows for copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry, allowing for orthogonal and efficient labeling. The peptide mass labels are generated via standard Fmoc solid phase peptide synthesis (Fmoc-SPPS). This approach ensures that each mass tag hasa unique m / z value and contains amino acid residues that are highly ionizable for easy MALDI MSI detection. Careful parameters were considered when designing the peptide mass tags to avoid potential side reactions, decreased signal intensity, and on-bead aggregation. One such parameter is the choice of amino acids. Proline (P) was avoided due to difficulty associated with synthesis. Cysteine (C) is prone the formation of disulfide bridges and was excluded. Lysine (K) and histidine (H) at the N-terminus or middle position have been reported to in decrease signal and were therefore not used. Tryptophan (W) and methionine (M) are sensitive to oxidation and were not used. Asparagine (N) and glutamine (Q) are susceptible to loss of ammonia during ionization and were used sparingly. Isoleucine (I) is isobaric with leucine (L) and therefore was not used. Of the remaining 10 amino acids, alanine (A), leucine (L), valine (V), phenylalanine (F), tyrosine (Y), aspartic acid (D), glutamic acid (E), serine (S), and threonine (T) were used, resulting in easier synthesis and greater purity during diethyl ether precipitation. Another parameter is the position of the amino acids. Arginine (R) on the C-term has been shown to promote signal intensity; therefore, all mass tags contain arginine in this position to enhance analysis. Repeating units of either charged residues or hydrophobic residues was avoided to prevent aggregation of peptides during and after synthesis. Mass tag diversity was achieved by using a combinatorial-like approach. Briefly, a 7-mer static peptide was made with a CEM Liberty Blue 2.0 microwave peptide synthesizer and fractioned into twelve different vessels. Three additional amino acid residues were then manually coupled via standard Fmoc-SPPS. Finally, the azide-modified photocleavable linker was added to the N-terminus of each peptide in a similar manner before cleavage from the resin, collection, and analysis.

[0146] Mass tag ionization efficiency is another critical component to having a sensitive and accurate assay. In this context, having a high ionization efficiency allows for the detection of mass tags (and therefore RNA) in low abundances at clear signal-to-noise ratio. This was accomplish by integrating a positively charged amino acid (e.g., arginine) within the designed static sequence such every synthesized mass tag will have a permanent positive charge. By including this in the mass tag’s design, the assay’s sensitivity is such that we can use a single mRNA probe sequence with a to locate endogenous RNA. This simplifies bioinformatic design significantly in comparison to traditional smFISH assays that use at least 25 individual probes labeled in the same fluorescence channel to achieve a clear signal-to-noise ratio. By only needing a single probe sequence per gene target, we reduce material cost, simplify the assay’s protocol, and are capable of probing for potential splicing variants. The simplified protocol also minimizes tissue degradation by using single hybridization step, compared to the fluorescent assays using a cyclic reporter strategy, that adds and removes buffers, enabling the tissue to be used and does not inherently require the removal of lipids or other endogenous molecules. Lastly, while a peptide mass tag wasuse here, it should be noted that anything that can be attached to the carboxylic acid of the azide-modified photocleavable linker can be used as a mass tag, including lipids, small molecules, etc.

[0147] The RNA binding sequences were designed using LGC Biosearch Technologies'Stellaris® Probe Designer to generate a set of 20mer sequences. The proposed RNA sequences were then filtered for guanine / cytosine composition, homopolymer regions, and potential dimerization interactions. After oligonucleotide synthesis, the MALDI ISH probe is synthesized as depicted in Scheme 1 (Fig. 10). Oligonucleotides are modified using 5-ethynyl-2’-deoxyuridine triphosphate (5-EdUTP) using terminal transferase (TdT) for template-independent tailing. Because of the structural similarity to thymidine, the TdT enzyme is highly efficient at incorporating the clickcompatible, 5-EdU. The length of the added tail was optimized to reach lengths greater than 200 bases, providing an amplification domain for the probe, allowing for single RNA detection by increasing the number of available sites for mass tag labeling. Following CuAAC labeling, the constructed probe was purified via ethanol precipitation and put into the ISH portion of the workflow.

[0148] After the conjugation reaction, the resulting triazole linkage is photolytically separated (with 365 nm light) from the DNA probe, such that the peptide mass tag is available for desorption / ionization. In addition to the efficiency of CuAAC chemistry, less purification is needed and enables combinatorial chemistry between mass tags and probe sequences. Triazoles are less common photocaged groups compared to o-nitrobenzylic alcohols or amines, however, have been shown to be photoreactive and stabile during photlysis. Because triazole leaving groups are less commonly used in photocleavable linkers, we verified efficiency of the photolytic separation, as well as the formation of potential side products within the peptide mass tag using MALDI TOF MS. The resulting photolysis products followed the expected cleavage mechanisms, yielding an aldehyde and nitroso group. While the molecular ion associated with the tag was detected, there was an additional ion associated with the loss of oxygen from the photolysis reaction. This has been previously observed and is noted in our sampled spectra. As a result of the photocleavage byproduct, each mass tag has two sets of four main m / z values associated with it. Because of the relatively high mass resolution of modern MALDI MSI instrumentation, this artifact does not significantly reduce the number of detectable labels or produce any overlapping m / z values associated with the protonation states (from arginine residues) and isotopic distribution of the peptide mass tags.

[0149] The MALDI ISH workflow follows a standard smFISH workflow with added NH4OAc post-hybridization washes to enable mass spectrometry analysis. In brief, the fresh frozen tissue is 4% PFA fixed, before the RNA is denatured and incubated with the customsynthesized photocleavable mass tagged probes. After incubating overnight, the unbound / nonspecific probes are washed away, and after air drying the tissue, the mass tags are photolytically cleaved with UV light (365 nm) to make the mass tags available for desorption / ionization by MALDI MSI. The tissue sections are coated in a 2,5- dihydroxyacetophenone (DHA) matrix and run through a MALDI imaging experiment to analyze the spatial distribution of the RNA paired with the constructed probes. One of the key parameters associated with these measurements is the amount of probe required for adequate signal. Using Foxp2 as a model probe sequence for preliminary experiments, probe concentration was adjusted across coronal serial sections (Figure 11 A). The resulting relationship between increasing probe concentration and the resulting intensity demonstrated a linear increase, where 50 ng / pL achieves satisfactory signal with distinct spatial localization across the entire tissue section (Figure 11B). The 50 ng / pL probe concentration was then applied to other unique mRNA probe constructs to examine Foxp2, Proxl , and Gpr17 expression and demonstrated successful signal detection for new RNA binding sequences and new mass tags (Figure 11C-11 E).

[0150] To expand the number of available genes that can be analyzed, the modular design of the MALDI ISH probes was leveraged to systematically evaluate the performance of new constructs. 20 neuroscience relevant genes were selected such that unique MADLI ISH probes could be multiplexed in sets of three across serial sections of mouse brain (Figure 12). New RNA binding sequences were paired with mass tags used in previous experiments, while new mass tags were paired with successfully visualized RNA binding sequences. Each gene shows discrete localization (except Gpr17 which was used twice) in each serial sagittal section imaged at 50 pm spatial resolution. For instance, Fas (CD95) and Slfn5 are mainly expressed within granular and Purkinje cell layers of the cerebellum, while Gfap shows widespread expression as an astrocyte marker. These images were visually inspected in accordance with the Allen Mouse Brain Atlas to verify the accuracy of the distribution of each probe detected.

[0151] Finally, this method enables serial multiomic data acquisition from the same tissue section on one instrument. Multiomic was carried out by first examining native lipids, followed by gene expression products were carried out in two separate MALDI MSI runs. First, the native tissue was analyzed for lipids by spraying DHA over the tissue for MALDI MSI run in negative mode at 10 pm. The matrix was then removed during the fixation and ethanol dehydration steps as part of the standard MALDI ISH workflow. Following the mass tagged probe hybridization, washing, and UV cleavage, the tissue was again sprayed with DHA for analysis in positive mode at 10 pm. The data from the two imaging runs was overlayed in SCiLS LabTM, using the combined average mass spectra across the tissue. (Figure 12). 156 lipids were detected, in addition to the two mass tags that were applied tothe tissue. With the combined data sets, it is possible to use the ISH profiles from individual pixels as a means of identifying cell types, such as in oligodendrocytes, and then extract a complete lipid profile from the same pixel (Figure 12).MethodsSynthesis of 4-Azidomethyl-3-Nitrobenzoic Acid

[0152] 4-Bromomethyl-3-nitrobenzoic acid (2.5 g, 9.61 mmol) was dissolved in 50 mL of methanol and stirred on ice. Sodium azide (0.75 g, 11.5 mmol, 1.2 eq) was added as a 0.38 M methanol solution. The mixture turned a dark yellow upon addition and after stirring on ice for 1 hour was removed to stir at room temperature overnight. The resulting mixture was evaporated dry and resuspended in a minimal amount of dichloromethane (DCM) (~300 mL). The organic layer was washed with 1 M HCI (50 mL), dH2O (50 mL) and a brine solution (50 mL) before being dried over sodium sulfate. The DCM was evaporated under vacuum and further dried under high vacuum overnight. The resulting solid, a light-yellow powder, (1.86 g, 87%) was weighed and examined using ESI-MS in negative mode (Exact Mass = 222.0389; [M-H]- = 221.0279).Synthesis of Photocleavable Peptide Mass Tags

[0153] A CEM Liberty Blue 2.0 microwave peptide synthesizer was used to synthesize 0.1 mmol variable static sequences prior to combinatorial manual synthesis. The first seven residues of the peptide were made on Fmoc-Arg(Pbf)-Wang resin (0.3 mmol / g). The resin was swollen in anhydrous N,N-dimethylformamide (DMF) for five minutes before deprotecting with 4-methylpiperidine (10% in DMF). Amino acid reagents (0.2 M) were coupled using 0.1 M diisopropylcarbodiimide (DIC) and 0.1 M OxymaPure for 2 min under microwave radiation at 90°C. After synthesizing the static region of the peptide, the resin was removed from the synthesizer and combinatorically exposed to manual solid phase synthesis using Thermo ScientificTM PierceTM 5 mL disposable columns. Briefly, peptides were swollen in DMF for 2 hours and washed with DMF, followed by deprotection of the Fmoc protecting groups by addition of 4-methylpiperidine (25% in DMF) for 10 minutes at room temperature. Amino acid coupling was performed by adding 5 molar equivalents of amino acid and 4.9 molar equivalents of DIC / OxymaPure to the column and heating at 95 °C for 20 minutes. The 4-azidomethyl-3-nitrobenzoic acid photocleavable linker is then coupled. Final deprotection and cleavage of the peptide was carried out by using trifluoroacetic acid (TFA), phenol, triisopropylsilane (TIPS), 88:5:5:2 for 2 hours at room temperature. The peptide was then precipitated in cold diethyl ether and spun down to remove excess deprotection cocktail. After washing the solid pellet 3 times, the ether was evaporated, and the peptide dried under vacuum. The peptide was solubilized to 5mM concentration in 50% acetonitrile and stored at -20 °C for conjugation.Photocleavage Efficiency Tests MALDI Spot Analysis

[0154] Synthesized peptide mass tags were diluted to 10 pM in water before being placed under UV-A light for 30 minutes. 2,5-dihyxdroxy acetophenone (DHA) matrix solution (10 mg / mL in 70% acetonitrile) was used to dilute the photocleaved products 10-fold before spotting the mixture onto an ITO slide. Because the photolysis reaction produces an aldehyde, the product is observed as a hydrate.Terminal Transferase Tailing with 5-Ethynyl-2’-deoxyuridine triphosphate (5-EdUTP)

[0155] In a 50 pL reaction volume, 3’OH oligonucleotide sequences (100 pmol, 2 uM) were incubated with terminal transferase (100 U, 2 U / pL) and 1X NEB TdT buffer (50 mM potassium acetate, 20 mM tris-acetate, 10mM magnesium acetate), cobalt chloride (0.25 mM), and 5-ethynyl-2’-deoxyuracil triphosphate (50 nmol, 1 mM) for 90 minutes at 37°C. Following the incubation, the enzyme was heat inactivated at 75 °C for 10 min. The reaction was cooled to 4 °C before proceeded to the mass tag labeling.Copper Click Coupling of Photocleavable Peptide Mass Tag

[0156] The alkyne-modified oligonucleotide (100 pmol, 1.667 pM) is mixed with the azide-modified peptide mass tag (50 nmol, 666.7 pM) and water. CuSO4(50 nmol, 666.7 pM), Tris(benzyltriazolylmethyl)amine (THPTA) (50 nmol, 666.7 pM) are premixed and allowed to incubate for 10 min before adding the the azide / alkyne. Sodium-L-ascorbate (200 nmol, 1.7 mM) is then added and mixed vigorously to a final volume of 75 pL. The mixture was degassed under N2, before allowing to incubate at 37 °C for 2 hours. The resulting mixture was purified by using the Monarch® PCR & DNA Cleanup Kit the resulting products concentration measured.Matrix-Assisted Laser Desorption / lonization In-Situ Hybridization Assay

[0157] Fresh frozen tissue was sectioned to 10 pm and mounted on an Indium Tin Oxide (ITO) coated glass slide. The mounted tissue was thawed in a vacuum desiccator for 10 minutes prior to being subjected to fixation with cold 4% PFA in neutral PBS. The tissue is fixed for 15 minutes at room temperature, followed by two washes with 1X PBS for 5 minutes each. The tissue was washed with nuclease free deionized water for 3 mins and incubated for 2 hours at 4 °C in 70% ethanol. The slide was then washed twice for 5 minutes each in 2X SSC and allowed to incubate at 37°C for 3 hrs in 30% formamide / 2X SSC. The mass-tagged oligonucleotide probes were hybridized (100 mg / mL dextran sulfate, 30% formamide, 2X SSC, 1 mg / mL BSA, 1 mg / mL Yeast tRNA, 200 pM RVC) at 30°C overnight. Following the incubation, the sample was washed with 30% formamide / 2X SSC, 10% formamide / 1X SSC, 10% formamide / 0.5X SSC for 5 minutes each at 37 °C, and 150 mM and 50 mM NH4OAc pH 7.0 for 5 minutes each at room temperature. The sample was then air-dried 15 minutes under darkness. Photocleavage was preformed using the Phrozen Tech UV illuminator, exposing the UV-A light for 30 minutes at full power.Mass Spectrometry Imaging Parameters / lmage Processing

[0158] The sample is then coated in 2,5 dihydroxy acetophenone (DHA) (10 mg / mL in 70% ACN) on an M3+ automated aerosolized sprayer. The sample was sprayed over 8 passes using N2 heated to 50 °C, a flow rate of 120 L / min, a nozzle velocity 1200 mm / min, tack spacing of 2.5 mm, and drying time of 2 seconds. The sample was then run on the Bruker timsTOF flex with experimental respective laser footprint. For RNA mass tag acquisitions, the mass spectrometer was run in positive ion mode, using the quadrupole as a mass filter for ions greater than 1 ,000 Da and less than 1 ,800 Da. Other pertinent MS parameters are shown in SI Table 2. The same tissue section can be imaged for lipids in either positive or negative polarity prior to the MALDI ISH assay. SI Table 3 contains instrumental parameters on the timsTOF fleX used for detecting lipids in these experiments.Conclusion

[0159] Disclosed in this Example is a method for performing MALDI ISH MSI on the murine brain for targeting 20 genes through a cost-effective, flexible and modular synthesis platform. A CuAAC-compatible linker was leverage that is capable of being incorporated with standard Fmoc peptide synthesis and undergoes photolysis efficiently. While the power of this technique was leveraged using a single hybridization sequence with a single peptide mass tag, the indiscriminate labeling chemistry could be applied in a traditional smFISH that uses a set of probes with a single label to increase detected signal even further. Additionally, this mass tagging approach could be used as a substitute for fluorescent oligonucleotide detection probes in more elaborate nucleic acid architectures, such as those used in MERFISH, Xenium, RNAscope and others.

[0160] Also demonstrated is the capability to integrate untargeted lipidomic and gene expression data, derived from the same tissue section. The use of a mass spectrometer enables multiomic detection of lipids and mRNA features that allows for cell type information to give additional context to cell’s lipid profile. This serial approach can easily be applied to incorporate the detection of small polar metabolites, drugs, and secondary metabolites. Additionally, the high resolving power of the mass spectrometer allows for simultaneous multiplexing like never before. While a small sampling has been shown here, the modular design allows for easy expansion to include any gene of choice. This approach can be further expanded and complemented further to simultaneously detect both gene and protein in a combined workflow MALDI IHC and MALDI ISH to cover entire biochemical pathways. Finally, it should be noted that MALDI ISH is not limited to MALDI ionization, and that different ionization sources may be easily integrated to this kind of photocleavable mass tagging strategy.

[0161] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosedinvention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0162] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method of generating a high resolution two-dimensional image of a tissue sample, the method comprising:(a) administering to the sample a plurality of probes that each bind distinct tissue targets, wherein each probe is conjugated to a unique mass tag by a cleavable linker, and wherein each of the unique mass tags are ionizable with a UV laser, charged, and have similar ionization efficiencies;(b) washing the sample under conditions suitable to remove unbound ISH probes;(c) treating the sample to cleave the linker and release the mass tag;(d) subjecting the sample to a mass spectrometry imaging method to generate a data set that comprises the identity and spatially-addressable abundance of each of the released mass tags across an area of said sample; and(e) generating an image from the data set with each unique mass tag being assigned a unique color.

2. The method of claim 1 , wherein the cleavable linker is a photo-labile linker, and wherein step (c) comprises treating the sample with UV light.

3. The method of claim 2, wherein the photo-labile linker is an o-nitrobenzyl-based linker.

4. The method of any one of claims 1 to 3, wherein the plurality of ISH probes bind at least 10, 100, 1 ,000, or 10,000 distinct RNA targets.

5. The method of any one of claims 1 to 4, wherein each of the unique mass tags is a peptide having a molecular weight from 500 Da to 3,000 Da, wherein the peptide comprises at least one charged residue selected from the group consisting of Arginine, Lysine, Histidine, Aspartate, Glutamate.

6. The method of any one of claims 1 to 4, wherein each of the unique mass tags is a lipid between 400 Da and 1200 Da.

7. The method of any one of claims 1 to 4, wherein each of the unique mass tags is a small molecule greater than 250 Da.

8. The method of any one of claims 1 to 7, wherein mass tag has a molecular weight at least 0.1 Da higher or lower than any other mass tag.

9. The method of any one of claims 1 to 8, wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) section, fresh frozen section, or fixed section.

10. The method of any one of claims 1 to 9, wherein the image has at least a 1 ppm accuracy.11 . The method of any one of claims 1 to 10, wherein the probe is an in situ hybridization (ISH) probe comprising a single stranded DNA or RNA oligonucleotide having a first portion that is complementary to a target single stranded nucleic acid.

12. The method of claim 11 , wherein the oligonucleotide further comprises a second portion that is complementary to a fluorescently labeled detection probe, wherein the method further comprises administering to the sample the detection probe and imaging the sample for fluorescence.

13. The method of any one of claims 1 to 10, wherein the probe is an antibody, antibody fragment, or aptamer.

14. The method of any one of claims 1 to 13, wherein the target is endogenous RNA or an administered therapeutic.

15. The method of any one of claims 1 to 14, wherein the mass spectrometry imaging method is carried out using an ionization source selected from the group consisting of matrix-assisted laser desorption ionization (MALDI), nanoparticle initiator mass spectrometry (NIMS), secondary ion mass spectrometry (SIMS), laser desorption, desorption electrospray ionization (DESI), probe electrospray ionization (PESI), laser spray, and laser ablation electrospray ionization (LAESI).

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