Conjugates for mass spectrometry imaging

Abstract

The present invention pertains to the provision of conjugates for the detection of target molecules using mass spectrometry imaging (MSI).

Description

[0001] Description

[0002] Conjugates for Mass Spectrometry Imaging

[0003] Technical Field

[0004] [1] The present invention pertains to the provision of conjugates for the detection of target molecules using mass spectrometry imaging (MSI).

[0005] Background Art

[0006] [2] Immunohistochemistry (IHC) and In Situ Hybridization (ISH) remain so far, the most used methods by biologists and pathologists to image multiple biomarkers in tissue specimens. IHC and ISH analyses are generally performed on tissue samples, for example collected by biopsying or surgical resection of a tumor. IHC / ISH are performed, from thin tissue-sections (e.g., 3 pm) mounted onto glass slides and generally coated to improve the adhesion of the sections. They can be performed from tissues with different preservations, namely fresh frozen (FF) and formalin-fixed and paraffin embedded (FFPE) tissues. A molecular probe or binding molecule is used to recognize the marker of interest thanks to the affinity of this probe against the target. For IHC the probes are antibodies while for ISH the probe are oligonucleotides. Various methods exist to enable the detection of the probes. Fluorophore and chromogenic agents conjugated to antibody or nucleic acid probes are the most common methods used to visualize the spatial distribution of targeted biomolecules using microscopy (e.g., protein antigens or transcripts such as mRNA or miRNA) [Katikireddy and O'Sullivan (2011 ) Methods Mol Biol 784: 155-67].

[0007] [3] However, IHC using standard fluorescence microscopy only allows the simultaneous detection of 3-5 different fluorophores (up to 8 for hyperspectral or multispectral methods that require more advanced equipment), which is a strong limitation in clinical contexts that may require the monitoring of a large panel of biomarkers in a single sample. Over the past two decades, mass spectrometry imaging (MSI) has appeared as an interesting alternative technology for readout, offering greater multiplexing capabilities than fluorescence for mapping antigen distribution in tissues. Interestingly, MS overcomes spectral limitations inherent to photonic techniques and offers multiplexing potential that is only constrained by the spectral resolution of the implemented MS technique, theoretically paving the way to detect and distinguish several tens or hundreds of molecular probes simultaneously. It was indeed shown that MS could be used up to 10-plex and allowed to detect proteins, transcripts and glycans using IHC / ISH methods based on MS detection by Matrix-Assisted Laser Desorption / lonization (MALDI) called Tag-Mass (Lemaire et al. Journal of proteome research 6, 2057-2067 (2007); US patent No.68784805; Gagnon, Prog Histochem Cytochem 47, 133-174 (2012); Stauber et al., Methods Mol Biol 656, 339-361 (2010)). According to this technique, antibodies (Ab) for IHC and oligonucleotides (ON) for ISH were modified to incorporate a photocleavable reporter bearing a peptide tag, which is released by the laser of the MALDI during the MSI experiment. More recently, this concept was extended, allowing for the development of up to 100-plex MALDI-IHC (Yagnik et al., J Am Soc Mass Spectrom 32, 977-988 (2021 ) in relation to the international patent application published under reference WO2022 / 093357). In this most recent version of the Tag-Mass, probe cleavage is achieved before the MALDI-MS Imaging (MSI) acquisition in a dedicated system. In a similar approach, reporters with Tags detectable from tissues by Laser Desorption / lonization (LDI) were also studied (Thiery et al. Proteomics 8, 3725-3734 (2008)). Because the spatial resolution in MSI is not only determined by laser focusing but also by the sample preparation, and more specifically the size of the matrix crystals, developing reporters detectable by LDI is of interest to improve the spatial resolution of MALDI-IHC. Interestingly, up to 4- plex sensitive detection (down to a few fmol) with LDI Tags based on dithioacetal conjugates (Kang et al., Tetrahedron 72, 5612-5619 (2016)) was demonstrated. Higher sensitivity was shown by employing an enzyme / substrate system that catalyzes the conversion of a substrate into an insoluble product, which precipitates at the level of the associated complex detected in LDI (Hong et al., Analytical chemistry 86, 1459-1467 (2014)). This method was validated on FFPE tissues using well- known cancer markers (i.e., Ki67). New reporters for LDI imaging based on Ru(ll)-polypyridine complexes were also demonstrated (Han et al. Chemical Communications 56, 5941-5944 (2020)) for the detection of avp3 integrins. Similar approaches were developed for Secondary Ion Mass Spectrometry (SIMS) with metal isotopes labelling as reporters (Wilson et al. Bioconjugate chemistry 23, 450-460 (2012)). Contrarily to MALDI, the Tag in SIMS is released by fragmentation during the surface sputtering process. These strategies were also integrated with flow cytometry leading to well- known mass cytometry techniques. Mass cytometry is based on metal-coupled Abs which are detected by Laser Ablation-lnductively coupled plasma mass spectrometry (LA-ICP MS - Giesen et al. Nature methods 11 , 417-422 (2014)). Single cells are detected by mass cytometry and the multiplexing can reach up to 18-plex for onco-immunology in routine, but it is limited to the available metals and lanthanides which present a sensitive detection in ICP-MS. Besides, the mass cytometry approach is extremely costly as it requires the acquisition of a highly specific instrumentation.

[0008] [4] All these technologies rely on labelling approaches that involve antibodies (Ab), which are known to present some cross-reactivity. Cross-reactivity occurs when two distinct epitopes are structurally similar and are thus recognized by the same antibody, which can be a technical issue. Indeed, it may lead to non-specific signals or false positive signals when an Ab binds to a biomolecule that is not its intended epitope target.

[0009] [5] Ab production further involves a significant risk of batch-to-batch variation. Their synthesis comprises preparing antigen samples that are subsequently injected into animals to induce high expression levels of antigen-specific Abs in the serum, which can then be recovered from the animal. While polyclonal Abs are recovered directly from the serum, Monoclonal Abs (mAbs) are produced by fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cells to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant. These procedures are cost- and time-consuming and limit the type antigens that can be targeted. It is indeed not possible to obtain Abs directed against toxic molecules as their injection into animal hosts would be lethal. Furthermore, antibodies have a size ranging from 150 to 180 kDa (15 nm diameter), preventing them from recognizing very small targets, from accessing hidden binding sites and from penetrating tissues efficiently. Another drawback in the use of antibodies as binding molecules for mass spectrometry imaging is the need for several complex purifications during the synthesis of the conjugates which may lead to reduced yields.

[0010] [6] There is thus a need for developing new tissue imaging techniques that would not require Abbased bioconjugates for detecting and mapping target molecules. Such techniques must provide high specificity and high sensitivity, avoid cross-reactivity, remain flexible and cost-effective, and enable the detection a larger panel of targets including small molecules (e.g., drugs, small metabolites, toxins, xenobiotics).

[0011] Summary of the invention

[0012] [7] The inventors have devised an innovative conjugate based on a nucleotide sequence that binds to a target molecule, in particular an aptamer, for detecting target molecules via mass spectrometry imaging (MSI). This conjugate is efficiently and flexibly synthesized using click chemistry or bioorthogonal chemistry.

[0013] [8] The first object of the invention is thus a conjugate of formula (A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)P- zq-)nD, wherein

[0014] - A is a tag,

[0015] - u, v, w, x, y, z are linkers,

[0016] - Li , l_2 and L3 are spacer arms,

[0017] - B is a cleavable moiety,

[0018] - C is a linker comprising a triazole moiety or a diazine moiety,

[0019] - n is an integer of 1 to 25,

[0020] - e, f, g, h, i, j, k, p and q are independently selected from 0 or 1 , and

[0021] - D is a nucleotide sequence that binds to a target molecule, in particular an aptamer.

[0022] [9] The invention also pertains to a method for detecting a target molecule in a tissue section, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)p-zq-)nD according to the invention; c) cleaving the cleavable moiety B of the conjugate so as to release the tag A; and d) detecting the tag A released by mass spectrometry imaging.

[0023]

[0010] The use of a nucleotide sequence D, in particular an aptamer, in the conjugate allows for the detection of target molecules to be highly selective, with high reproducibility since the nucleotide sequence, in particular the aptamer, is obtained by chemical synthesis and of wider application range in terms of detectable targets. The use of click-chemistry and bioorthogonal chemistry allows for the synthesis of the conjugate to be modular and flexible, and the conjugate is obtained with good production yield.

[0011] This method based on the conjugate according to the invention is suited for the detection of various targets which include drugs, peptide molecules, proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA-protein, lipids, free or associated glycans, volatile elements, metabolites, mRNAs, circulating tumor cells, non-coding RNAs and proteins issued from alternative open reading frames (ORF). This method presents i) remarkable specificity, ii) remarkable sensitivity, while remaining flexible and cost-effective since it does not require the acquisition of a specific mass spectrometry instrumentation.

[0024] Detailed Description of the invention

[0025]

[0012] Mass Spectrometry Imaging (MSI) is a technology with which the skilled person is completely familiar (see for review Buchberger et al., Analytical chemistry 90.1 (2018): 240).

[0026]

[0013] The inventors have developed innovative conjugates based on a nucleotide sequence that binds to a target molecule, in particular an aptamer, for MSI, which are efficiently and flexibly synthesized using click chemistry or bioorthogonal chemistry.

[0027]

[0014] The first object of the invention is thus a conjugate of formula (A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk- (Lsjp-Zq-jnD, wherein

[0028] - A is a tag,

[0029] - u, v, w, x, y, z are linkers,

[0030] - Li , l_2 and L3 are spacer arms,

[0031] - B is a cleavable moiety,

[0032] - C is a linker comprising a triazole moiety or a diazine moiety,

[0033] - n is an integer of 1 to 25,

[0034] - e, f, g, h, i, j, k, p and q are independently selected from 0 or 1 , and

[0035] - D is a nucleotide sequence that binds to a target molecule, in particular an aptamer.

[0036]

[0015] A nucleotide sequence that binds to a target molecule refers to a sequence that is capable of specifically recognizing and binding to a designated molecule. According to the invention, the nucleotide sequence D is a nucleotide sequence that binds to a target molecule. According to the invention, the nucleotide sequence D may be an aptamer, a cDNA, a siRNA, in particular an aptamer or a cDNA, more particularly an aptamer. According to the invention, D, the nucleotide sequence that binds to a target molecule, in particular an aptamer, may also be understood as a nucleotide sequence that binds to a target molecule, in particular an aptamer, to which a tail (that has reacted by click-chemistry, as explained below in the description) has been appended either synthetically or enzymatically.

[0037]

[0016] In order to obtain the conjugate of the invention :

[0038] - a reactive tail may be appended either synthetically or enzymatically to a nucleotide sequence that binds to a target molecule, in particular an aptamer. The reactive tail consists of the linear assemblage of multiple monomers bearing a reactive chemical function for reactions of clickchemistry or bio-orthogonal chemistry such as Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron- Demand Diels-Alder). A click-chemistry reaction (as explained below) is then carried out between at least one monomer of the tail appended to the nucleotide sequence, in particular an aptamer, and at least one block comprising a tag A and a cleavable moiety B;

[0039] - alternatively, a click-chemistry reaction is carried out between at least a monomer of a reactive tail and at least one block comprising a tag A and a cleavable moiety B; then the tail (that has reacted) is appended to the nucleotide sequence, in particular an aptamer.

[0040]

[0017] Aptamers are short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules. They exhibit specific and unique three-dimensional structures allowing the recognition and binding to specific target biomarkers with high affinity and specificity. In such a way, they are referred to as “chemical Abs”. While aptamers have equal or higher binding affinity than Abs, they display other advantages. Firstly, they are chemically synthesized, reducing batch-to-batch variation in production compared to Ab. Secondly, they exhibit minimal cross-reactivity. Moreover, they demonstrate a broader recognition range of target molecules. Lastly, aptamers can be synthesized without the need for animals. Indeed, the selection of aptamers directed against toxic that cannot be tolerated by the animal system as well as non-immunogenic molecules is possible. Compared to the production of a monoclonal Ab, the procedure to synthesize an aptamer is also faster and cheaper. Moreover, aptamers have a size ranging from 6 to 30 kDa (2nm diameter) while Ab range from 150 to 180 kDa (15 nm diameter). This allows them to recognize very small target molecules, to access hidden binding sites and to penetrate in tissues efficiently. Aptamers also display reversible denaturation property and can be easily functionalized. According to the invention, the term aptamer or aptamer refers to short single-stranded RNA or DNA. The aptamer in the conjugate may also be understood as an aptamer to which a tail (that has reacted by click-chemistry, as explained in the description) is appended either synthetically or enzymatically. Before the click-chemistry reaction, the tail is reactive as it consists of the linear assemblage of multiple monomers bearing a reactive chemical function for reactions of click-chemistry or bio-orthogonal chemistry such as Copper- catalyzed Azide-Alkyne Cycloaddition (CuAAC) or Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron-Demand Diels-Alder).

[0041]

[0018] In a particular embodiment, when the nucleotide sequence, in particular the aptamer, has been modified by an enzyme to bear a reactive tail, then the enzymatic reaction can have produced a mixture of aptamers bearing tails of different lengths. In that case, the number n in the formulas of the invention must be understood as a number average n.

[0042]

[0019] The use of nucleotide sequences, in particular aptamers enables a remarkably selective detection of target molecules, extending to a variety of substances such as drugs, peptide molecules, proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA-protein, lipids, free or associated glycans, volatile elements, metabolites, mRNAs, circulating tumor cells, non-coding RNAs and proteins issued from alternative open reading frames (ORF).

[0043]

[0020] In the context of the present invention, the conjugates are constructed from 2 main building blocks: a block comprising the nucleotide sequence, in particular an aptamer, D, and at least one block comprising a tag A and a cleavable moiety B, considering that these 2 blocks can be easily assembled by click chemistry or bioorthogonal chemistry. This convergent strategy ensures versatility, modularity, and adaptability in the conjugate design, enabling facile structural changes on the block comprising a tag A, allowing each tag A to be applied to various nucleotide sequences that binds to a target molecule, in particular aptamers, without the need for long syntheses, and most importantly it avoids potential technical issues such as loss of recognition due to side reactions or need for complex purification of nucleotide sequences that binds to a target molecule, in particularaptamers.

[0044]

[0021] The two building blocks can be connected via a Click Chemistry or Bioorthogonal Chemistry reaction, in particular via a Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or a Strain- Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron-Demand Diels-Alder). These key reactions can typically be carried out between a building block of formula A-ue-(Li)f-vg-B- Wh-(L2)i-Xj-N3 bearing an azide moiety and a modified nucleotide sequence, in particular an aptamer, D bearing n alkyne moieties, n being comprised between 1 and 25, or between a building block of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-Alkyne bearing an alkyne moiety and a modified nucleotide sequence, in particular an aptamer, D bearing n azide moieties, n being comprised between 1 and 25, thus forming a (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)P-zq-)nD conjugate with C comprising a triazole moiety resulting from the cycloaddition reaction between said azide and alkyne moieties, whereby a single nucleotide sequence, in particular an aptamer, D is conjugated to n A tags, n being comprised between 1 and 25.

[0045]

[0022] The same strategy can be followed by implementing an Inverse Electron-Demand Diels-Alder reaction between a building block of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj-Tetrazine bearing a tetrazine moiety and a modified nucleotide sequence, in particular an aptamer, D bearing n alkene moieties, n being comprised between 1 and 25, or between a building block of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i- Xj-Alkene bearing an alkene moiety and a modified nucleotide sequence, in particular an aptamer, D bearing n tetrazine moieties, n being comprised between 1 and 25. In both cases, the IEDDA reaction results in the formation of a (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)P-zq-)nD conjugate with C comprising a diazine moiety resulting from the IEDDA reaction.

[0046]

[0023] These highly efficient, selective and specific reactions exhibit fast kinetics in water in physiologically relevant conditions of temperature and pH. The use of these types of reactions allows to avoid altogether the exposure of nucleotide sequences that binds to a target molecule, in particular aptamers to reaction conditions that could lead to partial degradation or loss during the production, or loss of target recognition, while allowing the linkage of multiple molecules or tags to a single nucleotide sequence that binds to a target molecule, in particular an aptamer, thus dramatically increasing the sensitivity of the detection. Indeed, the only chemical reactions needed on the nucleotide sequence that binds to a target molecule, in particular an aptamer, after the oligonucleotide synthesis, are an enzymatic reaction and a biorthogonal reaction in an aqueous medium, which is fully compatible with such structures.

[0024] According to a particular embodiment, the nucleotide sequence, in particular an aptamer can typically be modified by introduction of a linear or cyclic alkyne moiety, such as a dibenzocyclooctyne (DBCO) or a BCN (Bicyclononyne), via a linker to avoid steric hindrance.

[0047]

[0025] According to the invention, a target molecule means a molecule of interest that is capable to specifically bind to the nucleotide sequence, in particular an aptamer, D of the conjugate according to the invention.

[0048]

[0026] In one embodiment, the target molecule is chosen in the group consisting of drugs, small molecules, peptide molecules, proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA-protein, lipids, free or associated glycans, volatile elements, metabolites, mRNAs, circulating tumor cells, extra cellular vehicles, non-coding RNAs and proteins issues from alternative ORF.

[0049]

[0027] According to the invention, a metabolite refers to an intermediate or end product of metabolism.

[0050]

[0028] According to the invention, a tag refers to a tag molecule of known molecular weight that is detectable in mass spectrometry profiling and imaging.

[0051]

[0029] The skilled person is completely familiar with the synthesis of tags suitable for detection by MSI. The skilled person knows how to select the most appropriate tag depending on the mass spectrometry technology used. For instance, when MALDI mass spectrometry is used, tags preferably have a m / z <5,000 u. Alternatively, when DESI is used, tags preferably have a m / z <2,000 u. Alternatively, when LAESI is used, tags preferably have a m / z < 5,000 u. Alternatively, when SpiderMass is used, tags preferably have a m / z <5,000 u.

[0052]

[0030] According to an embodiment, in the conjugate of the invention the tag A is selected from any molecules known to presents a good MS detection with the according ion source i.e. a molecule with heteroatoms in the composition and chemical groups such as amines, amides, carboxylic acids, alcohols, phosphates, sulfates, iminium... Thus, this includes peptides, amino-acids, nucleic acids, guanidinium derivatives, sugars, polymers, lipids, metabolites, fluorophores (such as cyanines or rhodamines) and derivatives thereof. In particular, arginine and derivatives thereof can be advantageously used as tags.

[0053]

[0031] Arginine derivatives that can be used as tags are for example L-Arginine alkyl ester dihydrochlorides, in particular L-Arginine methyl ester di hydrochlorides, L-Arginine ethyl ester dihydrochlorides, L-Arginine isopropyl ester dihydrochlorides, L-Arginine butanyl ester dihydrochlorides or L-Arginine isoamyl ester dihydrochlorides.

[0054]

[0032] Alternatively, instead of arginine derivatives, dipeptide or tripeptide derivatives can be used as tags (see Figure 1 ).

[0055]

[0033] According to a particular embodiment, isotopic forms of the tags can be used, in particular deuterated compounds, or13C or15N forms of tags. For example, isotopic forms of arginine and arginine derivatives can be used as tags. For example, isotopic forms of dipeptide and tripeptide derivatives can be used as tags.

[0034] In one embodiment, the u, v, w, x, y, z linkers comprise ester, amide, phosphate, ether, amine, carbonyl, succinimide, thiourea, carbamate or carbonate moieties and derivatives thereof.

[0056]

[0035] In the context of the invention, the term “ester” refers to a compound that carries a functional group of formula -COOR. The term “amide” refers to a compound that carries a functional group of formula -CONRR’. The term “phosphate” refers to a compound that carries a functional group of formula -PC>3(OH). The term “ether” refers to a compound of formula R-O-R’. The term “amine” refers to a compound of formula R-NR’-R”. The term “carbonyl” refers to a compound that carries a functional group of formula -C=O. The term “succinimide” refers to a compound that carries a cyclic functional group of formula (CH2)2(CO)2NR. The term “thiourea” refers to a compound that carries a functional group of formula RHN-C(=S)-NHR’. The term “carbamate” refers to a compound that carries a functional group of formula -0-C(=0)-NHR. The term “carbonate” refers to a compound that carries a functional group of formula -0-C(=0)-0.

[0057]

[0036] In one embodiment, the Li , L2, L3 spacer arms comprise a linear or branched C2 to C20 alkyl chain, said alkyl chain being optionally substituted by at least one functional group selected from amide, carbamate, sulfonamide, ester or ether, or substituted by at least one substituent that may increase the hydrophilic character of the chain, such as alcohols, or sulfonates. The Li , L2, L3 spacer arms can also comprise a a polyethylene glycol (PEG) chain with a number m of repeating units from 1 to 20, preferably of 1 to 12, said PEG chains being optionally substituted with hydrophilic substituents such as alcohols or sulfonates.

[0058]

[0037] In the context of the invention, the term “alkyl” refers to C1-20 linear (i.e., "straight-chain"), branched, or cyclic, saturated hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, groups. The term “polyethylene glycol (PEG) chain” refers to a polyether compound with a structure commonly expressed as R-(O-CH2-CH2)m-R’, m being the number of repeating units. The term “alcohol” refers to a compound that carries an hydroxyl group (-OH) bound to a saturated carbon atom. The term “sulfonate” refers to a compound that carries a functional group of formula -SOs-.

[0059]

[0038] According to the invention, C is a linker comprising a triazole moiety or a diazine moiety. Said triazole or diazine moieties can be advantageously synthesized via a Click Chemistry or Bioorthogonal Chemistry reaction, in particular via a Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or a Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron- Demand Diels-Alder).

[0060]

[0039] According to the invention, a "target molecule" means a molecule of interest that is capable to specifically bind to the nucleotide sequence, in particular an aptamer, D of the conjugate according to the invention.

[0061]

[0040] In one embodiment, the target molecule is chosen in the group consisting of drugs, peptide molecules, proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA- protein, lipids, free or associated glycans, volatile elements, metabolites, mRNAs, circulating tumor cells, extra cellular vehicles, non-coding RNAs and proteins issued from alternative ORF.

[0041] In one embodiment, the B cleavable moiety is a photocleavable moiety, in particular a UV-visible photocleavable moiety (UV-Vis-photocleavable moiety) or an IR-photocleavable moiety.

[0062]

[0042] In one embodiment, the photocleavable moiety is selected from o-nitrobenzyl, arylcarbonylmethyl, coumarin-4-ylmethyl, arylmethyl, arylsulfonyl, arylsilyl groups, orthonitrophenethyloxycarbonyl groups, ortho-nitroanilide groups, p-hydroxyphenacyl and heptamethine cyanine and derivatives thereof.

[0063]

[0043] The UV-Vis-photocleavable moiety may be cleaved by the MALDI Laser or prior to the MALDI- MSI experiment in a dedicated system. Using UV-Vis-photocleavable moiety in the conjugate makes it compatible with MALDI-MSI for the detection of target molecules.

[0064]

[0044] The skilled person is completely familiar with MALDI-MSI technique (see for review: Tuck and al.; Front. Chem. 2022, 10, 904688 doi.org / 10.3389 / fchem.2022.904688). The IR-photocleavable moiety may be cleaved by the IR-MALDI Laser or prior to the MSI experiment in a dedicated system. Using IR-photocleavable moiety in the conjugate makes it compatible with IR-MALDI-MSI for the detection of target molecules.

[0065]

[0045] The skilled person is completely familiar with IR-MALDI-MSI technique (see for review: 10.1021 / acs.analchem.2c03247).

[0066]

[0046] The IR-photocleavable moiety is cleavable by the SpiderMass Laser. Using IR-photocleavable moiety in the conjugate makes it compatible with SpiderMass for the detection of target molecules.

[0067]

[0047] Recently, the SpiderMass technique, a mini-invasive IR laser-based MS technique designed for in vivo real-time molecular analysis, has been described (WO2016046748, Fatou, B., Saudemont, P., Leblanc, E. et al. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Sci Rep 6, 25919 (2016). https: / / doi.org / 10.1038 / srep25919).

[0068]

[0048] The UV-Vis-photocleavable moiety and the IR-photocleavable moiety may be cleaved by the LAESI Laser or prior to the LAESI-MSI experiment in a dedicated system. Using UV-Vis- photocleavable moiety or IR-photocleavable moiety in the conjugate makes it compatible with LAESI- MSI for the detection of target molecules.

[0069]

[0049] The skilled person is completely familiar with LAESI-MSI technique (see for review: doi: 10.1007 / S00425-018-2989-4 ).

[0070]

[0050] In one embodiment, the B cleavable moiety is a chemically cleavable moiety.

[0071]

[0051] In one embodiment, the chemically cleavable moiety is selected from o-nitrobenzyl, imine, azobenzene, disulfide, linear or cyclic acetal, hydrazone, semicarbazone, silylated ether, boronate esters, boronic acids, carbamate and carbonate derivatives

[0072]

[0052] The chemically cleavable moiety may be cleaved by the DESI-MSI electrospray. Using chemically cleavable moiety in the conjugate makes it compatible with DESI-MSI for the detection of target molecules.

[0053] The chemically cleavable moiety may be cleaved by the nano-DESI-MSI nanospray. Using chemically cleavable moiety in the conjugate makes it compatible with nano-DESI-MSI for the detection of target molecules.

[0073]

[0054] The skilled person is completely familiar with DESI-MSI technique (see for reviews: doi.org / 10.1016 / j.ijms.2006.08.003 ; doi.org / 10.1002 / mas.21360; doi:

[0074] 10.1021 / acs. accounts.3c00382) and with nano-DESI-MSI technique (see for review: doi:10.1039 / C0AN00312C).

[0075]

[0055] According to an embodiment, the nucleotide sequence, in particular an aptamer, D is a nucleotide sequence, in particular an aptamer, to which a tail is appended.

[0076]

[0056] According to an embodiment, the nucleotide sequence, in particular an aptamer, D is a nucleotide sequence, in particular an aptamer, to which a tail is appended either synthetically or enzymatically.

[0077]

[0057] According to the invention, the reactive tail consists of a linear assemblage of multiple reactive monomers. These monomers are reactive as they bear reactive chemical function for click-chemistry or bio-orthogonal chemistry such as Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron-Demand Diels- Alder). The reactive chemical function allows to click the reactive monomers to the building block comprising the tag A and the cleavable moiety B. The reactive tail of the invention may comprise at least 7 reactive monomers, in particular at least 10 reactive monomers, more particularly at least 15 reactive monomers. The ratio between the number of monomers clicked to the building block comprising the tag A and the cleavable moiety B, and the total number of monomers of the reactive tail is at least 5%, in particular at least 50%, more particularly at least 80%. Accordingly, from one nucleotide sequence, in particular an aptamer, binding a target molecule, several tags A can be detected by MSI. The remarkable advantage is therefore a signal amplification for the detection of target molecules in tissue sections.

[0078]

[0058] According to an embodiment, the reactive tail is constituted of natural or modified nucleosides / nucleotides, in particular uridine nucleotides, thymine nucleotides or adenine nucleotides, i.e. the reactive tail may be a poly-U tail, a poly-T tail, or a poly-A tail. In this embodiment, to form the nucleotide sequence, in particular an aptamer, D, several strategies may be used. For example, the reactive tail may be enzymatically synthesized by TdT enzyme directly following the synthesis of a nucleotide sequence, in particular an aptamer. For example, a bioconjugation may occur between the reactive tail equipped with a maleimide and a nucleotide sequence, in particular an aptamer equipped with a thiol group. For example, a biorthogonal reaction may occur between the reactive tail equipped with a trans-cyclooctene (TCO), and a nucleotide sequence, in particular an aptamer equipped with a tetrazine.

[0079]

[0059] According to an embodiment, the reactive tail constituted of natural or modified nucleosides / nucleotides may be synthesized by PCR technique.

[0060] According to an embodiment, the reactive tail constituted of natural or modified nucleosides / nucleotides may be synthesized using primers.

[0080]

[0061] According to a particular embodiment, the reactive tail is a poly-U tail.

[0081]

[0062] According to a particular embodiment, the reactive tail is a poly-U tail wherein uridine nucleotides are reactive as they bear a reactive group to perform the click-chemistry, in particular a dibenzocyclooctyne (DBCO). The click chemistry or biorthogonal chemistry reaction is a cycloaddition reaction carried out between at least one uridine nucleotides bearing an alkyne, in particular a DBCO, or an azide and at least a building block comprising the tag A and the cleavable moiety B bearing an azide moiety or an alkyne group and complementary to A, thus forming a conjugate according to the invention.

[0082]

[0063] According to an embodiment, the reactive tail is constituted of modified amino-acids, in particular Lysine, i.e. the reactive tail may be a poly-Lys tail. In this embodiment, to form the nucleotide sequence, in particular an aptamer, D, a bioconjugation occurs between the reactive tail equipped with a maleimide and a nucleotide sequence, in particular an aptamer equipped with a thiol group.

[0083]

[0064] According to an embodiment, the reactive tail is constituted of other synthetic monomers, in particular modified peptidic nucleic acids. In this embodiment, to form the nucleotide sequence, in particular an aptamer, D a bioconjugation occurs between the reactive tail equipped with a maleimide and nucleotide sequence, in particular an aptamer equipped with a thiol group.

[0084]

[0065] According to an embodiment, the reactive tail is selected from a poly-U tail, a poly-A tail, a poly- T tail, a poly-C tail, a poly-G tail and a poly-Lys tail.

[0085]

[0066] According to an embodiment, in order to reach very high sensitivity, the inventors have adapted the SABER amplification method (Saka, S. et al. (2019). Nature Biotechnology, 37(9), 1080-1090) to perform the amplification on a nucleotide sequence, in particular an aptamer, for a detection by MSI. This in situ signal amplification is based on the controlled in vitro synthesis of long repetitive concatemer sequences by primer exchange reaction (PER), followed by programmed in situ assembly. A concatemer is a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series. In the present method, for the synthesis of concatemers, PER uses a catalytic hairpin for controlled extension of a short primer sequence in an iterative fashion. The primer designs are based on a three-letter code (consisting only of A, T and C nucleotides), where G nucleotides are avoided in the primer sequence and in the reaction mixture (Kishi, J.Y. et al. Nature chemistry 10, 155-164 (2018)). Therefore, the absence of this G nucleotide allows the C nucleotide to be used as a plug for the polymerase by locating a CCC motif at the concatemer after the template. To hybridize the concatemer with the nucleotide sequence, in particular an aptamer, D according to the invention, like in the SABER method, it is necessary to implement a 3' DNA sequence or RNA sequence at their extremities. It serves as an orthogonal bridge to carry out specific hybridization of the concatemers thanks to their sequence complementaries. This method thus allows, after recognition of the nucleotide sequence, in particular an aptamer, to specifically hybridize a complementary concatemer. By using this amplification method, the addition of the building block -72- comprising the tag A to the reactive tail is not done directly on the nucleotide sequence, in particular an aptamer, as described above, but upstream. Indeed, in this approach, the addition of the building block comprising the tag A is performed on a reactive tail by the click chemistry approach described above, with the difference that this reactive tail is linked to an orthogonal nucleotide primer. This primer must be complementary to a concatemer already hybridized to the nucleotide sequence, in particular an aptamer, to obtain a specific hybridization. Once this assembly is pre-programmed, a simple exposure of the sample to the appropriate cleavage conditions (for the type of cleavable moiety B used) releases the tag A for analysis by MS. The SABER method adapted to nucleotide sequences, in particular aptamers allows the nucleotide sequence, in particular an aptamer, D to comprise at least two reactive tails attached to tags. In this embodiment, the nucleotide sequence, in particular an aptamer, D is a nucleotide sequence, in particular an aptamer, to which at least two reactive tails are appended. Thus, from one nucleotide sequence, in particular an aptamer, binding a target molecule, even more tags can be detected by MSI. The advantage is therefore an even greater signal amplification for the detection of target molecules in tissue sections. In particular, the reactive tails are selected from poly-U tails, poly-A tails, poly-C tails, poly-G tails, poly-T tails and poly-Lys tails. Optionally, concatemers of intermediate branching may be included to further amplify the signal.

[0086]

[0067] Another object of the invention is a reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-N3, wherein

[0087] - A is a tag,

[0088] - u, v, w, x, are linkers,

[0089] - Li and L2 are spacer arms,

[0090] - B is a cleavable moiety,

[0091] - e, f, g, h and i are independently selected from 0 or 1 , and

[0092] - N3 is an azide moiety,

[0093] A, u, v, w, x, Li, L2, e, f, g, h, i and B being as previously defined.

[0094]

[0068] Another object of the invention is a reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj- Alkyne, wherein

[0095] - A is a tag,

[0096] - u, v, w, x are linkers,

[0097] - Li and L2 are spacer arms,

[0098] - e, f, g, h and i are independently selected from 0 or 1 , and

[0099] - B is a cleavable moiety,

[0100] A, u, v, w, x, Li, L2, e, f, g, h, i and B being as previously defined.

[0101]

[0069] In the context of the invention, the term “alkyne” refers to an unsaturated hydrocarbon containing at least one carbon-carbon triple bond.

[0070] The structure of the reaction intermediates of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-N3 or A-ue-(Li)f- vg-B-Wh-(L2)i-Xj-Alkyne makes them useful as building blocks for the synthesis of a conjugate of the invention of formula (A-ue-(Li )f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)p-zq-)nD . Indeed, said conjugate can be synthesized via a Click Chemistry or Bioorthogonal Chemistry reaction, in particular via a Copper- catalyzed Azide-Alkyne Cycloaddition (CuAAC) or a Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC). These reactions have the major advantages of being highly efficient, selective and specific, while exhibiting fast kinetics in water in physiologically relevant conditions of temperature and pH. By reacting a reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-N3 with a nucleotide sequence, in particular an aptamer, D modified with n alkyne moieties, n being comprised between 1 and 25, a conjugate of formula (A-Ue-(Li )f-vg-B-Wh-(L2)i-xrC-yk-(L3)p-zq-)nD can be easily synthesized. In the same manner, by reacting a reaction intermediate of formula A-ue-(Li )f-vg-B-Wh- (l_2)i-Xj-Alkyne with a nucleotide sequence, in particular an aptamer, D derivatized with n azide moieties, n being comprised between 1 and 25, a conjugate of formula (A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj-C- yk-(L3)P-Zq-)nD can be easily synthesized. Said (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)P-zq-)nD conjugate would then bear a C linker comprising a triazole moiety resulting from the cycloaddition reaction.

[0102]

[0071] Another object of the invention is a reaction intermediate of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj - Tetrazine, wherein

[0103] - A is a tag,

[0104] - u, v, w, x, are linkers,

[0105] - Li and L2 are spacer arms,

[0106] - B is a cleavable moiety, and

[0107] - e, f, g, h and i are independently selected from 0 or 1 ,

[0108] A, u, v, w, x, Li , L2, e, f, g, h, i and B being as previously defined.

[0109]

[0072] In the context of the invention, the term “tetrazine” refers to a compound comprising a sixmembered aromatic ring containing four nitrogen atoms of molecular formula C2H2N4.

[0110]

[0073] Another object of the invention is a reaction intermediate of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj- Alkene, wherein

[0111] - A is a tag,

[0112] - u, v, w, x, are linkers,

[0113] - Li and L2 are spacer arms,

[0114] - B is a cleavable moiety, and

[0115] - e, f, g, h and i are independently selected from 0 or 1 ,

[0116] A, u, v, w, x, Li , L2, e, f, g, h, i and B being as previously defined.

[0117]

[0074] In the context of the invention, the term “alkene” or “olefin” refers to an unsaturated hydrocarbon containing at least one carbon-carbon double bond.

[0075] The structure of the reaction intermediates of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-Tetrazine orA- Ue-(Li)f-vg-B-Wh-(L2)i-Xj-Alkene makes them useful as building blocks for the synthesis of a conjugate of the invention of formula (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)p-zq-)nD. Indeed, said conjugate can be synthesized via Bioorthogonal Chemistry reaction, in particular via an Inverse Electron-Demand Diels-Alder (IEDDA) reaction. This reaction has the major advantages of being highly efficient, selective and specific, while exhibiting fast kinetics in water in physiologically relevant conditions of temperature and pH. By reacting a reaction intermediate of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj- Tetrazinewith a nucleotide sequence, in particular an aptamer, D modified with n alkene moietes, n being comprised between 1 and 25, a conjugate of formula (A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)P- zq-)nD can be easily synthesized. In the same manner, by reacting a reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-Alkene with a nucleotide sequence, in particular an aptamer, D derivatized with n tetrazine moieties, n being comprised between 1 and 25, a conjugate of formula (A-ue-(Li)f-vg- B-Wh-(L2)i-Xj-C-yk-(L3)P-Zq-)nD can be easily synthesized. Said (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(l_3)P- zq-)nD conjugate would then bear a C linker comprising a diazine moiety resulting from the IEDDA reaction.

[0118]

[0076] Another object of the invention is a method for detecting a target molecule in a tissue section, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate according to the invention; c) cleaving the cleavable moiety B of the conjugate so as to release the tag A; and d) detecting the tag A released by mass spectrometry imaging.

[0119]

[0077] According to the invention, a "tissue section" preferably has the following properties: it may be frozen or paraffin-embedded, its thickness is preferably in the order of a mammalian cell diameter, thus comprised between 3 and 20 pm. In the case of a frozen section that was obtained from a frozen tissue using a cryostat, OCT (optimal cutting temperature polymer) is preferably used only to fix the tissue, but the frozen tissue is not embedded in OCT, so that tissue sections were not brought into contact with OCT to avoid contamination by polymers.

[0120]

[0078] In one embodiment, the mass spectrometry imaging used in the method of the invention is MALDI-MSI, DESI-MSI, nanoDESI, LAESI-MSI or SpiderMass.

[0121]

[0079] When using MALDI-MSI, at step c) of the method of the invention, the MALDI laser cleaves the UV-Vis-photocleavable moiety of the conjugate so as to release the tag A. Usually, the MALDI laser operates within a wavelength spectrum spanning from 200 nm to 500 nm, in particular within the range of 300 nm to 400 nm, more particularly 337 nm or 355 nm. The cleavage can occur directly or indirectly. In case of direct cleavage, the laser directly fragments the cleavable moiety. In case of indirect cleavage, the cleavable moiety is cleaved by fragmentation due to desorption / ionization process.

[0080] When using LAESI-MSI, at step c) of the method of the invention, the laser cleaves the UV-Vis- photocleavable or IR-photocleavable moiety of the conjugate so as to release the tag A. The cleavage can occur directly or indirectly. In case of direct cleavage, the laser directly fragments the cleavable moiety. In case of indirect cleavage, the cleavable moiety is cleaved by fragmentation due to desorption / ionization process.

[0122]

[0081] When using DESI-MSI, at step c) of the method of the invention, the electrosprayed charged solvent droplets cleaves the chemically cleavable moiety B of the conjugate so as to release the tag

[0123] A. The skilled person knows which solvents or conditions for the solvent (for example adding a solution of organic molecules) are suitable depending on the chemically cleavable moiety B of the conjugate.

[0124]

[0082] When using nano-DESI-MSI, at step c) of the method of the invention, the nanospray capillary charged solvent droplets cleaves the chemically cleavable moiety B of the conjugate so as to release the tag A. The skilled person knows which solvents or conditions for the solvent (for example adding a solution of organic molecules) are suitable depending on the chemically cleavable moiety B of the conjugate.

[0125]

[0083] Alternatively, according to an embodiment, the cleavable moiety B of the conjugate may be cleaved by a specific device depending on the nature of the cleavable moiety B prior to MALDI-MSI, DESI-MSI, nano-DESI-MSI or LAESI-MSI analysis. For example, for a UV-Vis-photocleavable moiety

[0126] B, the specific device may be a UV lamp having a wavelength in the range from 200 nm to 500 nm, in particular in the range from 300 nm to 400 nm, more particularly 365 nm. For example, for a chemically cleavable moiety B, the specific device may be a sprayer or micro-spotter using solvents. The skilled person knows which solvents or conditions for the solvent (for example adding a solution of organic molecules) are suitable depending on the chemically cleavable moiety B of the conjugate.

[0127]

[0084] As mentioned above, recently, the SpiderMass technique, a mini-invasive IR laser-based MS technique designed for in vivo real-time molecular analysis, has been described (WO2016046748, Fatou, B., Saudemont, P., Leblanc, E. et al. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Sci Rep 6, 25919 (2016). https: / / doi.org / 10.1038 / srep25919). SpiderMass is a water- assisted laser desorption / ionization mass spectrometry (WALDI-MS) technique that enables in vivo and real-time analysis of biological tissues. It uses a fibered infrared laser that excites the water molecules in the tissue, resulting in the desorption and ionization of target molecules that are transferred to a mass spectrometer through a tubing transfer line.

[0128]

[0085] The detailed protocol for SpiderMass is described in the following publication: Ogrinc et al. Nat Protoc 14, 3162-3182 (2019). doi.org / 10.1038 / s41596-019-0217-8.

[0129]

[0086] When using SpiderMass, at step c) of the method of the invention, the SpiderMass Laser cleaves the IR-photocleavable moiety of the conjugate so as to release the tag A. The cleavage can occur directly or indirectly. In case of direct cleavage, the Laser directly fragments the cleavable moiety. In case of indirect cleavage, the cleavable moiety is cleaved by fragmentation due to the desorption / ionization process. Typically, the SpiderMass Laser has a wavelength is the range from 2800 nm to 3000 nm, in particular 2900 nm to 3000 nm, more particularly 2940 nm.

[0130]

[0087] The conjugate according to the invention can be used in SpiderMass, therefore it can be used in in vivo and real-time surface analyses of biological tissues.

[0131]

[0088] According to an embodiment, the method of the invention can be used in multiplexed mass spectrometric imaging of tissues.

[0132]

[0089] As described in W02007000669A2, in the context of a multiplexed mass spectrometric imaging of tissues, multiple target molecules can be detected in a single mass spectrometric imaging readout. Specifically, more than 100 distinct target molecules may be mapped simultaneously in the same tissue section. In this context, the tissue section is placed into contact with multiple conjugates, each specific for one target molecule. The conjugates used in this context comprise distinct binding molecule (each specific for one of the target molecules) and distinct tags, thus displaying distinct molecular weights, to allow for the detection of several distinct target molecules. Using tag with widely dispersed molecular weights, it is thus possible to map simultaneously the expression of many distinct target molecules in the same tissue section. In specific embodiments, the detection encompasses at least 2, at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 target molecules.

[0133]

[0090] The method according to the invention may comprise the use of several a conjugate wherein the tag A for MS detection has the same molecular weight but a different isotopic composition. The tags are differentiated at the time of fragmentation, as in the TMT / SILAC approach (DOI: 10.1002 / mas.21709 ; dx.doi.org / 10.1021 / pr500880b).

[0134]

[0091] The method according to the present invention may be applied to biology- and clinics- related applications. It may find utility in various applications such as clinics, pharmacokinetics, forensic, toxicology and animal / plant biology.

[0135]

[0092] The method according to the invention can e.g. allow to determine the amount of drugs reaching a specific target and the amount of a specific marker that can be found in body tissues at a very low amount for an early diagnosis. As care of patient with heavy and chronic diseases, e.g., inflammatory bowel disease (IBD) or various cancers, represent high costs, the method according to the invention thus may contribute to improved diagnosis and prognosis and thus consequently improved patient management.

[0136]

[0093] The method according to the invention can also be used to quantify specific biomarkers, such as cytokines or other immune markers.

[0137]

[0094] The method according to the invention may be used to quantify the cellular heterogeneity and spatial organization in various samples, such as tumor samples, using a panel of nucleotide sequence, in particular aptamers, specific to immune cells associated with specific cell markers.

[0138]

[0095] The method according to the invention can also be used to detect specific markers, such as cancer markers and evaluating the impact of a drug and its localization as compared to said markers - I- using a tagged nucleotide sequence, in particular an aptamer, directed against the drug. Surface Ghost protein and its corresponding ncRNA may also be tracked.

[0139]

[0096] The method according to the present invention may be adapted for MS Profiling, i.e. the detection of a target of interest in a region without necessarily making the image.

[0140] Description of figure

[0141]

[0097] Figure 1. : Structures of azides wherein the tag is an arginine derivative, a dipeptide derivative or a tripeptide derivative.

[0142] Examples

[0143]

[0098] Chemicals and Instruments

[0144]

[0099] PC Azido-NHS Ester is available from Clickchemistry Tools (reference CCT-1161 ). Water (H2O), ethanol (EtOH), acetic acid, dimethyl sulfoxide (DMSO), methanol (MeOH), ammonium bicarbonate, and 96-well ELISA plates (439454) were obtained from Thermo Fischer Scientific (Courtaboeuf, France). The 99% pure trifluoroacetic acid (TFA), HEPES, Tween 20, 5-DBCO-PEG4-dUTP linker and 2,5-dihydroxybenzoic acid (DHB) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Acetonitrile (ACN) with HPLC Plus grade was purchased Carlo ERBA Reagent. Isopropanol HPLC was purchased from VWR Chemicals. The tris used was supplied by Interchim, the PBS by Gibco and the milk powder by Regilait. The DBCO-PEG5-NHS ester linker was supplied by Click Chemistry Tools. The rtdt enzyme and 5X tdt buffer were purchased from Promega (Madison, Wl, USA).

[0145]

[0100] Thin layer chromatography was performed on MERCK silica gels, KIESELGEL 60 GF254 and revealed under UV light (254 nm and 365 nm). The different conjugates were purified using KIESELGEL 60 GF254 1.0 mm SIL G-100 I UV 254 pre-coated TLC plates. Proton NMR spectra were performed on a BRUKER AM 300 WB (at 300 MHz) using tetramethylsilane as internal reference. Tissues were cut using a CryoStat (Leica Microsystems, Nanterre, France). The ITO (Indium Tin Oxide) slides were purchased from LaserBio Labs (Valbonne, France), while the polylysine coated slides came from EprediaTM (Braunschweig, Germany). The matrix to perform MALDI MS imaging was deposited on tissue using an HTX M5-Sprayer™ (HTX Technologies, Carboro, NC). Conjugate cleavage was activated by exposure to UV light at 365 nm (LED Cube 100 IC from Honle UV Technology, Marlboro, MA). Mass spectrometry analyses were performed on a Rapiflex Tissuetyper MALDI TOF (Bruker Daltonics, Billerica, MA) equipped with a 3D Smart Beam laser.

[0146]

[0101] 1. Chemical synthesis

[0147]

[0102] Synthesis of Arginine Tags: Esterification of the carboxylic acid function.

[0148]

[0103] Arginine hydrochloride (1.0 eq) was dissolved in the alcohol of interest (0.01 M) before the solution was cooled to 0°C with an ice bath. A solution of thionyl chloride (2.0 eq) was added dropwise. The reaction mixture was stirred under reflux for 90 min until a yellow solution was obtained before being returned to room temperature for 16 hours. The solvent was removed in vacuo, and the crude reaction was transferred to 15mL of alcohol to repeat the procedure twice.

[0104] The residue was taken up in 1mL of methanol, and diethyl ether was added to the solution until it became cloudy. The mixture was kept at room temperature for 4-5 hours before being put under 4°C to complete the crystallisation. After removing the solvents, the arginine ester is obtained as a dihydrochloride salt (white solid).

[0105] Table 1 : Arginine tags synthesized from the corresponding alcohol.

[0149]

[0106] Azide synthesis.

[0150]

[0107] Azides are synthesized from the corresponding arginine tags.

[0151]

[0108] In an inert atmosphere, 1.5 equivalents of L-Arginine alkyl ester dihydrochloride and one equivalent of PC Azido-NHS Ester were dissolved in anhydrous dichloromethane. The reaction mixture was stirred at room temperature and 3 equivalents of triethylamine were added dropwise.

[0152] The reaction mixture was kept in the dark under stirring at room temperature for 18 hours.

[0153]

[0109] Excess L-Arginine alkyl ester dihydrochloride was filtered off and the solvent removed in vacuo. The triethylamine salts were removed by precipitation using cold acetone, then the reaction crude was purified by chromatography on silica with dichloromethane and methanol.

[0110] Purification: DCM / MeOH (95 / 5).

[0154]

[0111] Yellow oil.

[0155]

[0112] Table 2 : Azides synthesized from the corresponding arginine tags.

[0156]

[0157]

[0113] Data regarding sensitivity of the azides

[0158]

[0114] The inventors obtained data showing the sensitivity of the 10 MS reporters (T1-T0, see Table

[0159] 2) which show similar signal indicating an improvement in terms of quantification (by comparison to Myralis from Ambergen)

[0160]

[0115] The sensitivity of probes with closely related chemical formula (using stable isotope and various ester) is enhanced due to the intrinsic proximity of structural elements within the molecule. -27-

[0161] This arrangement facilitates efficient detection of the MS reporter after cleavage, thereby enabling the potential for quantification by mass spectrometry.

[0162]

[0116] Therefore, a correction factor could be applied, as is already done in fluorescence spectroscopy, particularly in FRET-based techniques or with internal reporter probes.

[0163]

[0117] By analogy, the use of calibrated tags or internal isotopic standards could allow for more accurate quantification in MSI or targeted MS, even in complex biological contexts.

[0164]

[0118] The inventors also showed the detection of the MS reporter after chemical cleavage in acid condition of one of the Tag normally used for photocleavage.

[0165]

[0119] The photo-cleavable probes (azides) can also be cleaved under mild acidic conditions, notably in the presence of 10% TFA (trifluoroacetic acid), which is compatible with the solvents used for sample ionization via electrospray.

[0166]

[0120] This ionization technique is commonly applied in mass spectrometry systems coupled with DESI or nano-DESI sources operating directly on tissue.

[0167]

[0121] This demonstrates the chemical and analytical compatibility of these probes (azides) with ambient ionization mass spectrometry platforms, thus supporting their use in molecular imaging workflows under near-native conditions.

[0168]

[0122] 2. Preparation of molecular conjugates

[0169]

[0123] Aptamers

[0170]

[0124] Aptamers activation

[0171]

[0125] Functionalisation of the aptamer is an enzymatic reaction that must be carried out on the aptamer allowing 3'-OH elongation with a specific enzyme (rTdT) to chemically add activated nucleotides from dUTP-PEG4-DBCO.

[0172]

[0126] To do this, the aptamer is thawed, then heated at 95°C for 5 min and placed 15 min at room temperature (this step allows the aptamer to be conformed correctly).

[0173]

[0127] The following reaction mix was then prepared, in which 4 pL of 5X TdT, 5 pL of 1 mM dUTP- PEG4-DBCO in DMSO, 0.7 pL of rTdT (30 U / pL) and 40.3 pL of H20 were added to 2 pL of 10 pM aptamer. The mixture was incubated at 37°C for 30 minutes to obtain the polyU tail on our aptamer.

[0174]

[0128] Coupling of derived aptamers with azides by click chemistry.

[0175]

[0129] 44pL of PBS and 4pL of 1 mM Tag in DMSO were added to the above reaction mix. The mixture was incubated overnight at room temperature.

[0176]

[0130] Checking conjugate quality using agarose gel

[0177]

[0131] The efficiency of aptamer elongation and click chemistry was checked on a 2% agarose gel: The samples were prepared in such a way as to deposit the same quantity and were placed in 1X Blue Silver buffer. 3 pL of 100 base pair mass control was deposited. The agarose gel was incubated for at least 10 minutes in a solution of Sybr Gold 1X before reading under UV in the Vilber Lourmat instrument using Gel Smart V7.5 software.

[0178]

[0132] Detection of the fluorescence emitted by the Alexa Fluor 488 tag is read directly on an AMERSHAM IMAGER 600 fluorescence reader.

[0179]

[0133] Gel control of aptamer modification

[0180]

[0134] In order to study the behavior of the aptamer with respect to elongation reactions, it was decided to first carry out tests with unmodified dNTPs. The bands visible on the agarose gel showed that elongation of the aptamer by the dNTPs was successful. In addition, the time taken for the elongation reaction influenced the number of nucleotides incorporated into the tail added to the aptamer, making it possible to control the size of the poly-nucleotide tail generated. At 5 minutes of reaction, no amplification was visible, at 30 minutes a band at 150 base pairs (bp) was observed, and a band at 200 bp at 60 minutes. However, when the aptamer was elongated by the dUTP-PEG4- DBCO nucleotide, it was found that the reaction time had no effect on the size of the poly-nucleotide tail synthesized. Indeed, the bands observed on the gel are all around 150 bp. This can be explained by reduced recognition of the dUTP-PEG4-DBCO modified nucleotide by the rTdT enzyme, or steric hindrance that blocks elongation. A second step was carried out by adding an Alexa Fluor 488 tag which binds to the DBCO group of the nucleotides added to the aptamer. The fluorescent tag was successfully attached to the modified aptamer, with the observation of fluorescent bands. This demonstrates the elongation and click chemistry of a tag on an aptamer. This conjugate aptamertag according to the invention can be used for various MSI imaging : MALDI imaging, DESI imaging, SpiderMass imaging, and nano-DESI imaging.

[0181]

[0135] Chemical synthesis of the aptamer-based conjugates : obtained results.

[0182]

[0136] Two aptamer-based conjugates were successfully synthesized. Azides T4 and T5 were coupled with aptamers via click-chemistry as described in the experimental section :

[0183] Conjugate A: azide T4 + aptamer against IL-6

[0184] Conjugate B: azide T5 + aptamer against IL-10

[0185]

[0137] 3. Multiplex immunohistochemistry imaging

[0186]

[0138] For this study, fresh-frozen rat brains were sectioned in a cryostat set at -20°C. Consecutive 12 pm sections were mounted onto ITO conductive slides for multiplex immunohistochemistry imaging by MALDI-TOF MS and on poly-lysine coated slide for DESI and SpiderMass MS Imaging. The slides were then stored at -80°C before use.

[0187]

[0139] The slides to be analysed underwent several tissue preparation steps, including thawing for 1 min in a desiccator, fixation with 2% PFA for 10 min at 4°C, and delipidation to make the protein sites more accessible. Delipidation was carried out by immersing the slides in a series of consecutive baths as follows: 30 sec EtOH 70%, 30 sec EtOH 100%, 2 min Carnoy (3 :6 :1 , v / v / v CHCI3:EtOH:Acetic Acid), 30 sec EtOH 100%, 30 sec H2O, and 30 sec EtOH 100%.

[0140] The tissue was then dried under vacuum before a hydrophobic barrier was applied around the tissue. A blocking buffer (5% rat and rabbit serum, 2% BSA, 0.3% triton X-100 and PBS) was next applied to the tissue for a minimum of 2 hours at room temperature. The buffer was then aspirated and the TAG-conjugated aptamer previously prepared was applied at a concentration of 0.5 pM in blocking buffer. After incubation overnight at 4°C in a humidity chamber, the aptamer solution was removed from the tissue before a series of washes. The slides were then immersed in two baths of PBS containing 0.1% Tween-20 for 1 min, followed by three 5-minute baths of miliQ water. The slides were then dried under vacuum for at least 1 hour.

[0188]

[0141] For the MALDI MS imaging, the slides were exposed to UV light at a wavelength of 365 nm for 20 min, to activate the photo-cleavage of the conjugates.

[0189]

[0142] A) MALDI IMAGING

[0190]

[0143] In order to carry out MALDI imaging, a 20 mg / mL DHB matrix, taken up in 0.1 % MeOH:TFA (70:30, v / v), was sprayed onto the tissue using the HTX M5 automated system. The parameters used for matrix deposition included a spray temperature of 65°C, a plate temperature of 55°C, a pressure of 10 psi and a flow rate of 0.1 mL / min for 12 passes. The slides were then analysed using a MALDI TOF Rapiflex for MS Imaging. The MS spectra were obtained in positive reflectron mode, in the m / z 140-800 range, with 500 laser shots per pixel at a frequency of 5000 Hz, for a spatial resolution of 20 pm and a continuous scan of 20 pm.

[0191]

[0144] The spectral data were processed using Fleximaging software (Bruker Daltonics, Billerica, MA), applying TIC normalisation. It was then possible to visualise the spatial distribution, individually or in multiplex, of each tag deposited on the tissue by selecting the corresponding masses.

[0192]

[0145] Multiplex Aptamer IHC with MALDI-MS Imaging : obtained results.

[0193]

[0146] The inventors have demonstrated that the aptamer-based conjugate of the invention provides great performance in multiplex immunohistochemistry (IHC) with MALDI -MS imaging.

[0194]

[0147] The experiment was performed on a section of rat brain sagittal section from a rat that were submitted to trauma brain injury (TBI), inducing a cerebral inflammatory response.

[0195]

[0148] The inventors used the two conjugates A and B according to the invention, as described above. In details, two aptamers against IL-6 anf 11-10 were functionalized respectively with the T4 probe (for IL-6) and the T5 probe (for IL-10), two key cytokines in immune inflammation processes which are expected to be found from TBI samples.

[0196]

[0149] The MALDI-MS images obtained illustrate the detection of these two markers despite their low relative concentration in tissues. Three images were obtained. The first image shows the distribution of IL-6 based on the distribution of the m / z 217 (T4) and the second image shows the distribution of the 11-10 based on the distribution of the m / z 231 signal in the MS spectra (T5). The third image corresponds to the overlay of the distribution of the T4 and T5 signal on the same image.

[0197]

[0150] Indeed, interleukins IL-6 and IL-10 are generally expressed at low levels in tissues in an acute inflammatory context, of the order of 500 to 1000 pg / mL, while constitutive proteins, such as GFAP, can reach concentrations of 1 ,000 to 100,000 pg / mL. Detection of these weakly expressed targets underlines the high sensitivity of the probes used, as well as their compatibility with other classes of biomolecules such as aptamers.

[0198]

[0151] Another image representing the data from this experiment after their importation in SCILS software for image processing has been obtained by the inventors. Very importantly the MS spectra obtained from the aptamer IHC MALDI MS imaging in the m / z range 200-245 shows that the elevated intensities at m / z 217 and 231 in the tissue treated with the functionalized aptamer are indeed due to specific target recognition by the labeled aptamers (conjugates according to the invention) and that these signals are not found in the MS spectra of the controls.

[0199]

[0152] B) DESI immunohistochemistry imaging

[0200]

[0153] DESI solvents: The solutions were prepared fresh, particularly for the DESI spray, before the analysis. MeOH / H2O:50 / 50, MeOH / H2O:90 / 10, MeOH / H2O:95 / 5, ACN / H20:50 / 50, ACN / H2O with 1% acetic acid:50 / 50, MeOH / H2O with 0,1% TFA:50 / 50, ACN / H2O with 0,1% TFA:50 / 50 were tested as solvent spray.

[0201]

[0154] DESI imaging

[0202]

[0155] The mass spectrometer is a Quadrupole Time-of-Flight (Q-TOF), Waters Synapt G2-Si ion mobility enabled. The calibration of the mass spectrometer is performed with the ESI source using sodium formate 0.5 mM diluted in 90 / 10: isopropanol / HPLC grade water. The ESI source is replaced by the DESI source from Waters, with the High-Definition Imaging (HDImaging) software. The syringe pump used is Harvard Apparatus, Pump 11 Elite. The syringes used is SGE Luer-lock of 1 mL or 2.5 mL with respectively internal diameter of 4.606 mm and 7.284 mm.

[0203]

[0156] The DESI source is installed, with all the connections. The nitrogen gas pressure is set to around 0.5 MPa. The initial setup of the DESI source was performed using methanol as spray solvent with a flow rate of 2pL / min. The gas used is nitrogen at a pressure of 5 bar. The spray angle was set to 55°. The MS inlet capillary, also named bazooka, is placed as close as possible to the sample surface. The spray tip is placed at approximatively 1 mm above the surface. The distance between the spray tip and the bazooka is approximatively 3-4mm.

[0204]

[0157] The MassLynx software is used to control the DESI and mass spectrometer during the analysis. Imaging are setup with the software High-Definition Imaging (HDI) or alternatively they can be exported in the imzML format to be processed using the SCILS software (SCILS multivendor). DESI images are always acquired in a rectangular shape. To acquire an image, different steps are necessary: select and scan the sample, load the image into HDI and select the area desired for imaging, select the parameters desired for this analysis. This information is saved and exported to Masslynx, the method of analysis is loaded into MassLynx and the experiment is started. For lipid analysis, the m / z 100-1000 range was chosen. The data was acquired in sensitivity mode, in both positive and negative mode. The MS inlet temperature was set up at T=150 °C. The voltage is constant at V=4.5 kV. The gas pressure was fixed at 5 bar but can be modified to enhance the analysis.

[0158] Image processing

[0205]

[0159] The visualization of DESI images of a selected mass is possible with the HDI software package. DESI images are processed from the raw data. The processing involves peak picking to preserve the high mass accuracy data, to reduce the low background peaks and to reduce the file size. After normalization of the data by total ion count, it is possible to visualize individual m / z images or overlay of images. HDI Software allows to export images with different processing methods and export mass list. Region of interest analysis of multiple images can be performed. Hierarchical clustering analysis can be implemented with HDI. Multivariate statistical analysis, namely Principal Component Analysis (PCA), can be performed, if there are many data points, to search regions of interest in the tissue. If imaging data are processed with SCILS then various multivariate and / or univariate statistical analysis can be performed including segmentation.

[0206]

[0160] C) Spider MASS immunohistochemistry imaging

[0207]

[0161] SpiderMass analysis

[0208]

[0162] The overall layout of the instrument setup has already been covered elsewhere (Ogrinc et al. Nat Protoc 14, 3162-3182 (2019). doi.org / 10.1038 / s41596-019-0217-8). In brief, the system is made up of three parts: the mass spectrometer itself, a laser system standing remotely from the MS instrument for micro-sampling of tissues and a transfer line allowing for the transfer of the microsampled material back to the MS instrument. The first component consists of a pulsed Nd:YAG laser (pulse duration: 5 ns, = 1064 nm, Quantel, Les Ulis, France) pumping a tunable wavelength OPO (Radiant version 1.0.1 , OPOTEK Inc., Carlsbad, CA, USA). A handpiece with a 4 cm focusing lens is attached to the end of the biocompatible laser fiber, which is connected to the laser system output and has an inner diameter of 400-500 microns and a length of 1 m. In these studies, the laser intensity was set to 4 mJ / pulse for a fixed irradiation time of 10 s, resulting in a laser fluence of approximately 3 J / cm2. The second component of the system is a 2 m transfer line made of Tygon ND 100-65 tubing (Akron, Ohio, USA, 2.4 mm inner diameter, 4 mm outer diameter). The transfer line is directly connected to the mass spectrometer (Xevo, Waters, Manchester, United Kingdom) from which the conventional electrospray source was removed and replaced by a REIMS interface on one side and is attached to the laser handpiece on the other. A 200 pL / min infusion of isopropanol was administered before each acquisition. 10-200 pg / mL of Leucine enkephalin was added to the infusion to play the role of a lockmass. The sampling position was determined based on the histopathological annotations. The acquisition was composed of a burst of 10 laser shots resulting in an individual spectrum. Spectral acquisition was performed both in positive and negative ion mode in sensitivity mode with a scan time of 1 s. The mass range was set to m / z 50-1000.

[0209]

[0163] SpiderMass MS Imaging

[0210] The SpiderMass setup was described in the previous section. To perform imaging analysis, the Spider-Mass microprobe (conjugate) was coupled to a stiff robotic arm described in a previous work (Ogrinc, N., Kruszewski, A., Chaillou, P., Saudemont, P., Lagadec, C., Salzet, M., Duriez, C., and Fournier, I. (2021 ). Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging. Anal. Chem. 93, 14383-14391 . 10.1021 / acs.analchem.1c01692). The spatial step size was set to 250 pm to achieve imaging by oversampling and reach 250 pm spatial resolution for the images. The final spatial resolution was divided by two thanks to interpolation algorithm. The mass-range was fixed between m / z 100-1000. The acquisition sequence was composed of 3 consecutive laser shots and 3 seconds between each step. The laser bursts and the spectrometer acquisition were automatically triggered through a MATLAB in-house user interface developed for the robotic WALDI-MSI (I .Ogrinc, N., Kruszewski, A., Chaillou, P., Saudemont, P., Lagadec, C., Salzet, M., Duriez, C., and Fournier, I. (2021 ). Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging. Anal. Chem. 93, 14383-14391.

[0211] 10.1021 / acs.analchem.1c01692). The data was acquired in positive and sensitivity ion mode.

[0212]

[0164] 4. ISH with oligonucleotide-based conjugate: obtained results.

[0213]

[0165] The strategy previously described for performing immunohistochemistry-mass spectrometry (IHC-MS) using aptamers was found to be compatible with a broad range of oligonucleotide structures. This approach was extended to in situ hybridization (ISH) to target transcriptomic content by employing complementary single-stranded DNA (cDNA).

[0214]

[0166] In this experiment, a cDNA targeting actin mRNA was enzymatically elongated via terminal deoxynucleotidyl transferase (TdT), incorporating click-compatible dUTP-DBCO units. A subsequent click chemistry reaction was carried out to attach the T6 probe (T6-Azide, see Table 2) (m / z 245.217) to the oligonucleotide.

[0215]

[0167] The inventors have thus synthesized a conjugate as follows :

[0216] Conjugate C : azide T5 + cDNA having a “poly-U-DBCO” tail.

[0217]

[0168] A control sample lacking cDNA (but containing all other reaction components) was processed in parallel. Images were obtained. The resulting images show that: the sagittal rat brain section (centered on the cerebellum) revealed a structured molecular signal consistent with actin expression in the experimental condition, whereas only background noise was observed in the control.

[0218]

[0169] These results demonstrate the adaptability of the clickable probe strategy to other recognition molecules, and more specifically to single-stranded oligonucleotides for targeted transcript imaging by ISH-MS.

Claims

Claims

1. Conjugate of formula (A-Ue-(Li)f-vg-B-Wh-(L2)i-Xj-C-yk-(L3)p-zq-)nD, wherein- A is a tag,- u, v, w, x, y, z are linkers,- Li , l_2 and L3 are spacer arms- B is a cleavable moiety,- C is a linker comprising a triazole moiety or a diazine moiety,- n is an integer of 1 to 25,- e, f, g, h, i, j, k, p and q are independently selected from 0 or 1 , and- D is a nucleotide sequence that binds to a target molecule, in particular an aptamer.

2. Conjugate according to claim 1 , wherein the tag A is selected from peptides, amino-acids, nucleic acids, guanidinium derivatives, sugars, polymers, lipids, metabolites, fluorophores and derivatives thereof.

3. Conjugate according to claim 1 or 2, wherein the cleavable moiety B is a photocleavable moiety or a chemically cleavable moiety.

4. Conjugate according to anyone of claims 1 to 3, wherein the photocleavable moiety is selected from o-nitrobenzyl, arylcarbonylmethyl, coumarin-4-ylmethyl, arylmethyl, arylsulfonyl, arylsilyl, ortho- nitrophenethyloxycarbonyl, ortho-nitroanilide, p-hydroxyphenacyl and heptamethine cyanine moieties and derivatives thereof.

5. Conjugate according to anyone of claims 1 to 4, wherein the chemically cleavable moiety is selected from o-nitrobenzyl, imine, azobenzene, disulfide, linear or cyclic acetal, hydrazone, semicarbazone, silylated ether, boronate esters, boronic acids, carbamate and carbonate moieties and derivatives thereof.

6. Reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-N3, wherein- A is a tag,- u, v, w, x, are linkers,- Li and L2 are spacer arms,- B is a cleavable moiety,- e, f, g, h, i and j are independently selected from 0 or 1 , and- N3 is an azide moiety.

7. Reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-Alkyne, wherein- A is a tag,- u, v, w, x are linkers,- Li and L2 are spacer arms,- e, f, g, h, i and j are independently selected from 0 or 1 , and- B is a cleavable moiety.

8. Reaction intermediate of formula A-Ue-(Li)f-vg-B-Wh-(l_2)i-Xj-Tetrazine, wherein- A is a tag,- u, v, w, x, are linkers,- Li and L2 are spacer arms,- B is a cleavable moiety, and- e, f, g, h, i and j are independently selected from 0 or 1.

9. Reaction intermediate of formula A-ue-(Li)f-vg-B-Wh-(L2)i-Xj-Alkene, wherein- A is a tag,- u, v, w, x, are linkers,- Li and L2 are spacer arms,- B is a cleavable moiety, and- e, f, g, h, i and j are independently selected from 0 or 1.

10. A method for detecting a target molecule in a sample, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate according to any one of claims 1 to 5; c) cleaving the cleavable moiety B of the conjugate so as to release the tag A; and d) detecting the tag A released by mass spectrometry imaging.

11. The method according to claim 10 wherein said mass spectrometric imaging is MALDI-MSI, DESI- MSI, nano-DESI-MSI, LAESI-MSI, or SpiderMass.

Images