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WO2025099313A1 - Réduction de la fluorescence ambiante dans des applications d'hybridation in situ en fluorescence à molécule unique - Google Patents

Réduction de la fluorescence ambiante dans des applications d'hybridation in situ en fluorescence à molécule unique Download PDF

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WO2025099313A1
WO2025099313A1 PCT/EP2024/081851 EP2024081851W WO2025099313A1 WO 2025099313 A1 WO2025099313 A1 WO 2025099313A1 EP 2024081851 W EP2024081851 W EP 2024081851W WO 2025099313 A1 WO2025099313 A1 WO 2025099313A1
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black
dye
background
blue
sample
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Lena-Louise DEPNER
Tobias Otto
Christian Korfhage
Cynthia FABER
Disha SHAH
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Resolve Biosciences GmbH
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Resolve Biosciences GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • G01N2474/20—Immunohistochemistry assay

Definitions

  • the present invention relates to a method of reducing background fluorescence in single molecule fluorescence in situ hybridization (in the following referred to smFISH) applications and other hybridization approaches as well as to the use of dyes such as Sudan Black B.
  • smFISH single molecule fluorescence in situ hybridization
  • a common method for tissue fixation is the use of chemical cross-linkers such as formalin. These fixatives work by creating covalent bonds between proteins that bind them together, forming an insoluble mesh that preserves tissue structure.
  • aldehyde fixation combines aldehydes with amines to form Schiff bases which results in autofluorescence.
  • Autofluorescence from such fixation has a broad emission spectrum, occurring across the blue, green and red spectral range. Heat and dehydration of samples can also increase autofluorescence, the effect of which is greater in the red spectrum.
  • Autofluorescence or general background fluorescence can be problematic in fluorescence microscopy. Light-emitting sources, such as fluorescently labeled probes, are applied to samples to enable the visualization of specific molecules. Background fluorescence of the tissue interferes with the detection of specific fluorescent signals, especially when the signals of interest are very dim.
  • Sudan Black B a lipophilic dye
  • Lipofuscin is a granular, lipophilic pigment that accumulates in lysosomes with age in many tissues including skeletal muscles, neurons, and the heart. Lipofuscin fluoresces across the spectra, occurring most strongly at 500-695 nm, and thus can be another problematic compound as its granular appearance can be mistaken for specific staining. To negate these effects, Sudan black can effectively eliminate this autofluorescence (PMID: 10330448). Usually, Sudan black is used to eliminate lipofuscin autofluorescence in monkey, human, or rat neural tissues, using a concentration of 1% (doi: 10.1177/002215549904700211).
  • the technical problem underlying the present invention is to reduce the background fluorescence of FFPE sections significantly without affecting the probe signal detection in fluorophore detection, in particular for fluorescence in situ hybridization applications and in situ protein detections.
  • the present disclosure pertains to methods of removing background fluorescence from a sample comprising: removing lipids from the sample, and contacting the sample to a lipophilic dye.
  • compositions comprising a background reducer dye treated FFPE sample.
  • the said before mentioned background reducer dye is preferably selected from the group consisting of Sudan Black B, TrueBlack®, TrueBlack® Plus, Brilliant blue, Amido black, Eriochrome Black, Trypan Blue, Fast Sulphon Black, Brilliant Black, Evans Blue, Chicago Blue and Toluidine Blue, or mixtures thereof.
  • the background reducer dye is Sudan Black B.
  • FIG. 1 shows various tissue fluorescence intensities.
  • Y axis indicates grey valuses from 0 to 14000, in intervals of 2,000.
  • tissue fluorescence intensities in channel 1, grey, and channel 2, black, for Sudan Black at 0% (control), 0.1%, 0.2% and 0.3%.
  • tissue fluorescence intensity decreases upon Sudan Black treatment, indicating a reduction in background florescence.
  • FIG. 2 shows transcript counts per tile. Number of transcripts is indicated on the y axis from 0 to 50,000 in intervals of 5,000. Transcript counts per tile are indicated for Sudan Black destaining treatments of 0% (control), 0.1%, 0.2% and 0.3%. Sudan blac treatment increases transcript counts per tile, indicating a reduction of background that would otherwise interfere with transcript counts per tile.
  • FIG. 3A and FIG. 3B show microscope images of Sudan Black (FIG. 3A) and TrueBlack (FIG. 3B) during a molecular cartography experiment with a fluidics channel (“Proto-0”).
  • FIG. 4 shows the molecular cartography experiment schematics for a molecular cartography experiment without a fluidics system (“Standard MCI”) and with a fluidics system employed before imaging (“Proto-0 FL”) and the accompanying microscopic images resulting from the molecular cartography assay.
  • the molecular cartography experiment without a fluidics system (“MCI”) and with a fluidics system employed before imaging (“FL01” and “FL02”) images with TrueBlack as a background reducer are shown in Channel 1 and Channel 2.
  • FIG. 5 shown the signal to noise ratio (SNR) for each of the molecular cartography experiment without a fluidics system (“MCI colorization”) and with a fluidics system employed before imaging (“Fluidic engine FL01” and “Fluidic engine FL02”).
  • SNR signal to noise ratio
  • FIG. 5 shows that the signal to noise ratios for each of the molecular cartography experiments with TrueBlack are comparable with and without the fluidics system employed.
  • FIG. 6A shows a TLC analysis of Sudan Black (“SB”) in ethanol and TrueBlack (“TB”) in DMF ran in an ethanol solvent system for the TLC experiment.
  • SB Sudan Black
  • TB TrueBlack
  • FIG. 6B shows a TLC analysis of Sudan Black (“SB”) in ethanol and TrueBlack (“TB”) in DMF ran in a DMF solvent system for the TLC experiment.
  • SB Sudan Black
  • TB TrueBlack
  • FIG. 7A shows a TLC analysis of Sudan Black (“SB”) and TrueBlack (“TB”) dissolved in the same solvent system of ethanol/DMF ran in an ethanol solvent system for the TLC experiment.
  • FIG. 7B shows a TLC analysis of Sudan Black (“SB”) and TrueBlack (“TB”) dissolved in the same solvent system of ethanol/DMF ran in a DMF solvent system for the TLC experiment.
  • FIG. 8 shows absorption spectra of Evans blue and Sudan Black (“Sudan B).
  • FIG. 9 shows absorption spectra of Chicago blue and Sudan Black (“Sudan B).
  • FIG. 10 shows absorption spectra of Trypan Blue and Sudan Black (“Sudan B).
  • FIG. 11 shows absorption spectra of Amido Black and Sudan Black (“Sudan B).
  • FIG. 12 shows absorption spectra of Brilliant Black and Sudan Black (“Sudan B).
  • FIG. 13 shows absorption spectra of Eriochrome and Sudan Black (“Sudan B).
  • FIG. 14 shows absorption spectra of True Black and Sudan Black (“Sudan B).
  • FIG. 15 shows absorption spectra of Toluidine blue and Sudan Black (“Sudan B).
  • FIG. 16 shows absorption spectra of Eriochrome in water (“Eriochrome in H20”) and Sudan Black (“Sudan B).
  • FIG. 17 shows absorption spectra of Eriochrome 1 mM MgCh, Eriochrome 2 mM MgCh, Eriochrome 5 mM MgCh, Eriochrome 10 mM MgCh, and Sudan Black (“Sudan B).
  • FIG. 18 shows absorption spectra of Eriochrome 1 mM CaCh, Eriochrome 2 mM CaCh, Eriochrome 5 mM CaCh, Eriochrome 10 mM CaCh, and Sudan Black (“Sudan B).
  • FIG. 19 shows absorption spectra of Eriochrome at pH 5, Eriochrome at pH 6, Eriochrome at pH 7, Eriochrome at pH 8, and Sudan Black (“Sudan B).
  • FIG. 20 shows absorption spectra of Eriochrome in water (“Eriochrome/H20”) and Sudan Black in water (“Sudan B/H2O).
  • FIG. 21 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 6 (“Eriochrome/SSC, pH6”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • FIG. 22 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 7 (“Eriochrome/SSC, pH7”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • FIG. 23 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 6 with Ca 2+ (“Eriochrome/SSC, pH6, Ca2+”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • FIG. 24 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 7 with Ca 2+ (“Eriochrome/SSC, pH7, Ca2+”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • FIG. 25 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 6 with Cu 2+ (“Eriochrome/SSC, pH6, Cu2+”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • FIG. 26 shows absorption spectra of Eriochrome in saline-sodium citrate (SSC) at pH 7 with Cu 2+ (“Eriochrome/SSC, pH7, Cu2+”) and Sudan Black in water (“Sudan B/H2O).
  • SSC saline-sodium citrate
  • a method for reducing background fluorescence, in particular autofluorescence, in single molecule fluorescence in situ hybridization (smFISH) applications and/or and in situ protein detection wherein first a background reducer dye or a plurality of different background reducer dyes like Sudan Black B is applied to formalin-fixed, paraffin-embedded tissue (FFPE) sections and thereafter the smFISH application is carried out.
  • smFISH single molecule fluorescence in situ hybridization
  • reducing background signals means that the background signal is reduced when the method according to the present invention is applied compared to the background signal produced without applying the method according to the present invention.
  • tissue autofluorescence can interfere with the detection of specific fluorescent signals to enable the visualization of specific molecules. This is reduced by the method according to the present invention.
  • the background reducer dye is selected from the group consisting of Sudan Black B, TrueBlack®, TrueBlack® Plus, Brilliant blue, Amido black, Eriochrome Black, Trypan Blue, Fast Sulphon Black, Brilliant Black, Evans Blue, Chicago Blue and Toluidine Blue, or mixtures thereof, in particular wherein the background reducer dye is Sudan Black B.
  • the background reducer dye is selected from the group consisting of an Indoline dye like Indigo or Indoline dye D149, an Anthrachinon dye like Remazol Black B, an Polymethine dye, an Azo dye like Direct black 19, 22, 28, 36, 38, 56, NY ANZA BLACK or Brillant Black BN, a Polymer dye or a Polymeric Colorant and Toluidine Blue, or mixtures thereof.
  • Sudan Black B used in the method according to the present invention is a lipophilic dye, known to be an efficient eliminator of autofluorescence, such as lipofuscins in neural tissues.
  • it is commonly used for Immunohistochemistry approaches, but to our knowledge, it is not yet used for smFISH applications.
  • Sudan Black B can be efficiently used for smFISH applications of FFPE tissues.
  • Sudan Black B was originally used as a lipid dye to stain lipids, i.e., Sudan Black B binds to lipids.
  • Sudan Black B (2,3-Dihydro-2,2-dimethyl-6- ⁇ [l-naphthyl-4-(phenylazo)]azo ⁇ -lZZ- perimidin) has the following formula:
  • Sudan Black B is a member of the class of perimidines that is 2,2-dimethyl-2,3-dihydro- IH-perimidine carrying a [4-(phenyldiazenyl)naphthalen-l-yl]diazenyl substituent at position 6.
  • TrueBlack® is a reagent developed by Biotium Inc. that quenches lipofuscin autofluorescence in tissue sections for immunofluorescence staining (see https://biotium.com/product/trueblack-lipofuscin-autofluorescence-quencher).
  • TrueBlack® Plus is a next-generation lipofuscin quencher developed by Biotium Inc. (see https://biotium.com/product/trueblack-plus-lipofuscin-autofluorescence-quencher-40x-in-dmso). TrueBlack® Plus is water-soluble, so quenching can be performed in PBS instead of ethanol. In some embodiments, TrueBlack may display an absorption spectra of FIG. 14.
  • the background reducer dye Brilliant blue includes Brilliant blue FCF (Blue 1), a synthetic organic compound used primarily as a blue colorant for processed foods, medications, dietary supplements, and cosmetics ("FD&C Blue 1 (Brilliant Blue)". International Association of Color Manufacturers. Archived from the original on 2019-05-06. Retrieved 2019-05-06). It is classified as a triarylmethane dye and is known under various names, such as FD&C Blue No. 1 or acid blue 9.
  • the background reducer dye Brilliant blue includes Coomassie brilliant blue that is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from Coomassie brilliant blue R-250 by the addition of two methyl groups. The name "Coomassie” is a registered trademark of Imperial Chemical Industries.
  • the background reducer dye Amido black includes Amido black 10B (Sodium 4-amino- 5-hydroxy-3-((E)-(4-nitrophenyl)diazenyl)-6-((E)-phenyldiazenyl)naphthalene-2,7-disulfonate), an amino acid staining azo dye used in biochemical research to stain for total protein on transferred membrane blots, such as the western blot (Kurien, B. T., & Scofield, R. H. (2015). Western Blotting: An Introduction. In B. T. Kurien & R. H. Scofield (Eds.), Western Blotting: Methods and Protocols (pp. 17-30). Springer.
  • Amido black 10B sodium 4-amino- 5-hydroxy-3-((E)-(4-nitrophenyl)diazenyl)-6-((E)-phenyldiazenyl)naphthalene-2,7-disulfonate
  • Amido Black can be either methanol or water based as it readily dissolves in both. With picric acid, in a van Gieson procedure, it can be used to stain collagen and reticulin. In some embodiments, Amido black may display an absorption spectra of FIG. 11.
  • the background reducer dye Eriochrome Black may comprise Eriochrome Black A, an azo dye that can be removed from water by an adsorbent made of magnetic NiFe2O4 nanoparticles and Eriochrome Black T (Sodium l-[l-Hydroxynaphthylazo]-6-nitro-2 -naphthol -4-sulfonate), a complexometric indicator that is used in complexometric titrations, e.g. in the water hardness determination process.
  • Eriochrome may have an absorption spectra as shown in any one of FIG. 13, or FIG. 16 - FIG. 26.
  • the background reducer dye Trypan blue ((3Z,3'Z)-3,3'-[(3,3'-dimethylbiphenyl-4,4'- diyl)di(lZ)hydrazin-2-yl-l-ylidene]bis(5-amino-4-oxo-3,4-dihydronaphthalene-2,7-di sulfonic acid) is an azo dye. It is a direct dye for cotton textiles. In biosciences, it is used as a vital stain to selectively colour dead tissues or cells blue. Trypan blue is derived from toluidine, that is, any of several isomeric bases, C14H16N2, derived from toluene.
  • Trypan blue is so-called because it can kill trypanosomes, the parasites that cause sleeping sickness.
  • An analog of trypan blue, suramin, is used pharmacologically against trypanosomiasis.
  • Trypan blue is also known as diamine blue and Niagara blue. In some embodiments, Trypan blue may display an absorption spectra of FIG.
  • the background reducer dye Fast Sulphon Black includes Fast Sulphon Black F (1- hydroxy-8-(2 -hydroxy-1 -naphthylazo)-2-(4-sulfo-l-naphthylazo)-naphthalene-3,6-di sulfonic acid), a complexometric indicator used with EDTA, almost exclusively used in copper complexation determination.
  • the background reducer dye T-1824 or Evans blue (tetrasodium (6E,6'E)-6,6-[(3,3'- dimethylbiphenyl-4, 4'-diyl)di(lE)hy drazin-2 -yl-l-ylidene]bis(4-amino-5-oxo-5, 6- dihydronaphthalene-l,3-disulfonate) is an azo dye that has a very high affinity for serum albumin. Because of this, it can be useful in physiology in estimating the proportion of body water contained in blood plasma. It fluoresces with excitation peaks at 470 and 540 nm and an emission peak at 680 nm. In some embodiments, Evans blue may display an absorption spectra of FIG. 8. Evans blue
  • the background reducer dye Chicago Blue includes Chicago Sky Blue 6B (6,6'-[(3,3'- dimethoxy[l,l'-biphenyl]-4,4'-diyl)bzs(2,l-diazenediyl)]bzs[4-amino-5-hydroxy-l,3- naphthalenedisulfonic acid, tetrasodium salt), a dye used as counterstain in histochemistry; also potent inhibitor of L-glutamate uptake into synaptic vesicles; also inhibits MIF.
  • Chicago blue may display an absorption spectra of FIG. 9.
  • the background reducer dye Chicago Blue ((7-amino-8-methylphenothiazin-3- ylidene) -dimethylammonium chloride), also known as TBO or tolonium chloride (INN) is a blue cationic (basic) dye used in histology (as the toluidine blue stain) and sometimes clinically (Sridharan, G; Shankar, AA (2012). "Toluidine blue: A review of its chemistry and clinical utility”. J Oral Maxillofac Pathol. 16 (2): 251-5. doi:10.4103/0973-029X.99081).
  • Toluidine blue may display an absorption spectra of FIG. 15. Toluidine blue
  • Indoline is an aromatic heterocyclic organic compound with the chemical formulation H9N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a fivemembered nitrogen-containing ring. The compound is based on the indole structure, but the 2-3 bond is saturated. By oxidation/dehydrogenation it can be converted to indole (Katritzky, A. R.; Pozharskii, A. F. (2000). Handbook of Heterocyclic Chemistry (2nd ed.). Academic Press). Indoline
  • Anthraquinone dyes are an abundant group of dyes comprising a anthraquinone unit as the shared structural element. Anthraquinone itself is colourless, but red to blue dyes are obtained by introducing electron donor groups such as hydroxy or amino groups in the 1-, 4-, 5- or 8-position (Hunger, Klaus, ed. (2003), Industrial Dyes: Chemistry, Properties, Applications, Weinheim: WILEY-VCH Verlag, pp. 35 ff). Anthraquinone dyestuffs are structurally related to indigo dyestuffs and are classified together with these in the group of carbonyl dyes. Anthraquinone dyes include red insect dyes derived from scale insects such as carminic acid, kermesic acid, and laccaic acids. Anthraquinone
  • Methine dyes are dyes whose chromophoric system consists of conjugated double bonds (polyenes) flanked by two end groups: an electron acceptor A and an electron donor D (Zollinger, Heinrich (2003), Color Chemistry: Syntheses, Properties, and Applications of Organic Dyes and Pigments (in German) (3 ed.), Weinheim: WILEY-VCH Verlag, p. 68, ISBN 3-906390-23-3).
  • the background reducer dye like Sudan Black B is applied to FFPE tissue sections.
  • Fixation of tissues with formalin and subsequent embedding in paraffine is one of the most common methods for the preservation and stabilization of biological tissues prior to microsection and histological examination under a microscope.
  • Many clinical specimens are stored as FFPE-treated specimens.
  • the FFPE alterations of the specimen must be reversed, e.g., for extraction and subsequent analysis of the DNA or RNA by sequencing or by microarray for purposes of personalized medicine or proteomics. Therefore, various extraction and renaturation methods have been developed to allow FFPE specimens to be used for other diagnostic procedures years later.
  • the method according to the present invention can be applied to the FFPE tissue sections immediately obtained after the forming of the tissue blocks.
  • the method according to the present invention can also be applied to FFPE tissue blocks which are further processed, for example deparaffinization and/or rehydration, which are usual steps for processing FFPE tissue blocks.
  • tissues can be fixed for several hours (mostly 6-72 h) in formaldehyde of various concentrations (mostly 3,7%, 4% or 10%) at various temperatures (4°C to room temperature). Afterwards, tissues can be dehydrated with alcohol and embedded in paraffin wax. This process leads to a cross-linking of proteins, RNA, and DNA molecules to proteins in the tissue, which is important for the preservation of tissue architecture and keeping the molecules in the physiological position in the tissue.
  • FFPE tissue section (optionally further processed as explained above) is bought into contact with the background reducer dye compound like Sudan Black B.
  • a second step is carried out in which the smFISH application for detecting RNA is carried out with the tissue section obtained after the first step.
  • the method according to the present invention is used for reducing background fluorescence in single molecule fluorescence in situ hybridization applications.
  • single molecule fluorescence in situ hybridization applications are described.
  • smFISH single molecule fluorescence in-situ hybridization'
  • smFISH single-cell in situ RNA profiling by sequential hybridization
  • the mRNAs of interest are detected via specific directly labeled probe sets.
  • the set of mRNA specific probes is eluted from the mRNAs and the same set of probes with other (or the same) fluorescent labels is used in the next round of hybridization and imaging to generate gene specific color-code schemes over several rounds.
  • the technology needs several differently tagged probe sets per transcript and needs to denature these probe sets after every detection round.
  • oligonucleotides of the probe sets provide nucleic acid sequences that serve as initiator for hybridization chain reactions (HCR), a technology that enables signal amplification; see Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), p. 342-357.
  • HCR hybridization chain reactions
  • RNA imaging Spatially resolved, highly multiplexed RNA profiling in single cells, Science 348(6233):aaa6090.
  • the mRNAs of interest are detected via specific probe sets that provide additional sequence elements for the subsequent specific hybridization of fluorescently labeled oligonucleotides.
  • Each probe set provides four different sequence elements out of a total of 16 sequence elements. After hybridization of the specific probe sets to the mRNAs of interest, the so-called readout hybridizations are performed.
  • each readout hybridization one out of the 16 fluorescently labeled oligonucleotides complementary to one of the sequence elements is hybridized. All readout oligonucleotides use the same fluorescent color. After imaging, the fluorescent signals are destroyed via illumination and the next round of readout hybridization takes place without a denaturing step. As a result, a binary code is generated for each mRNA species. A unique signal signature of 4 signals in 16 rounds is created using only a single hybridization round for binding of specific probe sets to the mRNAs of interest, followed by 16 rounds of hybridization of readout oligonucleotides labeled by a single fluorescence color.
  • a technology referred to as 'intron seqFISH' is described in Shah et al. (2016), Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH, Cell 117(2), p. 363-376. There, the mRNAs of interest are detected via specific probe sets that provide additional sequence elements for the subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides one out of 12 possible sequence elements (representing the 12 ‘pseudo colors’ used) per color-coding round. Each color-coding round consists of four serial hybridizations.
  • EP 0 611 828 discloses the use of a bridging element to recruit a signal generating element to probes that specifically bind to an analyte.
  • a more specific statement describes the detection of nucleic acids via specific probes that recruit a bridging nucleic acid molecule. This bridging nucleic acids eventually recruit signal-generating nucleic acids.
  • This document also describes the use of a bridging element with more than one binding site for the signal generating element for signal amplification like branched DNA.
  • Player et al. (2001), Single-copy gene detection using branched DNA (bDNA) in situ hybridization, J. Histochem. Cytochem. 49(5), p. 603-611, describe a method where the nucleic acids of interest are detected via specific probe sets providing an additional sequence element.
  • a preamplifier oligonucleotide is hybridized to this sequence element.
  • This preamplifier oligonucleotide comprises multiple binding sites for amplifier oligonucleotides that are hybridized in a subsequent step.
  • These amplifier oligonucleotides provide multiple sequence elements for the labeled oligonucleotides. This way a branched oligonucleotide tree is build up that leads to an amplification of the signal.
  • RNAscope a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J. Mol. Diagn. 14(1), p.22-29, which uses another design of the mRNA-specific probes.
  • two of the mRNA-specific oligonucleotides have to hybridize in close proximity to provide a sequence that can recruit the preamplifier oligonucleotide. This way the specificity of the method is increased by reducing the number of false positive signals.
  • a further development of the technology uses so called split initiator probes that have to hybridize in close proximity to form the initiator sequence for HCR, similarly to the RNAscope technology, this reduces the number of false positive signals; see Choi et al. (2016), Third- generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145(12).
  • EP 2 992 115 Bl describes a method of sequential single molecule hybridization and provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms through sequential barcoding.
  • Spatial transcriptomics (or Spatial *omics) according to the present disclosure means any kind of analysis where data from the sample are derived in a spatial manner from in-situ samples of tissues or whole organisms.
  • the in-situ sample may be a section of an organ or an organism.
  • the in-situ sample may be not pretreated or pretreated in a way that is required for improving the result.
  • Spatial*omics may include the detection of small molecules compounds of tissues or cells, proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to proteins, DNA, and/or RNA. More preferentially, spatial*omics is restricted to DNA and / or RNA. Even more preferentially, spatial*omics is restricted to smFISH.
  • the spatial transcriptomics detecting comprises a multiplex method for detecting different analytes in a sample by sequential signalencoding of said analytes as described in WO 2020/254519 Al, WO 2021/255244 and WO 2021/255263.
  • the smFISH application is a technique known in this field as “Molecular Cartography, described for example in WO 2020/254519 Al, WO 2021/255244 and WO 2021/255263, the disclosures of which are each hereby incorporated by reference in their respective entireties.
  • Molecular Cartography is based on combinatorial single-molecule fluorescent in situ hybridization (smFISH).
  • smFISH combinatorial single-molecule fluorescent in situ hybridization
  • the present disclosure pertains to a method for detecting an analyte in a sample with a single molecule fluorescence in situ hybridization application comprising first applying Sudan Black B to formalin-fixed, paraffin-embedded tissue sections and thereafter carrying out the single molecule fluorescence in situ hybridization application and/or and in- situ protein detection.
  • the method according to the present invention is used for detecting analytes by spatial transcriptomics as the single molecule fluorescence in situ hybridization application.
  • spatial transcriptomic analysis of biosafety samples is done after RNA or DNA is isolated. This is typical for a scenario using the Visium technology (lOx genomics) or the GeoMx system (Nanostring; https://www.nature.com/articles/s41467-021- 21361-7).
  • the analysis and detection of small quantities of analytes in biological and non- biological samples has become a routine practice in the clinical and analytical environment. Numerous analytical methods have been established for this purpose. Some of them use encoding techniques assigning a particular readable code to a specific first analyte which differs from a code assigned to a specific second analyte.
  • the applied background reducer dye like Sudan Black B is dissolved in a liquid solvent, for example an alcohol, like ethanol.
  • a liquid solvent for example an alcohol, like ethanol.
  • an alcohol like ethanol
  • the alcohol concentration of the alcoholic solution can be about 40 % to about 99 %, for example about 60 % to about 80 %, such as about 63 %.
  • the background reducer dye like Sudan Black B concentration dissolved in the liquid solvent can be about 0.05 % to about 5 %, for example about 0.1 % to about 1 %, such as about 0.1 % to about 0.7 %, in particular about 0.2 % to about 0.5 % percent by weight (wt%).
  • the background reducer dye like Sudan Black B is incubated with the FFPE tissue sections during about 1 second to about 5 hours, for example during about 1 minute to about 30 minutes, such as about 2 minutes to about 15 minutes. This incubation can be carried out at about room temperature.
  • the background reducer dye like Sudan Black B can be applied to FFPE tissue sections which were already further processed. That is, in one embodiment, the background reducer dye like Sudan Black B is applied to FFPE tissue sections after deparaffmization and/or rehydration of said tissue and before fluorescence detection. [0100] In another embodiment, there is used an aqueous solution/alcoholic solution of a lower concentration than used for dissolving the background reducer dye like Sudan Black B for a washing step after incubation of the FFPE tissue with Sudan Black B.
  • the present invention further relates to the use of a background reducer dye like Sudan Black B for reducing background fluorescence in single molecule fluorescence in situ hybridization applications. That is, the present invention is direct also to the use of a background reducer dye like Sudan Black B in the method according to the present invention. Since this method is described in detail above regarding the method steps and the materials used, it is referred thereto in its entirety.
  • kits are a combination of individual elements useful for carrying out the use and/or method of the disclosure, wherein the elements are optimized for use together in the methods.
  • the kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the disclosure.
  • Such kits unify all essential elements required to work the method according to the disclosure, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the present disclosure.
  • quencher or “quencher dye” or “quencher molecule” refers to a dye or an equivalent molecule, such as nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which is capable of reducing the fluorescence of a fluorescent reporter dye or donor dye.
  • a quencher dye may be a fluorescent dye or non-fluorescent dye. When the quencher is a fluorescent dye, its fluorescence wavelength is typically substantially different from that of the reporter dye and the quencher fluorescence is usually not monitored during an assay.
  • sample as referred to herein is a composition in liquid or solid form suspected of comprising the analytes to be encoded.
  • a sample described herein may be a biological sample.
  • a sample may comprise biological tissue, biological cells, and/or extracts and/or part of cells, or any combination thereof.
  • the sample may comprise eukaryotic or procaryotic cells.
  • the sample may derive from any kind of animal (including Homo, rat, mouse, mammalia, birds, fish, insects, worms), plant, or fungi.
  • the sample may comprise a mammalian cell.
  • the sample may comprise a human cell.
  • the sample can be selected from any organ, any tissue, any kind of culture, any specimen taken for diagnostic purposes (e.g. smear, liquid biopsy, tissue biopsy etc.).
  • the sample may comprise DNA and/or RNA. Samples may taken from alive or dead organisms. Cells may not be complete and samples may contain only partial cells.
  • the sample may be frozen, fixed or embedded.
  • the biological tissue, biological cells, extracts and/or part of cells are fixed.
  • the analytes are fixed in a permeabilized sample, such as a cell-containing sample.
  • the term “cell” as used herein is the smallest unit of life and comprises a number (for example, at least one, at least two, at least 5, at least 10, at least 20) genome elements that can be differentiated.
  • the cell may be dead or alive.
  • the cell may be eukaryotic or prokaryotic.
  • the cell may not be complete and contain only a part of the cell (e.g., due to preparation or fixation of the sample or cell).
  • genomic as used herein is a complete identity or a part of this. It can be DNA or RNA.
  • transcription is used herein for a process during which one strand of the genome sequence of the genome element is copied into a complementary RNA (e.g., an mRNA) strand.
  • RNA e.g., an mRNA
  • These single-stranded copies may be independent molecules (not covalently connected to another molecule) but may be connected for a certain time to the genome element by a non- covalently binding (e.g., by hydrogen bonding).
  • the connection can be stabilized by a fixation method (e.g., methanol, formalin etc.).
  • biological sample is defined as a material that is derived from an organism and at least contains detectable nucleic acids, cells or parts of cells. These cells may originate from the same or different organs or even organisms.
  • tissue is used herein for any kind of a sample material that is formed by a certain number of cells of the same or different type with a meaningful structural relationship (or the lack thereof), and thus does comprise genome elements.
  • tissue section is used herein for a thin section of a tissue favorably done by a cryotome or a microtome.
  • an “analyte” may be any molecule (e.g., a biomolecule) of interest. Sometimes herein the term “analyte” is replaced by “target.”
  • an analyte may comprise a biomolecule (e.g., a protein, a nucleic acid, a biomolecule, a lipid, or any combination thereof).
  • an analyte may be a nucleic acid (e.g., DNA, PNA, LNA, RNA, or any combination thereof).
  • an analyte may be a DNA molecule (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA, or any combination thereof).
  • an analyte may be an RNA molecule (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combination thereof).
  • an analyte may be an mRNA (e.g., a transcript).
  • an analyte may be a “coding sequence”, “encoding sequence”, “structural nucleotide sequence”, or “structural nucleic acid molecule” which refers to a nucleotide sequence that is translated into a polypeptide, e.g., via mRNA, when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.
  • cell As used in the present disclosure, “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny.
  • the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included.
  • An “encoding scheme” may describe a set of code words that are associated with the analytes to be detected. Each code word refers to one of the analytes and can be distinguished from all other code words.
  • a code word hereby is a sequence of signs provided by the detection cycles of the method. A sign within a code word is a detectable signal or the absence of a signal. A code word does not need to comprise of all different signals used in the method. The number of signs in a code word is defined by the number of detection cycles.
  • oligonucleotide refers to a nucleic acid molecule, such as DNA, PNA, LNA or RNA.
  • the length of the oligonucleotides may be within the range of 10-10,000 nucleotides (nt), 10-15,000 nt, 10-10,000 nt, 10-5,000 nt, 10-2,000 nt, 10-1,500 nt, 10-1,000 nt, or 10-800 nt. In some embodiments, the length of oligonucleotides may be within the range of 100-1,500 nt, 100-1,200 nt, 100-1,000 nt, or 100-800 nts.
  • the length of oligonucleotides may be within the range of 400-1,500 nt, 400-1,200 nt, 400-1,000 nt, or 400-800 nts.
  • the nucleic acid molecule can be fully or partially single-stranded.
  • the oligonucleotides may be linear or may comprise hairpin or loop structures.
  • the oligonucleotides may comprise modifications such as biotin, labeling moieties, blocking moieties, or other modifications.
  • “Essentially complementary” means, when referring to two nucleotide sequences, that both sequences can specifically hybridize to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule with a sense and an antisense strand connected to each other via hydrogen bonds (Watson-and-Crick base pairs). “Essentially complementary” includes not only perfect base-pairing along the entire strands, e.g., perfect complementary sequences but also imperfect complementary sequences which, however, still have the capability to hybridize to each other under stringent conditions. Among experts it is well accepted that an “essentially complementary” sequence has at least 88% sequence identity to a fully or perfectly complementary sequence.
  • percent sequence identity describes the similarity between two or more sequences (e.g., a nucleic acid sequence or an amino acid sequence). Sequence similarity calculations may be performed using the BLAST algorithm for sequence alignment, which is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). Percent sequence identity compares a given sequence to a claimed or described sequence after alignment of the given sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”).
  • the term “about” in the context of a number in some cases refers to a range spanning 10% below to 10% above that number. In the context of a range, the term refers to an expanded range spanning 10% blow the listed lower limit to 10% above the listed upper limit. In cases where the number is small and cannot be subdivided, the term in some cases refers to a range spanning one unit below to one unit above the number referred to.
  • FIG. 1 shows the rreduction of tissue autofluorescence (measured in grey values) in FFPE mouse heart tissue using different concentrations of Sudan Black B (0.1-0.3%). In contrast to 0% Sudan Black B, tissue fluorescence was significantly reduced with all applied Sudan Black B concentrations.
  • FIG. 2 shows MC transcript detection (measured in transcript counts per tile) in FFPE mouse heart tissue after usage of Sudan Black B staining. Correlating with decrease in background, the transcript detection in MC was increased when using Sudan Black B for background reduction.
  • FIG. 3A and FIG. 3B show microscope images of Sudan Black (FIG. 3A) and TrueBlack (FIG. 3B) during a molecular cartography experiment with a fluidics channel (“Proto-0”).
  • FIG. 3A and FIG. 3B TrueBlack showed better reduction in background fluorescence than Sudan Black during the molecular cartography experiment with the fluidics system (“Proto-0”).
  • FIG. 3A and FIG. 3B TrueBlack showed better reduction in background fluorescence than Sudan Black during the molecular cartography experiment with the fluidics system (“Proto-0”).
  • FIG. 4 shows the molecular cartography experiment schematics for a molecular cartography experiment without a fluidics system (“Standard MCI”) and with a fluidics system employed before imaging (“Proto-0 FL”) and the accompanying microscopic images resulting from the molecular cartography assay.
  • the molecular cartography experiment without a fluidics system (“MCI”) and with a fluidics system employed before imaging (“FL01” and “FL02”) images with TrueBlack as a background reducer are shown in Channel 1 and Channel 2.
  • FIG. 5 shown the signal to noise ratio (SNR) for each of the molecular cartography experiment without a fluidics system (“MCI colorization”) and with a fluidics system employed before imaging (“Fluidic engine FL01” and “Fluidic engine FL02”.
  • SNR signal to noise ratio
  • MCI colorization fluidics system
  • Fluidic engine FL01 fluidics system employed before imaging
  • Fluidic engine FL02 fluidics system employed before imaging
  • FIG. 5 shows that the signal to noise ratios for each of the molecular cartography experiments with TrueBlack are comparable with and without the fluidics system employed.
  • the performance of TrueBlack for reduction of background fluorescence was comparable between the molecular cartography experiment without a fluidics system (“Standard MCI”, as shown at the top left of FIG. 4) and with a fluidics system (“Proto-0 FL”, “FL01”, and “FL02, as shown at the top right of FIG. 4).

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Abstract

La présente invention concerne un procédé de réduction de la fluorescence ambiante dans des applications d'hybridation in situ en fluorescence à molécule unique, consistant tout d'abord à appliquer du Noir Soudan B à des sections de tissu biologique fixées par la formaline et noyées dans la paraffine et ensuite à mettre en œuvre l'application d'hybridation in situ en fluorescence à molécule unique. En outre, l'invention concerne l'utilisation de Noir Soudan B pour réduire la fluorescence ambiante dans des applications d'hybridation in situ en fluorescence à molécule unique.
PCT/EP2024/081851 2023-11-10 2024-11-11 Réduction de la fluorescence ambiante dans des applications d'hybridation in situ en fluorescence à molécule unique Pending WO2025099313A1 (fr)

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