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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
  • C07F5/02—Boron compounds
  • C07F5/025—Boronic and borinic acid compounds
• • 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
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • 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
  • C12Q1/6844—Nucleic acid amplification reactions
• • 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
  • C12Q2523/00—Reactions characterised by treatment of reaction samples
  • C12Q2523/10—Characterised by chemical treatment
  • C12Q2523/101—Crosslinking agents, e.g. psoralen
• • 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

Definitions

• PCR polymerase chain reaction
• cross-linking of nucleic acids to cellular components by the action of formaldehyde thus presents challenges to the detection of various cellular components, including detection of nucleic acids and proteins.
• improvements generally involve merely degrading protein in the sample or providing detergents that do not generally change the covalent bonds forming the cross-links. The present invention addresses this and other problems.
• the present invention provides methods for analyzing one or more components of a formaldehyde cross-linked biological sample.
• the methods comprise contacting the sample with a sufficient amount of a catalyst to release at least a portion of the cross-linked component, thereby improving the accessibility of the one or more components for analysis.
• the biological sample is a tissue sample from an animal.
• the amount of catalyst is between about 0.2 mM and about 5.0 mM. In some embodiments the sample and catalyst are heated for a period of at least 10 minutes.
• the methods further comprise detecting the component.
• the catalyst is substantially removed from the sample prior to the detecting step. In some embodiments, the concentration of catalyst is reduced to less than about 0.1 mM prior to the detecting step.
• the detecting step comprises quantifying the component.
• the component is a nucleic acid.
• the nucleic acid is DNA.
• the component is RNA.
• the methods further comprise detecting the nucleic acid.
• the detecting step comprises amplifying the nucleic acid.
• the nucleic acid component is contacted to a probe under conditions to allow for formation of the probe and nucleic acid, and detecting the presence of the duplex.
• the probe is linked to a solid support.
• the amplifying step comprises the polymerase chain reaction.
• the component is protein.
• the methods further comprise detecting the protein.
• the detecting step comprises mass spectrometry or electrophoresis.
• the mass spectrometry comprises matrix-assisted laser desorption/ionization (MALDI).
• MALDI matrix-assisted laser desorption/ionization
• the sample is embedded in paraffin prior to the contacting step.
• the catalyst is selected from the group consisting of (2-Amino-5- fluorophenyl) boronic acid, 2-Aminophenyl boronic acid, (2-Amino-5-methylphenyl) boronic acid, l,3-Dihydro-l-hydroxy-2,l-benzoxaborol-7-amine, 3,4-Dihydro-l-hydroxy- lH-2,l-benzoxaborin-7-amine, (Aminophenylmethyl) phosphonic acid diethyl ester, Bismuth (III) bromide, Bismuth (III), iodide, Bismuth (III) citrate and Bismuth (III) salicylate.
• the portion of the component that is available for analysis is increased at least about two-fold compared to the portion accessible for analysis if the contacting step is not performed. In some embodiments, the portion of the component that is available for analysis is increased at least about ten-fold compared to the portion accessible for analysis if the contacting step is not performed.
• the methods further comprise contacting the sample with a protease to degrade the protein in the sample, thereby rendering the nucleic acids more available for analysis.
• the present invention also provides a kit for improving the availability of one or more components of a formaldehyde cross-linked biological sample.
• the kit comprises a catalyst; and a protease or a reagent or device for removal of the catalyst from a biological sample, wherein the catalyst is selected from the group consisting of an aminophenyboronic acid, a cyclic boronic acid ester, a phosphonic acid ester, and a bismuth salt.
• the kit comprises a reagent or device for removal of the catalyst from a biological sample.
• the device is a column for purification of nucleic acids.
• the kit comprises a protease.
• the protease is proteinase K.
• the kit further comprises nucleotides and/or a thermostable polymerase.
• the thermostable polymerase is Taq polymerase.
• the catalyst is selected from the group consisting of (2-Amino-5- fluorophenyl) boronic acid, 2-Aminophenyl boronic acid, (2-Amino-5-methylphenyl) boronic acid, l,3-Dihydro-l-hydroxy-2,l-benzoxaborol-7-amine, 3,4-Dihydro-l-hydroxy- lH-2,l-benzoxaborin-7-amine, (Aminophenylmethyl) phosphonic acid diethyl ester, Bismuth (III) bromide, Bismuth (III), iodide, Bismuth (III) citrate and Bismuth (III) salicylate.
• the present invention also provides reaction mixtures.
• the reaction mixtures comprise a formaldehyde cross-linked biological sample; and a sufficient amount of a catalyst to release at least a portion of the cross-linked component, wherein the catalyst is selected from the group consisting of an aminophenyboronic acid, a cyclic boronic acid ester, a phosphonic acid ester, and a bismuth salt.
• the amount of catalyst is between 0.2 mM and 5.0 mM.
• the catalyst is selected from the group consisting of (2-Amino-5- fluorophenyl) boronic acid, 2-Aminophenyl boronic acid, (2-Amino-5-methylphenyl) boronic acid, l,3-Dihydro-l-hydroxy-2,l-benzoxaborol-7-amine, 3,4-Dihydro-l-hydroxy- lH-2,l-benzoxaborin-7-amine, (Aminophenylmethyl)phosphonic acid diethyl ester, Bismuth (III) bromide, Bismuth (III), iodide, Bismuth (III) citrate and Bismuth (III) salicylate.
• the biological sample is a tissue sample from an animal. DEFINITIONS
• a “formaldehyde cross-linked biological sample” refers to a biological sample that has been treated with formaldehyde such that cross-linking is formed between a nitrogen in proteins or nucleic acids to other nitrogen-containing proteins and/or nucleic acids.
• a biological sample will typically contain cells.
• the biological sample can be, for example, a tissue sample from an animal, for example, from a human. Many formaldehyde-treated samples are stored by embedding them in paraffin.
• the term "catalyst” refers to an agent that catalyzes the removal of adducts and/or crosslinks from fixed biomolecules (e.g., aldehyde fixed biomolecules, formaldehyde fixed biomolecules), upon contact with a sample having aldehyde fixed biomolecules (e.g. a FFPE biological sample).
• a catalyst can be said to remove crosslinks as well as other adducts (e.g. aldehyde fixation relate adducts, formaldehyde fixation related adducts).
• the pKa of the catalyst is in a range of from 2.5 to 9.0.
• the catalyst is selected from the compounds in TABLE 1.
• the phrase "detecting the component” refers to determining at least the presence or absence of the component and can include further quantification or other characterization of the component or part of the component.
• a “component” of a biological sample refers to a class of molecules (e.g., proteins, nucleic acids, etc.) or a specific target such as a specific protein or nucleic acid sequence that one wishes to detect.
• nucleic acid refers to polymers of deoxyribonucleotides (containing 2-deoxy-D-ribose) (i.e., DNA), polyribonucleotides (containing D-ribose) (i.e., RNA), and any other N-glycosidic analogs of a purine or pyrimidine base, or modified purine or pyrimidine bases.
• the phrase "to release at least a portion of the cross-linked component” refers to altering the covalent bonds forming a cross-linkage between two components (e.g., a nucleic acid and a protein) of the biological sample such that the two components are no longer linked by a covalent bond.
• the phrase encompasses, but is not limited to, a complete reversal of the cross-linking process.
• the phrase "accessibility for analysis” as used herein refers to the ability of a detection method to determine the presence or absence and/or quantity of a particular target molecule. For example, numerous detection methods are at least partly inhibited from detecting protein or nucleic acids in a formaldehyde cross-linked biological sample and thus certain cross-linked components are not "accessible” for detection.
• an increased amount e.g., at least about 10% more and typically at least about 2-fold more, or sometimes about at least 10 or 100-fold more
• an increased amount e.g., at least about 10% more and typically at least about 2-
• Figure 1 illustrates the reaction of formaldehyde to convert a free amine to its hemiaminal adduct and imine intermediate which can further react with other amines to form aminal crosslinks.
• Figure 2 illustrates an example of the "de-crosslinking" by catalysts of the present invention.
• Figure 3 illustrates the reversal of the dAMP dimer to the dAMP monomer under reaction conditions described in Example 2 in the absence of catalyst or in the presence of Compound 2 (2APB), Compound 3 (2A5MPB), Compound 6 (APMPDE) and Compound 7 (BiBrs).
• the present invention provides for methods of rendering formaldehyde cross-linked components of a biological sample more accessible for detection by contacting the sample with a catalyst.
• the quantity of catalyst used to render the components more accessible can vary and will depend in part on the specific catalyst used, the component to be detected, and the detection method to be used as different detection methods have different sensitivities and so may require more or less of the component to be accessible.
• the amount of a component rendered accessible to a particular detection method will be the entire amount of the component in the sample. However, generally, the amount of component rendered accessible for detection will be less than the entire quantity of the component in the sample.
• a sufficient amount of catalyst is used under conditions to render at least about two times the amount of the component accessible for detection as would be accessible (using the same detection method) if the sample was not treated with the catalyst.
• a sufficient amount of catalyst is used under conditions to render at least about 5, 10, 20, 100 times the amount of the component accessible for detection as would be accessible (using the same detection method) if the sample was not treated with the catalyst.
• the concentration of catalyst used to release the cross-linking of the sample is between about 0.2 mMand about 5.0 mM (or more).
• the conditions in which the sample and catalyst are combined will affect the ability and amount of cross-linkage reversal.
• Catalyst treatment is effective at ambient (e.g., between 20-40 or 50°C) temperature and thus does not necessarily require a heating step to release cross-linkages. This can be particularly useful when detecting components that are relatively labile, such as R A. Nevertheless, higher temperature (e.g., 80-100°C, 90- 100°C, 90-99°C, etc.) may further improve the accessibility of nucleic acids or proteins for detection.
• the amount of time the catalyst is incubated with the sample will affect the amount of the components rendered accessible for detection.
• the samples can be incubated with the catalyst for at least about 5, 10, 20, 30, 60, 120 minutes or more. While a longer time of incubation may increase the amount of component that is released from cross-linking, this may need to be balanced with how labile a particular component may be. For example, it may be desirable to use a shorter incubation time when a labile component such as RNA is to be detected. On the other hand, a less labile component, such as protein or DNA, can be exposed to a longer incubation without harming the component.
• the selected catalyst will generally be capable of releasing the components from the formaldehyde -induced cross linkages and reverting the components (e.g., nucleic acids and/or protein) to substantially the same component as existed prior to the formaldehyde cross-linking.
• the cross-linking reaction is reversible process that proceeds by reaction of formaldehyde and a first amine to form a hemiaminal, followed by dehydration to afford an imine.
• the imine reacts with a second amine to afford the product aminal.
• the process reverts to the starting materials by reaction of the imine with water instead of a second amine.
• the catalyst of the present invention releases the components from the formaldehyde -induced cross linkages by acting as a competitive reactant in the formation of the imine and the aminal.
• the cross- linkages release as part of the equilibrium process, the imine intermediate and the formaldehyde react with the catalyst, thereby releasing the components from the formaldehyde-induced cross linkages.
• Catalysts used to practice the methods of the invention may include aminophenylbornoic acids, cyclic boronic acid esters (benzoxaborines), phosphonic acid esters, and bismuth salts and complexes. These catalysts accelerate the cleavage of formaldeyde adducts and crosslinks at mild reaction conditions, thereby increasing the quality of nucleic acids from FFPET samples. Specific embodiments of catalysts useful for the present invention are selected from the compounds shown in TABLE 1.
• the key properties for the catalysts described in the present invention are: Catalysis of aminal cleavage (i.e. the reverse reaction or "de-crosslinking"), no detrimental effects on nucleic acids and other biomolecules, sufficient water solubility, compatibility with nucleic acid purification protocols, amenability to automation, and safe handling (i.e. non- toxicity). Furthermore, the catalysts should operate at reaction conditions that minimize nucleic acid, protein or biomolecule impairment (e.g. pH range of 4.5-8.0, temperature range of 25°C-60°C, time period of at least 10 minutes and less than 60 minutes). The addition of a catalyst that allows the extraction of nucleic acids and other biomolecules to be performed at mild reaction conditions is expected to significantly improve the quality of the extracted material. As such, these compounds may find broad application in manual and automated nucleic acid and protein extraction and recovery protocols.
• the tissue samples will be derived from animal tissues.
• the samples will be embedded in paraffin.
• the samples can be formalin fixed paraffin embedded tissue (FFPET).
• FFFPET formalin fixed paraffin embedded tissue
• the samples have been obtained from an animal (e.g., a human) and then stored in a formaldehyde-containing solution to stabilize the sample prior to analysis, thereby cross- linking the nucleic acids and/or protein in the sample.
• a cervical or other gynecological swab e.g., for detection of sexually transmitted disease
• a solution containing formaldehyde e.g., formaldehyde
• the cross-linking can be subsequently reversed using a catalyst according to the methods of the invention.
• sample components accessible to detection
• additional purification or other steps may be included in the methods of the invention.
• a nucleic acid component of the sample is to be detected, it can be helpful to treat the sample (e.g., before or following catalyst treatment) with a protease, or otherwise degrade the protein in the sample.
• a protease is proteinase K, though it will be appreciated that various other proteases could be substituted.
• the inventors have found it helpful to purify the nucleic acids in the sample from other components of the sample as well as from the catalyst by using a reagent or device such as a spin column to purify nucleic acids from other parts of the sample.
• a reagent or device such as a spin column to purify nucleic acids from other parts of the sample.
• An exemplary device is a silica-based spin column with affinity for nucleic acids (such as the QiaquickTM spin column from Qiagen, Valencia, CA), though of course other purification methods may also be used to remove the catalyst.
• any detection method may be used in combination with the catalyst treatment described above to detect a component of the previously cross-linked sample.
• exemplary components of the sample for which cross-linking interferes with detection include nucleic acids and proteins. Detection of components can involve simply determining the presence or absence of a particular component or part (e.g., a particular protein or nucleic acid sequence) of the component. Alternatively, detection can involve quantification of the component and/or characterization of the component. Characterization can include, for instance, peptide or nucleic acid sequencing and/or determination of post- transcriptional or translational modifications, including, e.g., glycosylation, phosphorylation, etc.
• RNA or RNA can be detected. Detection can include quantification of a particular sequence or RNA, and/or characterization of a nucleic acid, for example, by nucleotide sequencing or sequence-specific hybridization techniques (e.g., such as those used to detect single nucleotide polymorphisms (SNPs) and the like).
• SNPs single nucleotide polymorphisms
• amplification methods to amplify a particular nucleic acid to assist in detection of nucleic acids.
• Any type of amplification method may be used, including exponential amplification methods, linear amplifications, thermocycling or isothermal methods, etc.
• Suitable amplification methods include, but are not limited to, the polymerase chain reaction (PCR) (Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.
• thermostable enzyme isolated from Thermus aquaticus is described in U.S. Pat. No. 4,889,818 and a method for using it in conventional PCR is described in Saiki et al., 1988, Science 239:487-91.
• Another representative thermostable enzyme includes Thermus species Z05 DNA polymerase. See, e.g., U.S. Patent No. 5,674,738.
• real-time PCR or other quantitative amplification techniques can be used to quantify a particular nucleic acid sequence. Methods of quantitative amplification are disclosed in, e.g., U.S. Patent Nos.
• RNA levels for one or more gene can be measured within a sample.
• RT-PCR methods are well known to those of skill (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds., 2002)) and are readily adapted for quantitative amplification methods.
• Other methods can also be used to detect nucleic acids.
• nucleic acids can be isolated from a sample and hybridized to a probe. In some instances, the probe will be linked to a solid support (e.g., a microarray).
• Protein components of a sample can also be detected following treatment with a catalyst. Any of a variety of protein detection and characterization methods may be employed according to the method of the present invention.
• An exemplary protein detection method is mass spectrometry. Exemplary mass spectrometry methods include, but are not limited to, electrospray ionization and matrix- assisted laser desorption/ionization (MALDI), including MALDI time of flight (MALDI- TOF) methods. See, e.g., Karas, M.; Hillencamp, F. Anal. Chem. 60:2301 (1988); Beavis, R. C. Org. Mass Spec. 27:653 (1992); Creel, H. S. Trends Poly. Sci. 1(11):336 (1993).
• MALDI matrix- assisted laser desorption/ionization
• MALDI- TOF MALDI time of flight
• kits useful for employing the above-described methods of the invention can comprise one or more of the reagents described herein.
• the kits can include written (paper) or electronic instructions for their use.
• kits of the invention will include a catalyst with at least one additional reagent for detection or improving detection of a nucleic acid or protein.
• the kits comprise a catalyst and a protease (including but not limited to proteinase K) for degrading protein and rendering nucleic acids even more accessible to detection.
• reagents for detection or improving detection of a nucleic acid or protein include, e.g., reagents useful for amplifications.
• a typical polymerase chain reaction can include, without limitation, as reagents upstream and downstream primers, at least one template, deoxyribonucleoside triphosphates (including dATP, dCTP, dGTP, TTP, dUTP), a polymerase enzyme, buffers, metal cations and salts.
• a kit for an RT-PCR reaction can also include a reverse transcriptase and/or primers.
• one or more polynucleotide probes are employed to hybridize to the desired target.
• the probes are typically labeled with a detectable label, e.g., a fluorescent label.
• An exemplary probe is a TaqmanTM probe, though it will be appreciated that other types of probes can be used to monitor a target in a quantitative amplification reaction.
• a nucleic acid sequence-based amplification (NASBA) reaction can include primers, reverse transcriptase, RNase H, and a DNA polymerase.
• a transcription-mediated amplification (TMA) reaction can include primers, reverse transcriptase, and an RNA polymerase.
• TMA transcription-mediated amplification
• SDA strand displacement amplification
• SDA a modified nucleotide and a restriction endonuclease.
• Certain amplification reactions can also include deoxyUridine N-Glycosylase (UNG) as an ancillary amplification reagent (e.g., Amperase®, Roche Molecular Sciences, Alameda, CA) (see, Kleiboeker, Virol J (2005) 11 :29).
• UNG deoxyUridine N-Glycosylase
• reagents for detection or improving detection of a nucleic acid or protein include, e.g., reagents or devices for purifying proteins or nucleic acids, for example as described herein.
• the present invention also provides reaction mixtures.
• An exemplary reaction mixture will comprise a formaldehyde-fixed sample, optionally including paraffin, and a catalyst as described herein.
• the reaction mixtures can include the concentrations of catalyst that are described above. Further, the reaction mixtures are optionally at the temperatures recited above.
• Reaction mixtures can optionally further include a protease (e.g., proteinase K).
• Example 1 This example illustrates the preparation and analysis of formaldehyde adducts of mononucleotides.
• dAMP Deoxyadenosine monophosphate
• VWR Deoxyadenosine monophosphate
• EM grade was purchased from Thermo Fisher Scientific.
• Solvents and reagents were purchased from Sigma-Aldrich or VWR.
• the names, CAS numbers and Molecular Weights of the compounds (catalysts) are as follows: Compound 1 : (2-Amino-5-fluorophenyl)boronic acid; CAS number 1040400-87-00; Molecular Weight 154.93.
• Compound 2 2-Aminophenylboronic acid; CAS number 5570-18-3/863753-30-4 (HCI); Molecular Weight 173.40.
• Compound 3 (2-Amino-5-methylphenyl)boronic acid; CAS number 948592-72-1 ; Molecular Weight 150.97.
• Compound 5 3, 4-Dihydro-l -hydroxy- lH-2,l-benzoxaborin-7-amine; CAS number N/A; Molecular Weight 162.98.
• Compound 10 Bismuth (III) salicylate; CAS number 14882-18-9; Molecular Weight 362.09.
• This example illustrates reversal of cross-linking chemistry of the dAMP dimer with catalysts.
• Reverse crosslinking of the methylene-bis-dAMP dimer were monitored by HPLC. Reactions were carried out on in 50 mM phosphate buffer, pH 5.0 at 60°C. dAMP dimer at 1.0 mM concentration was mixed with either no catalyst or with 5.0 mM concentration of Compounds 2, 3, 6, and 7. For each reaction mixture, an aliquot (10 ⁇ ) was taken at 10 minutes, 20 minutes, 40 minutes, 60 minutes and 90 minutes.
• Figure 3 shows the results of this experiment and demonstrated that all the tested catalysts were able to reverse cross-linking (shown as % of dAMP produced) at rates that are much faster than when no catalyst was added.

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