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  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• the invention relates to the field mapping the epigenetic modifications of DNA and RNA, which has become increasingly important, because these modifications play a role in several biological processes and diseases including development, aging and last but not least, cancer.
• C cytosine
• 5mC 5-methylcytosine
• oxidation can also be performed by chemical means using e.g. potassium perruthenate (KRuCh).
• KRuCh potassium perruthenate
• WO 2022/096751 also disclosed a method for generating a dihydrothymine (DHT) or a dihydrouracil (DHU) residue from a nucleoside or a polynucleotide containing 5-methylcytosine (5mC) or 5-carboxyl-cytosine (5caC) by using a radical initiator which may be used together with a nucleophilic compound.
• DHT dihydrothymine
• DHU dihydrouracil
• the present invention is based on the basic idea that the introduction of a nucleophile at 5fC, 5hmC or caC and the subsequent intramolecular addition at C- 6 of a 5-substitued cytosine is enabling a deamination at C-4 of a cytosine derivative.
• the present invention is directed to a method comprising the steps of a) providing a nucleic acid comprising 5fC, 5hmC, or 5caC b) providing one reactant comprising two reactive groups wherein the first reactive group is capable of reacting with the formyl, hydroxymethyl, or carboxyl group, and the second reactive group is a nucleophilic group, c) reacting said first reactive group with the formyl, hydroxymethyl or carboxyl group thereby resulting in a modified 5fC, 5hmC, or 5caC d) reacting said second reactive group with the C6 position of said modified 5fC, 5hmC, or 5caC, thereby obtaining a bicyclic or tricyclic molecule comprising a 5,6 di-hydro Cytosine entity, and e) deaminating said 5,6 di-hydro Cytosine entity to a 5,6 di-hydro-Uracil entity.
• step d) The newly formed cyclic structures in step d) are 5-7 membered rings
• the method further comprises step f), i.e. reversing the ring formation of step d) thereby obtaining a 5-substituted Uracil.
• the method may also comprise a step of converting 5mC to 5fC, 5hmC or 5caC prior to step a).
• the present invention is facilitating the methylation status analysis of naturally occurring DNA.
• said nucleic acid comprises at least one 5fC residue.
• the first reactive group may be a C-H acidic group, an amine or a phosphorus ylide.
• said nucleic acid comprises at least one 5hmC residue. Then, the first reactive group may constitute a Glycosyl Transferase substrate.
• said nucleic acid comprises a 5caC residue. Then, the first reactive group may be an amine.
• the said second reactive group may be a nucleophilic group selected from a group consisting of Thiol or Sulfinate.
• All methods as disclosed above may further comprise a subsequent step of amplifying said nucleic acid, which may be preferably executed by means of PCR amplification.
• all methods disclosed above may further comprise the step of a subsequent step of sequencing said nucleic acid either with or without a prior amplification step, which is preferably a PCR amplification.
• Fig. 1 provides a scheme for detection of methylated sequences according to the present invention.
• Fig. 2 shows a reaction scheme for modification of 5fC by means of a Knoevenagel condensation using a reactant with a C-H acidic group
• Fig. 3 shows a reaction scheme for modification of 5fC by means of an aldol condensation reaction with a CH-acidic reagent comprising a sulfinate moiety
• Fig. 4 shows a reaction scheme for modification of 5fC by means of a Wittig reaction.
• Fig. 5 shows a specific example for a Wittig reaction according to Fig. 4 with a sulfinate modified Wittig reagent.
• Fig: 7 shows a specific example for a reductive amination as in Fig. 6, where 5fC is reacted with 2-aminomethyl sulfinate
• Fig. 8 shows a reaction scheme for the modification of hmC using UDP-glucose substituted at the 2-position of the glucose moiety with a nucleophile as a reagent and executing a subsequent beta-glucosyl-transferase reaction.
• Fig. 9 shows a similar reaction scheme as example 8, wherein 2-thio-glucose-UDP is used.
• Fig. 10 shows an example for modification of the carboxyl group of caC.
• a nucleophilic group is a functional group that is attractive to electron-poor or positively charged centers and donates electrons, in particular donating an electron pair to an electrophile or an electrophilic center to form a covalent bond.
• Nucleophilic groups are for example alcohols, alcoholates, thiols, thiolates, amines, sulfinates or carbanions.
• deamination refers herein to a substitution reaction of an exocyclic amino group by a hydroxyl group, in particular substitution of the amino group at the C-4 position of a cytosine nucleobase to convert into a uracil nucleobase. Deamination at C-4 position is enhanced in 5,6-dihydrocytosines to give 5,6-dihydrouracil derivatives, as e.g. being applied after bisulfite addition to the C5- C6 double bond of cytosine.
• C-H acidic group is used herein for a carbon atom within a compound which carries at least one electron-withdrawing group and thus being more easily deprotonated by a base.
• Electron-withdrawing groups are for example carbonyl containing groups, a nitro group, a cyano group, a sulfoxide or sulfone group.
• Wittig reagent or phosphorus ylide refers to neutral dipolar molecules containing a formally negatively charged carbon atom (a carbanion) directly attached to a phosphorus atom with a formal positive charge.
• phosphorus ylides phosphonium ylides
• Phosphorus ylides are thus 1,2- dipolar compounds and a subclass of zwitterions.
• triphenylphosphonium ylides are reacted with aldehydes or ketones to give an alkene bond.
• a Homer-Wadsworth-Emmons reaction stabilized phosphonate carbanions are used as reagent.
• a Glycosyl Transferase Substrate refers to a reagent comprising an activation group, e.g. a UDP moiety, and a sugar residue, for example a glucose or substituted glucose.
• Glycosyltransferases catalyze the transfer of the sugar moiety from an activated donor sugar (the glycosyl transferase substrate) onto a saccharide or non- saccharide acceptor.
• the non-saccharide acceptor can be for example 5- hydroxymethyl-dC.
• T4 Phage B-glucosyltransferase for example from New England Biolabs, specifically transfers a glucose moiety of uridine diphosphoglucose to the 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, making beta- glucosyl-5-hydroxymethylcytosine.
• FIG. 1 A general scheme, how methylated C-residues mC are detected is schematically shown in Fig. 1.
• the epigenetic dC modifications 5-methyl-dC (mC), 5-hydroxymethyl-dC (hmC), 5-formyl-dC (fC) and 5-carboxyl- dC (caC) are read as unmodified dC when amplified and/or sequenced.
• oxidation of mC by enzymatic and/or chemical means results in oxidized mC modifications hmC, fC and caC (general: xC).
• Oxidation or conversion of 5mC to 5fC can be achieved by using TET enzymes (ten- eleven translocation (TET) methylcytosine dioxygenases).
• TET ten- eleven translocation
• the same class of enzymes are suitable for converting 5mC to 5hmC.
• 5hmC can be further oxidized to 5fC enzymatically by a laccase enzyme or chemically using either KRuO4 or Cu(II)/TEMPO. Further details are disclosed in Pfeifer et al. Epigenetics & Chromatin 2013, 6: 10, p.1-9.
• a reagent of the invention comprising two reactive groups, the first reactive group forming a covalent bond with hmC, fC or caC and thus introducing the second reactive group which comprises a nucleophile.
• the present also provides a method for analyzing the methylation status of a naturally occurring DNA comprising the steps of a) converting 5mC to 5fC, 5hmC or 5caC b) providing one reactant comprising two reactive groups wherein the first reactive group is capable of reacting with the formyl, hydroxymethyl or carboxyl group, and the second reactive group is a nucleophilic group, c) reacting said first reactive group with the formyl, hydroxymethyl or carboxyl group thereby resulting in a modified 5fC, 5hmC, or 5caC, d) reacting said second reactive group with the C6 position of said modified 5fC, 5hmC, or 5caC, thereby obtaining a bicyclic or tricyclic molecule comprising a 5,6 di-hydro Cytosine entity, and e) deaminating said 5,6 di-hydro Cytosine entity to
• the sample is derived from a subject or a patient.
• the sample may comprise a fragment of a solid tissue or a solid tumor derived from the subject or the patient, e.g., by biopsy.
• the sample may also comprise body fluids that may contain nucleic acids (e.g., urine, sputum, serum, blood or blood fractions, i.e., plasma, lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric fluid, intestinal fluid, or fecal samples).
• nucleic acids e.g., urine, sputum, serum, blood or blood fractions, i.e., plasma, lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid,
• the sample is a cultured sample, e.g., a tissue culture containing cells and fluids from which nucleic acids may be isolated.
• the nucleic acids of interest in the sample come from infectious agents such as viruses, bacteria, protozoa or fungi.
• the present invention involves manipulating isolated nucleic acids isolated or extracted from a sample. Methods of nucleic acid extraction are well known in the art (see, Sambrook et al., & quot; Molecular Cloning: A Laboratory Manual, & quot; 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, N. Y.).
• kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples (e.g., KAPA Express Extract (Roche Sequencing Solutions, Pleasanton, Cal.) and other similar products from BD Biosciences Clontech (Palo Alto, Cal.), Epicentre Technologies (Madison, Wise.); Gentra Systems, (Minneapolis, Minn.); and Qiagen (Valencia, Cal.), Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Cal.); and more.
• the present invention involves detecting an epigenetic modification, specifically, an epigenetic cytosine modification in nucleic acids (including, but not limited to, cytosine methylation).
• the nucleic acid sequences that are subject to conditional epigenetic modification are the target sequences analyzed by the method disclosed herein.
• the same nucleic acid sequence may or may not have the epigenetic modification characterized by methylation of cytosines at the 5-position (5mC or 5hmC).
• a set or a panel of target nucleic acids are probed for the presence of methylation. For example, as shown in Patai, et al.
• the method of the instant invention includes detecting methylation in all sites throughout the genome of an organism to diagnose a disease or condition or predisposition to a disease or condition using the sequence analysis and artificial intelligence tools described, e.g., in Shull, et al. “Sequencing the cancer methylome,” Methods Mol Biol. 1238:627-635 (2015).
• the method of detecting epigenetic modifications includes sequencing.
• the nucleic acid processed as described herein is subjected to sequencing; preferably, massively parallel single molecule sequencing. Analyzing individual molecules by massively parallel sequencing typically requires a separate level of barcoding for sample identification and error correction.
• the use of molecular barcodes such as described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368.
• a unique molecular barcode is added to each molecule to be sequenced to mark molecule and its progeny (e.g., the original molecule and its amplicons generated by PCR).
• the unique molecular barcode has multiple uses including counting the number of original target molecules in the sample and error correction (Newman, et al., “An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage,” Nature Medicine doi: 10.1038/nm.3519 (2014)).
• Nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence. Nanopore-based sequencing technology detects the unique electrical signals of different molecules as they pass through the nanopore with a semiconductor-based electronic detection system. This technology makes for a high throughput, cost effective sequencing solution.
• the biological nanopore a protein pore embedded in a membrane, while the brains of the technology lie in the electronics of a semiconductor integrated circuit and proprietary chemistries.
• unique molecular barcodes are used for sequencing error correction.
• the entire progeny of a single target molecule is marked with the same barcode and forms a barcoded family.
• a variation in the sequence not shared by all members of the barcoded family is discarded as an artefact.
• Barcodes can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, et al. “Integrated digital error suppression for improved detection of circulating tumor DNA,” Nature Biotechnology 34:547 (2016)).
• the method involves forming a library comprising nucleic acids from a sample.
• the library consists of a plurality of nucleic acids ready for sequencing or another type of detection method, e.g., PCR.
• a library can be stored and used multiple times for further processing such as amplification or sequencing of the nucleic acids in the library.
• the library is the input nucleic acid in which methylation is detected by the method described herein.
• the library is formed from nucleic acids that have undergone the methylation detection reactions described herein.
• Non-limiting examples of sequence assays that are suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Patent Publication Nos. US2013/0244340, US2013/0264207, US2014/0134616, US2015/0119259 and US2015/0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears, et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman, et al., Methods Mol.
• sequencing-by- hybridization platforms from Affymetrix Inc. (Sunnyvale, Calif.), sequencing-by- synthesis platforms from Illumina/Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.).
• Other sequencing technologies include, but are not limited to, the Ion Torrent technology (ThermoFisher Scientific), and nanopore sequencing (Genia Technology, Roche Sequencing Solutions, Santa Clara, Cal.), Oxford Nanopore Technologies (Oxford, UK), and sequencing by expansion (Stratos Genomics, Roche Sequencing Solutions).
• the sequencing step involves sequence aligning.
• a consensus sequence is determined from a plurality of sequences all having an identical UID.
• the sequences having an identical UID are presumed to derive from the same original molecule through amplification.
• UID is used to eliminate artifacts, i.e., variations existing in the progeny of a single molecule (characterized by a particular UID). Such artifacts resulting from PCR errors or sequencing errors can be eliminated using UIDs.
• the number of each sequence in the sample can be quantified by quantifying relative numbers of sequences with each UID among the population having the same multiplex sample ID (MID).
• MID multiplex sample ID
• Each UID represents a single molecule in the original sample and counting different UIDs associated with each sequence variant can determine the fraction of each sequence variant in the original sample, where all molecules share the same MID.
• a person skilled in the art will be able to determine the number of sequence reads necessary to determine a consensus sequence.
• the relevant number is reads per UID (“sequence depth”) necessary for an accurate quantitative result.
• the desired depth is 5-50 reads per UID. Modification of 5fC
• Modification of 5fC according to the present invention can be achieved if the first reactive group of the reagent according to the present invention is a C-H acidic group, an amine or a phosphorus ylide.
• the reaction can be executed as a Knoevenagel condensation, which is schematically depicted in Fig. 2.
• the CH-acidic reagent comprising a nucleophile group is added via Knoevenagel conditions to fC, then subsequent intramolecular addition of the nucleophile to the C5-C6 double bond takes place, thereby enabling the deamination at C4 to obtain a “T-equivalent”.
• Yields of intramolecular addition of the nucleophile to the C5-C6 double bond can be increased by a photoisomerization of the double bond and/or by applying a radical initiation or a catalyst. If desirable, a reversal of the ring closure can be achieved under alkaline conditions.
• an aldol condensation reaction of 5fC with a CH-acidic reagent comprising a sulfinate moiety e.g. 1 -(methyl sulfonyl)methane sulfinate
• a subsequent addition of a sulfinate moiety at the C-6 position of the substituted cytosine followed by deamination and desulfmation (5fC to T conversion) can be executed (Fig. 3)
• a desulfmation step under alkaline conditions may even not be required.
• a thiol moiety can also be applied as nucleophile
• addition reaction to the C5-C6 double bond can be achieved by means of applying thiol-ene chemistry using a radical initiator or a catalyst.
• the yields may be increased by photoisomerization of the newly formed double bond.
• Modification of 5fC according to the present invention can also be done by means of a Wittig reaction (Fig. 4).
• the reagent according to the present invention is a phosphorus ylide comprising a nucleophilic group.
• the reagent is added via Wittig conditions to 5fC, then a subsequent intramolecular addition of the nucleophile to the C5-C6 double bond takes place, thereby enabling a deamination at the C4 position in order to obtain a “T-equivalent”.
• Yields of intramolecular addition of the nucleophile to the C5-C6 double bond may be increased by photoisomerization of the newly formed double bond, and/or by applying radical initiation or a catalyst. If desirable, a reversal of the ring closure can be achieved under alkaline conditions. Alternatively, the reaction can be performed using Horner-Wadsworth-Emmons reaction conditions, in particular using a sulfinate group as nucleophile.
• reagent of the invention comprising an amine and a further nucleophilic group is added via reductive amination to 5fC. Subsequently there is an intramolecular addition of the nucleophile to the C5-C6 double bond, which enables the deamination at the C4 position to obtain a “T- equivalent”. Reversal of ring closure can be achieved under alkaline conditions, if necessary. Yields can be increased by applying radical initiation or a catalyst.
• a more detailed example for implementing a reductive amination reaction is illustrated in Fig. 7.
• 5fC is reacted with 2-aminomethyl sulfinate, and subsequently addition of the sulfinate moiety at C-6 position of substituted cytosine takes place.
• This is followed by a deamination and desulfination step, which converts 5fC to T.
• a desulfination step under alkaline conditions may not be needed.
• the sulfinate moiety may be replaced by a thiol moiety using thiol-ene chemistry for the addition reaction to double bond C5-C6, which optionally includes the use of a radical initiator or a catalyst.
• Fig. 8 schematically shows a reaction scheme for the modification of hmC.
• the reagent used according to the present invention is a UDP -glucose, which is modified at the 2-position with a nucleophilic group. It is added via a B-glucosyl transferase enzyme to hmC. Then, a subsequent intramolecular addition of the nucleophile to the C5-C6 double bond takes place, thereby enabling the deamination at the C4 position in order to obtain a “T-equivalent”.
• yields of intramolecular addition of the nucleophile to the C5-C6 double bond can be increased by applying radical initiation or a catalyst and a reversal of the ring closure can be achieved under alkaline conditions, if necessary.
• Fig. 9 An alternative example is shown in Fig. 9.
• 5hmC is reacted with 2-thio- glucose-UDP and B-glucosyltransferase.
• an addition of the thiol moiety at the C6 position of substituted cytosine applying thiol-ene chemistry and optionally using a radical initiator or a catalyst is taking place, which is followed by a deamination reaction, resulting in a 5hmC to T conversion.
• 2- Sulfinato-glucose-UDP can also be applied as substrate for B-glucosyltransferase.
• the method of the present invention uses a reagent comprising an amine and a further nucleophilic group. It is added after an EDC/NHS activation of the carboxyl group of caC. This results in caC forming an amide bond, and a subsequent intramolecular addition of the nucleophile to the C5-C6 double bond, thereby enabling the deamination at the C4 position in order to obtain a “T-equivalent”.
• the yield of intramolecular addition of the nucleophile to the C5-C6 double bond can be increased by means of applying radical initiation or a catalyst and reversal of the ring closure can be achieved under alkaline conditions.

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