The present invention is related to a method for adding one or more L-nucleotides to the 3′end of a first L-nucleic acid, wherein the method comprises the step of reacting the one or more L-nucleotides with the first L-nucleic acid in the presence of a protein comprising a mutant enzymatic activity exhibiting moiety, wherein the enzymatic activity is capable of adding one or more L-nucleotides to the 3′ end of the first L-nucleic acid, wherein the mutant enzymatic activity exhibiting moiety comprises an amino acid sequence, wherein the amino acids of the amino acid sequence are D-amino acids, wherein the mutant enzymatic activity exhibiting moiety is a variant of an enzymatic activity exhibiting moiety, wherein the enzymatic activity exhibiting moiety consists of an amino acid sequence according to SEQ ID NO: 15 and wherein the amino acids of the amino acid sequence according to SEQ ID NO: 15 are D-amino acids, wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety differs from the amino acid sequence of the enzymatic activity exhibiting moiety consisting of an amino acid sequence according to SEQ ID NO: 15 at least at one amino acid position, preferably at three amino acid positions, and/or wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety is a truncated form of an amino acid sequence according to SEQ ID NO: 15, and wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety is different from an amino acid sequence according to any of SEQ ID NOs 15 to 22 and 51.

The invention relates to a method for analysing ribonucleic acid (RNA) interactions comprising: a) cross-linking base-paired nucleotides of at least one RNA molecule and/or at least one pair of RNA molecules using a tagged, reversible cross-linking agent (preferably tagged-psoralen) under ultraviolet irradiation; b) fragmenting the said cross-linked RNA molecule(s); c) using said tag to extract said cross-linked RNA fragment(s); d) ligating the said cross-linked RNA fragment(s) to produce cross-linked ligated RNA chimera(s); e) reversing the cross-linking of the said agent to the said RNA molecule(s); f) preparing a sequence library by sequencing the ligated RNA chimera molecule(s) or pair(s); and g) analysing the sequence library to determine RNA interactions. Also disclosed is a method of studying a subject by analysing RNA interactions and attributing them to a clinical picture, or a drug discovery method by attributing an efficacy score to the drug based upon determined RNA interactions.

The invention relates to a method for analysing ribonucleic acid (RNA) interactions comprising: a) cross-linking base-paired nucleotides of at least one RNA molecule and/or at least one pair of RNA molecules using a tagged, reversible cross-linking agent (preferably tagged-psoralen) under ultraviolet irradiation; b) fragmenting the said cross-linked RNA molecule(s); c) using said tag to extract said cross-linked RNA fragment(s); d) ligating the said cross-linked RNA fragment(s) to produce cross-linked ligated RNA chimera(s); e) reversing the cross-linking of the said agent to the said RNA molecule(s); f) preparing a sequence library by sequencing the ligated RNA chimera molecule(s) or pair(s); and g) analysing the sequence library to determine RNA interactions. Also disclosed is a method of studying a subject by analysing RNA interactions and attributing them to a clinical picture, or a drug discovery method by attributing an efficacy score to the drug based upon determined RNA interactions.

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxymethylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxymethylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

The present technology provides polynucleotide compositions and methods of using the same to detect circulating tumor DNA (ctDNA) in a patient. Kits for use in practicing the methods are also provided.

The present invention provides a TNA-based probe for detecting and imaging a target miRNA in living cells. TNA-based probe is composed of a fluorophore-labeled TNA reporter strand partially hybridizing to a quencher-labeled TNA recognition strand which is designed to be antisense to the target RNA transcript via pair pairing. Upon cellular entry without the need of harmful transfection treatment, the quencher-labeled TNA recognition strand binds to targeted transcript, and these target binding events displace the reporter strand from the quencher, resulting in a discrete “turning-on” of the fluorescence. The extent of fluorescence enhancement is quantifiably related to the target RNA expression level. Additionally, the TNA-based probe shows rapid detection response, excellent selectivity and specificity toward target miRNAs and is able to distinguish the target molecules with 1-2 base mismatches.

The present invention provides a TNA-based probe for detecting and imaging a target miRNA in living cells. TNA-based probe is composed of a fluorophore-labeled TNA reporter strand partially hybridizing to a quencher-labeled TNA recognition strand which is designed to be antisense to the target RNA transcript via pair pairing. Upon cellular entry without the need of harmful transfection treatment, the quencher-labeled TNA recognition strand binds to targeted transcript, and these target binding events displace the reporter strand from the quencher, resulting in a discrete “turning-on” of the fluorescence. The extent of fluorescence enhancement is quantifiably related to the target RNA expression level. Additionally, the TNA-based probe shows rapid detection response, excellent selectivity and specificity toward target miRNAs and is able to distinguish the target molecules with 1-2 base mismatches.

Provided are methods for identification of DNA repair locations in a genome of a non-dividing cell, by incorporating a reactive nucleoside analogs into the genome of the non-dividing cell, then sequencing the regions of the genome that incorporated the nucleoside analog.

Provided are methods for identification of DNA repair locations in a genome of a non-dividing cell, by incorporating a reactive nucleoside analogs into the genome of the non-dividing cell, then sequencing the regions of the genome that incorporated the nucleoside analog.