The present invention is directed to methods and kits for enzymatic synthesis of labeled polynucleotide probes using template-free DNA polymerases.

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. The oligonucleotide aptamers of the present invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5 or 3 single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3 ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5 ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5 or 3 single-strand protruding ends, along with any methylation present.

This disclosure relates to improved methods of identifying A-to-I RNA edits in a sample. In certain embodiments, this disclosure relates to methods of purifying RNA containing an inosine base comprising the steps of: exposing an RNA sample to endonuclease V or fusion thereof and calcium ions in the absence of magnesium ions providing an RNA and endonuclease V binding complex. In certain embodiments, the methods further comprise purifying the RNA and endonuclease V binding complex from unbound RNA in the sample; separating the RNA from endonuclease V providing separated RNA; sequencing the separated RNA; and identifying positions in the RNA sequences wherein A-to-I edits occur. In certain embodiments, the RNA is derived from a cell.