Methods and compositions for enriching nucleic acids

A method for enriching target single-stranded nucleic acids in a mixed population of single-stranded nucleic acids, said method comprising:

• a) providing a mixed population of single-stranded nucleic acids containing target single-stranded nucleic acids and non-target single-stranded nucleic acids, wherein the target single-stranded nucleic acids comprise a target sequence at or within 21 bp of their 5′ ends;
• b) contacting the mixed population of single-stranded nucleic acids with a blocking oligonucleotide, wherein the blocking oligonucleotide is capable of hybridising to the target sequence of the target single-stranded nucleic acids, resulting in the target nucleic acid lacking an affinity binding domain attached to a capture domain;
• c) contacting the mixed population of nucleic acids of step b) with a single-strand specific 5′ exonuclease;
• and
• d) incubating the mixed population of nucleic acids of step c) under conditions for the 5′ exonuclease to digest the non-target single-stranded nucleic acids, such that the target single-stranded nucleic acids are enriched,
• wherein the blocking oligonucleotide has a melting temperature (Tm) of at least 45° C., at least 47° C., or at least 49° C.