Novel self-cleaving dnazymes and selection process

A method of identifying a subset of single-stranded DNA strands that comprise self-cleaving DNAzymes among a plurality of single-stranded DNA strands, the method comprising:

• (a) providing the plurality of single-stranded DNA strands, wherein each of the single-stranded DNA strands of the plurality of single-stranded DNA strands comprises a core sequence of randomized nucleotides that is a candidate self-cleaving DNAzyme, wherein the core sequence is flanked by a first constant sequence at its 5′ end and a second constant sequence at its 3′ end, and wherein each the first constant sequence and the second constant sequence comprises a primer binding part,
• (b) subjecting the plurality of single-stranded DNA strands to self-cleaving conditions, resulting in cleavage of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme among the plurality of single-stranded DNA strands, thereby generating a first cleavage product and a second cleavage product of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme;
• wherein
• a. the first cleavage product comprises the constant sequence 1 or a portion of the constant sequence 1;
• and
• b. the second cleavage product comprises the constant sequence 2 and the core sequence;
• whereby when the first cleavage product comprises the portion of constant sequence 1, the second cleavage product further comprises a remaining portion of the constant sequence 1,
• (c) isolating the second cleavage product of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme,
• (d) producing complements of the second cleavage product of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme;
• (e) ligating the 3′ end of the complements of the second cleavage product to a complement of the first cleavage product, thereby obtaining a complement of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme;
• (f) amplifying the complements of the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme, thereby obtaining the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme;
• and
• (g) sequencing the subset of single-stranded DNA strands that comprises the self-cleaving DNAzyme, thereby identifying the subset of single-stranded DNA strands that comprise self-cleaving DNAzyme with a DNAzyme activity among the plurality of single-stranded DNA strands.