In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine–pyrimidine junctions

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine-pyrimidine (pyr-pyr) junctions, which are cleaved 1-3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr-pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited k(obs) values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited approximately 1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.