Abstract:

Previous experiments have identified numerous RNA ligase deoxyribozymes, each of which can synthesize either 2‘,5‘-branched RNA, linear 2‘−5‘-linked RNA, or linear 3‘−5‘-linked RNA. These products may be formed by reaction of a 2‘-hydroxyl or 3‘-hydroxyl of one RNA substrate with the 5‘-triphosphate of a second RNA substrate. Here the inherent propensities for nucleophilic reactivity of specific hydroxyl groups were assessed using RNA substrates related to the natural sequences of spliceosome substrates and group II introns. With the spliceosome substrates, nearly half of the selected deoxyribozymes mediate a ligation reaction involving the natural branch-point adenosine as the nucleophile. In contrast, mostly linear RNA is obtained with the group II intron substrates. Because the two sets of substrates differ at only three nucleotides, we conclude that the location of the newly created ligation junction in DNA-catalyzed branch formation depends sensitively on the RNA substrate sequences. During the experiment that led primarily to branched RNA, we abruptly altered the selection strategy to demand that the deoxyribozymes create linear 3‘−5‘ linkages by introducing an additional selection step involving the 3‘−5‘-selective 8−17 deoxyribozyme. Although no 3‘−5‘ linkages (≤1%) were detectable in the pool products at the point that the 3‘−5‘ selection pressure was applied, deoxyribozymes that specifically create 3‘−5‘ linkages quickly emerged within a few selection rounds. Our success in obtaining 3‘−5‘ linkages via this approach shows that the outcome of deoxyribozyme selection experiments can be dramatically redirected by strategic changes in the selection procedure, even at a late stage. These results relate to natural selection, in which abrupt environmental variation can provide a rapid change in selection pressure. Linear 3‘−5‘ RNA linkages are an important practical objective because the native backbone is desirable in site-specifically modified ribozymes assembled by ligation. Therefore, this new approach to obtain 3‘−5‘-selective RNA ligase deoxyribozymes is particularly important for ongoing selection efforts.