Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages.
We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.
Coppins
Rebecca L
RL
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Silverman
Scott K
SK
D001483:Base Sequence; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011118:Polynucleotide Ligases / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D021881:DNA, Catalytic; D012313:RNA; D011118:Polynucleotide Ligases