Recombination, RNA evolution, and bifunctional RNA molecules isolated through chimeric SELEX.
Exchange of RNA structural domains through recombination can be used to engineer RNAs with novel functions and may have played an important role in the early evolution of life. The degree of function an RNA element retains upon recombination into a new sequence context is a measure of how deleterious or beneficial recombination will be. When we fused pairs of aptamers previously selected to bind coenzyme A, chloramphenicol, or adenosine, the chimerae retained some ability to bind both targets, but with reduced binding activity both in solution and on affinity resins, probably due to misfolding. Complex populations of recombined RNAs gave similar results. Applying dual selection pressure to recombined populations yielded the combinations that were best suited to binding both targets. Most reselected RNAs folded into the active conformation more readily than chimerae built from arbitrarily chosen aptamers, as indicated both by solution Kd measurements and affinity resin binding activity. Deletion/selection experiments confirmed that the sequences required for binding are fully contained within the respective domains and not derived from interaction between the domains, consistent with the modular architecture of their original design. The combinatorial nature of the recombination methods presented here takes advantage of the full sequence diversity of the starting populations and yields large numbers of bifunctional molecules (10(6) to more than 1012). The method can be easily generalized and should be applicable to engineering dual-function RNAs for a wide variety of applications, including catalysis, novel therapeutics, and studies of long-range RNA structure.
Burke
D H
DH
Department of MCD Biology, University of Colorado, Boulder 80309-0347, USA. dhburke@indiana.edu
Willis
J H
JH
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002678:Chimera / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D019143:Evolution, Molecular; D005818:Genetic Engineering; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011995:Recombination, Genetic
D004274:DNA, Recombinant; C039919:RNA, recombinant; D012313:RNA