RECOMBINATION, RNA EVOLUTION, AND BIFUNCTIONAL RNA MOLECULES ISOLATEDTHROUGH CHIMERIC SELEX

Exchange of RNA structural domains through recombination can be used t o engineer RNAs with novel functions and may have played an important role in the early evolution of life. The degree of function an RNA ele ment retains upon recombination into a new sequence context is a measu re of how deleterious or beneficial recombination will be. When we fus ed pairs of aptamers previously selected to bind coenzyme A, chloramph enicol, or adenosine, the chimerae retained some ability to bind both targets, but with reduced binding activity both in solution and on aff inity resins, probably due to misfolding. Complex populations of recom bined RNAs gave similar results. Applying dual selection pressure to r ecombined populations yielded the combinations that were best suited t o binding both targets. Most reselected RNAs folded into the active co nformation more readily than chimerae built from arbitrarily chosen ap tamers, as indicated both by solution K-d measurements and affinity re sin binding activity. Deletion/selection experiments confirmed that th e sequences required for binding are fully contained within the respec tive domains and not derived from interaction between the domains, con sistent with the modular architecture of their original design. The co mbinatorial nature of the recombination methods presented here takes a dvantage of the full sequence diversity of the starting populations an d yields targe numbers of bifunctional molecules (10(6) to more than 1 0(12)). 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.