Recombination, RNA evolution, and bifunctional RNA molecules isolated through Chimeric SELEX

Abstract

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 K d 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 10 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.

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

“…Although the ribozyme variants isolated from this pool were not faster than the ribozyme used to build it, some were considerably smaller. It is also known that recombination between different aptamer domains can generate molecules with the binding properties of both parental aptamers (Burke and Willis 1998), and recombination by randompriming was used in combination with several other approaches to improve the activity of an RNA polymerase ribozyme (Wochner et al 2011). In this study, we found that recombination of point mutations by synthetic shuffling was an effective way to further improve a kinase ribozyme that had been previously improved by random mutagenesis and reselection: the activity of the most efficient kinase ribozyme isolated was 30-fold greater than that of the most efficient kinase used to build the synthetically shuffled pool and 1300-fold faster than that of the initial isolate (Fig.…”

The dawn of the RNA World: Toward functional complexity through ligation of random RNA oligomers

“…Indeed, in vitro selection experiments have shown that the ligation and exchange of RNA structural domains through recombination can be used to engineer new functional RNAs (Burke and Willis 1998;Joyce 2004). It is of particular interest how the evolution of the class I ligase ribozyme (Bartel and Szostak 1993), appended with a track of 76 random nucleotides at its 39 end, allowed the selection of a 189-nt-long template-dependent RNA polymerase ribozyme (Johnston et al 2001).…”

Selecting Molecular Recognition. What Can Existing Aptamers Tell Us about Their Inherent Recognition Capabilities and Modes of Interaction?

“…However, if the problematic sequence components cannot be readily identified the required optimization of fused properties can be addressed directly by SELEX itself. This concept was demonstrated early by Burke and Willis who optimized fused aptamers against very divergent cytoplasmic small molecule targets [14]. Another critical question is how much cargo needs to be delivered for efficacy if the delivered nucleic acid encoded functionality is catalytic of otherwise amplified in nature.…”