RNA based computing: Some examples from RNA catalysis and RNA editing

This report will focus on a combined treatment of the elds of RNA evolution and DNA computers. Using examples of in vitro selection of functional RNA molecules from large pools of random sequences, we show that these experiments can be viewed as an \RNA computer", in the language of DNA computation. By applying a strict set of criteria to a pool of heterogeneous DNA or RNA sequences, both types of experiments nd sequences that contain solutions to hard molecular computational problems. These can even be nondigital problems that an electronic computer cannot solve. For example, a problem we posed to a pool of RNA molecules was one of catalysis, or the ability to ligate a small substrate RNA molecule to itself. In vitro evolution searches for the best solution or class of solutions contained within a large library of approximately 1015 unique sequences. The advantage of these \molecular computers" is their ability to examine and eliminate billions of possible solutions in parallel. We also describe an example of a cellular paradigm for RNA-based computing: RNA editing. 1 Molecular Selection In vitro selection is a technique which, like the Polymerase Chain Reaction (PCR), has revolutionizedmolecular biology, because it allows the study and construction of novel nucleic acid molecules with speci c properties or functions. Like DNA computers, in vitro selection is a seiving process of collecting desired molecules and discarding unwanted ones from large combinatorial libraries of 1013 to 1016 unique sequences (Figure 1). Independently invented by three laboratories in 1989 and 1990 ([6], [4], [16]), in vitro selection allows ampli cation and isolation of rare molecules based solely on their speci c functional properties and not on their primary sequence. All random molecules in molecular selection experiments are anked by identical primer binding sites for PCR ampli cation; therefore it is essentially \PCR by function". The ability to pass a functional assay and to survive to the next step depends on either binding a target ligand or undergoing a selfmodi cation reaction. This leads to di erential ampli cation of the \ ttest" molecules. In a catalytic assay for RNA enzymes, or ribozymes, the active molecules themselves must become modi ed by the reaction in such a way as to provide a \ shing hook" to distinguish them from inactive ones and to allow their capture from a large sequence pool. This limits the types of RNA catalysts (ribozymes) which can be retrieved by in vitro selection to those that can act in cis, allowing \self-tagging" of active ribozymes. Any molecule which catalyzes the reaction only in trans, meaning it acts on another molecule but not on itself, will be lost during the ltering process. Through multiple rounds of selection and ampli cation, in vitro evolution allows amplication, mutation and isolation of single desired nucleic acid molecules from large pools of heterogeneous sequences, leading to puri cation of molecules present in as few as one in 1016 copies. Since these molecules can have novel catalytic properties and myriad applicationssuch as binding a speci c ligand or catalyzing a reaction, the ability to harness combinatorial chemistry to isolate new ribozymes has launched a whole new industry of generating designer RNA tools. By expanding our knowledge of RNA's functional repertoire, the technique has also revealed the possibility of uncovering early catalysts in RNA evolution and even resuscitating active RNA molecules that have been \extinct" for over 3.9-billion years. Normally the genetic carrier between DNA informational molecule and protein functional molecule, RNA is an ideal unit for in vitro evolution, because it can combine both genotype and phenotype into one molecule. Therefore selection can act directly on RNA or DNA, because the primary sequence is contained within the actual molecule. In addition, the sequence is easily mutated and recovered, allowing experimental evolution on test-tubes of nucleic acids. The basic concept of in vitro selection is the di erential ampli cation of molecules with desired properties from pools of random sequence (Figure 1). Ampli cation of selected molecules increases their frequency in subsequent rounds, or generations, leading to survival of the ttest molecules. Multiple rounds of in vitro selection plus evolution allow rapid ampli cation, mutation, and isolation of a single desired nucleic acid molecule that can bind a novel target or perform a speci c function, from a pool of as many as 1016 heterogeneous { 2 { Figure 1: Outline of in vitro evolution. The \winning" molecule or class of molecules at the end are the most e cient at surviving the selection cycle [11].