We have expanded the field of ''DNA computers'' to RNA and present a general approach for the solution of satisfiability problems. As an example, we consider a variant of the ''Knight problem,'' which asks generally what configurations of knights can one place on an n ؋ n chess board such that no knight is attacking any other knight on the board. Using specific ribonuclease digestion to manipulate strands of a 10-bit binary RNA library, we developed a molecular algorithm and applied it to a 3 ؋ 3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine ''bits'' or placeholders in a combinatorial RNA library. We recovered a set of ''winning'' molecules that describe solutions to this problem.DNA computing ͉ satisfiability ͉ RNA evolution ͉ in vitro selection ͉ SELEX A dleman (1) introduced DNA-based computing as an approach to solving mathematical problems, which uses DNA as a data carrier and techniques of molecular biology to operate on DNA. Since then, there have been relatively few experimental demonstrations of DNA-based computations (2, 3), although the theoretical foundation is strong (4, 5). Here we introduce a method for the construction of binary nucleic acid libraries, and we introduce RNA as a molecule for computation to present a general approach for the solution of the famous satisfiability (SAT) problems of propositional logic.Using a combination of a binary RNA library and ribonuclease (RNase) H digestion, we developed a destructive algorithm (6) that would hydrolyze RNA strands that did not fit the constraints of a chosen problem, instead of an algorithm that required efficient hybridization extraction (1, 2). The use of restriction endonucleases (3) would have also permitted a logical binary-style operation, as each restriction enzyme cleaves only in the presence of its recognition site, and cleaves the doublestranded DNA sufficiently to completion. However, by using RNase H in the context of different sets of oligonucleotides, one can go beyond the set of available restriction enzymes. Here, RNase H acts as a ''universal restriction enzyme'' because it allows selective marking of virtually any RNA strands for digestion in parallel, and use of a thermostable RNase H (7) ensures fidelity of hybridization between DNA and RNA strands, minimizing incorrect marking of noncognate strands. Materials and MethodsChemical Synthesis of DNA ''Half Libraries.'' The DNA library was prepared as two halves by using mix and split phosphoramidite chemistry with the sequence codes shown in Table 1. In brief, bit n set to 0 and spacer n were synthesized on one column, and bit n set to 1 and spacer n were synthesized on the other column. The columns were decrimped and the two resins poured together and mixed, and then half of this mixture (containing approximately equal proportions of 0's and 1's at bit position n) was returned to each column for synthesis of the next variable position, and the entire process was repeated (Fig. 1). Each of the four combinatorial synthesis...

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.