In the end, the most important parameter is life; i.e. how does the L3 basic thumb contribute to the fitness of the organism? While only some of the mutants had gross effects on cell growth, they all affected translational fidelity as monitored by decreased rates of À1 PRF (Figure 2 ). In fact, the effects on À1 PRF correlated well with the A-site aa-tRNA-binding data. For example, R247A and K241A/R244A, which had the most pronounced effects on À1 PRF, also promoted >3-fold decreases in affinity for aa-tRNA. In contrast, R232A and K236A, which had the smallest effects on aa-tRNA binding, also promoted the smallest decreases in À1 PRF. As noted above, the mutants investigated in this report are unique in that they are the first examples that promoted decreased À1 PRF. This trend had only previously been observed with anisomycin, a competitive inhibitor for aa-tRNA 3 0 binding to the PTC (41) . We have suggested that the majority of À1 PRF occurs after aa-tRNA accommodation into the A-site, and prior to peptidyltransfer, while a smaller fraction can occur during translocation [reviewed in (64) ], a view that is supported by a recent study coupling kinetic modeling of À1 PRF within the translation elongation cycle with mass spectroscopic analyses of frameshifted peptide products (P.-Y. Liao et al., submitted for publication). By this model, decreased rates of aa-tRNA accommodation into the A-site should decrease the steady state abundance of substrate for À1 PRF, thus inhibiting this reaction. Changes in À1 PRF in turn alter the relative amounts of viral protein products available for viral particle self assembly, a ratio that is critical for virus propagation (18, 40) . This illustrates how minute changes in ribosome structure at the atomic scale can propagate outward, affecting ribosome biochemistry, translational fidelity, and the ability of cells to replicate viruses. Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes. Microbial genomes can be highly variable because of high mutation rates. Because of this extreme variability, it is often difficult to identify regions within a specific virus genome that are sufficiently evolutionarily conserved to serve as targets for specific detection primers and probes. RNA viruses are especially variable. The influenza virus, a negative sense, single-stranded RNA (ssRNA) virus with a highly variable RNA genome, for example, has been known to cause the diagnostic problem that is at the basis of this article, because of a high rate of mutation and genetic drift. In such situations, optimal detection primers and probes would be broadly targeted yet specific, and remain functional even if the genome sequence changed because of genetic drift.