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Rapid Construction of Empirical RNA Fitness Landscapes

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Science  15 Oct 2010:
Vol. 330, Issue 6002, pp. 376-379
DOI: 10.1126/science.1192001

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  1. Fig. 1

    Population structure before and after one round of in vitro selection. (A) Histograms of RNA ligase ribozyme populations before (blue) and after (red) in vitro selection (6.7 × 106 sequences each). Sequences are binned according to their Hamming distance (28) from the a4-11 (11) master sequence (MS) (13). (B and C) Pre-selection mutant spectrum (13, 19). Each spot is a unique species. Projection axes 1 and 2 are hash scores to the master sequence and an arbitrary string, respectively. Genotype frequency is the number of times a sequence was observed (13). (D and E) Mutant spectrum after one 24-hour in vitro selection step.

  2. Fig. 2

    Changes in population structure during serial depletion. (A) Hamming-distance histograms from serial depletion, showing genotype frequencies from the pre-selection (blue), 1-min (green), and 24-hour (pink) time points. Frequencies of the master sequence in the three populations are indicated. The asterisk denotes a subpopulation that is dominated by the parental sequence (14) of the engineered pool. (B) Rates of depletion of genotypes most abundant in the 1-min (green) and 24-hour (magenta) time points as a function of their similarity to the master sequence.

  3. Fig. 3

    Genotype frequency correlates positively with experimental rate constants. (A) kobs (green, measured in triplicate, error bars represent SEM) and information content (black) for the entire populations from each serial depletion time point. (B) Correlation between biochemically measured kobs of individual point mutants (table S3) and observed frequencies of the mutations (table S4) in all sequence variants with a Hamming distance ≤ 8 from the master sequence (red line, r = 0.67) (17). The green line represents the kobs of the master sequence (14). The gray dashed lines denote two independent estimates of the lower detection limit of the biochemical assay (13). (C) Histogram of correlation coefficients of kobs (n = 135 point mutants) with randomly reassorted mutation frequencies. The real correlation (r = 0.67) between the mutant frequencies in the selection and the experimental kobs is 7.4 standard deviations from the mean.

  4. Fig. 4

    Analysis of the experimentally constructed fitness landscape as information content per position. Information content of a position in bits (15, 16) of genotypes with a projection 1 hash score ≥ 800 (a Hamming distance of ≤ 8 from the master sequence) and Dmax = 1 min (A) and 24 hours (B), depicted as a heat map. Analyses were based on 4,485,943 reads of 311,869 unique sequences and 586,606 reads of 117,507 unique sequences for (A) and (B), respectively (fig. S5 and tables S5 to S8). P1, P2, and P3 denote helices; L2 and L3 denote loops. (C) Change in information content between Dmax = 1 min and 24 hours. Positions 12, 22, 33, 38, and 39 appear to be selectively neutral (29). Black and red base-pair symbols indicate pairing predicted from aligning the 256 most common sequences and from analysis of the Watson-Crick covariation of all sequences with a projection 1 hash score ≥ 800 (13).