Selection scheme for the isolation of trans-acting ribozyme ligases.

FACS analysis of compartmentalized and noncompartmentalized ligation reactions. Reactions were conducted as described in the text using the b1-207t ribozyme. In the absence of compartmentalization, only a single fluorescent population was observed (A, gray). Compartmentalization resulted in the formation of two distinct populations, a highly fluorescent “reacted” population and a less fluorescent “unreacted” population (B). Decreasing the gene-to-bead ratio (B–D) resulted in a proportional decrease in size of the “reacted” population. The fraction of events that have a fluorescent signal greater than that of the “unreacted” population has been indicated as a percentage. The number of fluorescent events is indicated on the Y-axis and the relative level of fluorescence on the X-axis.

Progress of the compartmentalized in vitro selection. (A) Description of selection stringency and progress, by round. (B) Ligation activity of the pool. Activity was determined using a coupled transcription–ligation assay. A 5′ P³²-labeled RNA substrate (S^OH) was used in solution, without emulsification. Ligation products and substrates were at the correct molecular weight, as determined by their mobility relative to standards. The bands located between the “product” and the “substrate” were artifacts of T7 RNA polymerase acting on RNA substrates; these bands were also observed in the absence of a dsDNA template.

Active variant (r4.dom) isolated from the selection. The predicted secondary structure is based on that of the wild-type b1-207t ribozyme. All residues shown in uppercase were doped at a level of 70% during the selection, while those residues shown in lowercase were held constant. Invariant (red) and semi-conserved (blue) residues identified by Ekland and Bartel (1995) are indicated. Residues that differed from those originally found in the parental ribozyme are boxed in grey, with the inverted base pair boxed in yellow. The original sequence identity of each position is indicated in parentheses. The sequences of the 3′ constant region and the substrates ^PPPS (red line) and S^OH (blue line) are given in Materials and Methods.

Ligation kinetics for the selected clone r4.dom. (A) Rates of ligation. The observed rate of ligation (k_obs) was determined from purified RNA under selection-like conditions, without emulsification. For comparison, the rate of ligation of the parental ribozyme b1-207t and the parental ribozyme bearing the 3′ constant extension (b1-207t-full) are also shown. (B) Emulsified ligation reactions. Assays were conducted under the conditions employed during the selection and analyzed by FACS. Histograms have been normalized to the number of single bead events collected.