Model selection. (A) Gene constructs. Genes coding for the extended X-motif ribozyme and an inactive control RNA are shown. Both genes are of the same length and under the control of a T7 RNA polymerase promoter (ProT7) and code for RNA bearing 5′ and 3′ constant regions (cte). The position of the unique NcoI restriction found only in the inactive control RNA is indicated. (B) Electrophoretic analysis of the model selection. A mixture of one active for 49 inactive genes was subjected to the selection procedure. A PCR was performed on the reaction mixture before selection (−) or on the sorted droplets (+). PCR products were then digested by NcoI prior to being analyzed on 2% agarose gel. The X-motif gene did not possess an NcoI cleavage site and stayed intact while the gene for the inactive control RNA was cut by the enzyme into two fragments. X-motif genes were enriched 28-fold. (C) Activity assay. A PCR was performed on the reaction mixture before selection, on the sorted droplets, or with no template added. PCR products were incubated with an in vitro transcription (IVT) mixture for 1 h at 37°C. An aliquot of IVT mixture was then mixed with fluorogenic substrate and the fluorescence (Ex. 488 nm/Em. 525 nm) was monitored at 37°C. While only background activity was observed in the absence of PCR product (open triangles) and weak activity was obtained when DNA originated from unsorted droplets (open squares), the initial rate increased 26-fold after the sorting procedure (filled circles), compared with the unsorted droplets.
