Journal: Nucleic Acids Research

Article Title: Toehold-VISTA: a machine learning approach to decipher programmable RNA sensor-target interactions

doi: 10.1093/nar/gkag097

Figure Lengend Snippet: Generalization of toehold-VISTA across reporters and expression contexts. ( A ) Schematic of VISTA-designed toehold switches using a LacZ reporter for colorimetric output. ( B ) Endpoint photographs of cell-free transcription-translation reactions showing colorimetric responses for truncated, full-length, and non-cognate SARS-CoV-2 N gene targets. ( C ) Time-course absorbance measurements (570 nm) demonstrating activation kinetics for truncated and full-length targets relative to non-cognate controls. Reaction rate was determined at 60- and 70-min timepoints. Quantification of OFF-state ( D ) and ON-state absorbance for truncated ( E ) and full-length ( F ) RNA targets comparing VISTA-ranked and tsgen2-ranked toehold switches. Fold-change in LacZ reaction rates for truncated ( G ) and full-length ( H ) targets, showing improved fold changes for VISTA-ranked switches. Comparisons made with two-tailed t -test. Bars represent mean and whiskers represent SD.

Article Snippet: In vitro cell-free protein synthesis system (New England Biolabs E6800L) solutions A (0.4) and B (0.3) were combined, with the remaining volume composed of 0.005 U RNase inhibitor (New England Biolabs M0314L), 0.6 mg ml −1 enzymatic substrate chlorophenol red-β-D-galactopyranoside (CPRG, Roche 10884308001 distributed by Sigma), linear DNA templates comprising target or sensor-LacZ, and ultrapure DNase/RNase-free DI water to a total volume of 5 μl.

Techniques: Expressing, Activation Assay, Two Tailed Test