Figure 1

The GG library and assembly schematic. (a) Crystal structure of PUM-HD bound to RNA, adapted from reference [15], GenBank ID code 1M8Y. (b) Schematic of PUM-HD bound to RNA. Filled boxes, PUF modules. Circles, RNA bases. (c) Schematic of the main library components: 8 repeat modules, with matching overhangs colored in identical colors; 4 variations of module 2, with corresponding recognition nucleotide indicated above; the aa sequence of module 2, with mutant aa indicated in red; and two receiving vectors. (d) The GG assembly schematic. A one-pot reaction that contains 8 modules of choice, a receiving vector, and enzymes allows the creation of 9 unique overhangs. The exact matching of the overhangs results in the predetermined repeat order assembled in the receiving vector. (e) Schematic of the GG library. R, module; N, nucleotide; recognized nucleotides indicated in the top row. First and last letters in the module names represent aa residues 12 and 16, in each module, respectively. Middle letter, if present, represents the “stacking” aa 13. Black font, WT modules. Red font, mutant modules. Green, yellow, pink, and blue fillings for modules recognizing A, G, U, and C, respectively.