Thermostable Artificial Enzyme Isolated by In Vitro Selection
Figure 1
In vitro selection of artificial ligase enzymes with increased stability.
(A) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C [17], [22]. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated in vitro yielding a library of mRNA-displayed proteins [22]. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (B) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C [17], [22], and with (C) 20 nucleotides of each substrate during the current selection at 65°C. HEG4 represents the linker of four hexaethylene glycol units (red wavy line).