An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes.

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure.

Selection of 2'-Fluoro-Modified Aptamers with Optimized Properties.

RNA or single-stranded DNA aptamers with 2'-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2'-modifications, we now report the selection of 2'-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity.

The expanding world of DNA and RNA.

DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases.

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems.