Short, but significant, microRNAs (miRNAs) are an important class of gene regulators. Small‐molecule modifiers of miRNA function, such as 1 (see schematic representation), were identified in a cellular screen for miRNA‐pathway inhibitors. Such compounds are expected to be useful tools for the elucidation of detailed mechanisms of miRNA action and may serve as lead structures for the development of new therapeutic agents.

Our understanding of the complex processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to harness and reprogram the cellular machinery. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aaRSs against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the canonical twenty amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone, alkynyl, and hydrazino substituted amino acids. In an attempt to better understand this polyspecificity, the x-ray crystal structure of the aaRS/p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificitiy.

Tyrosyl radicals (Y•s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n=1–4) are promising tools for studying the mechanism of Y• formation and reactivity, as their pKas and peak potentials span four units and 300 mV, respectively, between pH 6–10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRS) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs, while maintaining high specificity over Y. An evolved aaRS is then used to site-specifically incorporate FnYs into the two subunits (α2 and β2) of E. coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y•s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s) and the resulting proteins isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y• in the wt RNR, generate long-lived FnY•s that are characterized by EPR spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-β2 is energetically and/or conformationally modulated such that the enzyme retains its activity, but that a new on-pathway Y• can accumulate. The distinct EPR properties of the 2,3,5-F3Y• facilitate spectral subtractions that make detection and identification of new Y•s straightforward.