Epitranscriptomic RNA modifications can regulate mRNA function; however, there is a major gap in our understanding of the biochemical mechanisms mediating their effects. Here, we develop a chemical proteomics approach relying upon photo-cross-linking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N-methyladenosine (mA), the most abundant internal modification in eukaryotic RNA. In addition to identifying YTH domain-containing proteins and ALKBH5, known interactors of this modification, we find that FMR1 and LRPPRC, two proteins associated with human disease, "read" this modification. Surprisingly, we also find that mA disrupts RNA binding by the stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42. Our work provides a general strategy for interrogating the interactome of RNA modifications and reveals the biochemical mechanisms underlying mA function in the cell.

Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes1, 2, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene3, 4, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide−/− mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction5, 6. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE’s physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

Researchers seeking to improve the efficiency and cost effectiveness of the bioactive small-molecule discovery process have recently embraced selection-based approaches, which in principle offer much higher throughput and simpler infrastructure requirements compared with traditional small-molecule screening methods. Since selection methods benefit greatly from an information-encoding molecule that can be readily amplified and decoded, several academic and industrial groups have turned to DNA as the basis for library encoding and, in some cases, library synthesis. The resulting DNA-encoded synthetic small-molecule libraries, integrated with the high sensitivity of PCR and the recent development of ultra high-throughput DNA sequencing technology, can be evaluated very rapidly for binding or bond formation with a target of interest while consuming minimal quantities of material and requiring only modest investments of time and equipment. In this review we describe the development of two classes of approaches for encoding chemical structures and reactivity with DNA: DNA-recorded library synthesis, in which encoding and library synthesis take place separately, and DNA-directed library synthesis, in which DNA both encodes and templates library synthesis. We also describe in vitro selection methods used to evaluate DNA-encoded libraries and summarize successful applications of these approaches to the discovery of bioactive small molecules and novel chemical reactivity.

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.

Abstract:The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNAtemplated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio-and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio-and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio-and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), and heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

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Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.