X-ray crystallography remains a powerful method to gain atomistic insights into the catalytic and regulatory functions of RNA molecules. However, the technique requires the preparation of diffraction-quality crystals. This is often a resource- and time-consuming venture because RNA crystallization is hindered by the conformational heterogeneity of RNA, as well as the limited opportunities for stereospecific intermolecular interactions between RNA molecules. The limited success at crystallization explains in part the smaller number of RNA-only structures in the Protein Data Bank. Several approaches have been developed to aid the formation of well-ordered RNA crystals. The majority of these are construct-engineering techniques that aim to introduce crystal contacts to favor the formation of well-diffracting crystals. A typical example is the insertion of tetraloop–tetraloop receptor pairs into non-essential RNA segments to promote intermolecular association. Other methods of promoting crystallization involve chaperones and crystallization-friendly molecules that increase RNA stability and improve crystal packing. In this review, we discuss the various techniques that have been successfully used to facilitate crystal packing of RNA molecules, recent advances in construct engineering, and directions for future research in this vital aspect of RNA crystallography.

Smoking-cessation drugs bind many off-target nicotinic acetylcholine receptors (nAChRs) and cause severe side effects if they are based on nicotine. New drugs that bind only those receptors, such as α6β2* nAChR, implicated in nicotine addiction would avoid the off-target binding. Indolizidine (-)-237D (IND (-)-237D), a bicyclic alkaloid, has been shown to block α6β2* containing nAChRs and functionally inhibit the nicotine-evoked dopamine release. To improve the affinity of indolizidine (-)-237D for α6β2*, we built a library of 2226 analogs. We screened virtually the library against a homology model of α6β2 nAChR that we derived from the recent crystal structure of α4β2 nAChR. We also screened the crystal structure of α4β2 nAChR as a control on specificity. We ranked the compounds based on their predicted free energy of binding. We selected the top eight compounds bound in their best pose and subjected the complexes to 100 ns molecular dynamics simulations to assess the stability of the complexes. All eight analogs formed stable complexes for the duration of the simulations. The results from this work highlight nine distinct analogs of IND (-)-237D with high affinity towards α6β2* nAChR. These leads can be synthesized and tested in in vitro and in vivo studies as lead candidates for drugs to treat nicotine addiction.

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This paper describes the synthesis of enamino carbonyl compounds by the copper(i)-catalyzed coupling of acceptor-substituted diazo compounds and tertiary thioamides.

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.