Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM.

RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.

Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analog, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 >M. NMR characterization of a soluble analog, 2, revealed a non-canonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important implications for RNA ligand design.

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (-)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (-)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (-)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb2+ ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region.

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem–loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.

Keywords:RNA structure and function RNA interactions Epitranscriptomics Small molecules RNA-based therapeutics RNA in disease Drug target A B S T R A C T The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets. RNA as a drug targetThe vast diversity of RNAs expanding beyond coding transcripts to several classes of ncRNAs that vary in length, biogenesis, polarity, and putative functions, increases the repertoire of druggable targets. 19,20 Here, specific RNA properties contribute to its attractive, yet challenging makeup as a target molecule. RNA can form complex three-dimensional structures through canonical Watson-Crick base pairing and complex tertiary interactions that are mediated by non-canonical bonds. Such structures can be as intricate and stable as those formed by proteins and can recognize small-molecule ligands, other nucleic acids, and/or proteins with high affinity and specificity. [21][22][23][24] At the same time, the highly dynamic conformation and repetitive character of its surface presents difficulties for drug design. 25,26 In addition, many putative small molecule-binding pockets in RNA are much more polar and solvent exposed than binding sites on proteins, complicating ligand design efforts. Compounding these issues, most target RNAs are expressed at low levels, with the exception of ribosomal (rRNA) and transfer (tRNA) RNAs, which constitute 80-90% and 10-15% of total cellular RNA, respectively. 27 Other abundant RNAs, such as messenger (mRNA), small nuclear (snRNA), and small nucleolar (snoRNA) are present at levels that are about 1-2 orders of magnitude lower than rRNA and tRNA. Certain small RNAs, such as micro (miRNA) and piwi (piRNAs) can be present at very high levels; however, this appears to be cell type dependent. One should keep in mind that if the RNA has catalytic activity or if it acts as a scaffold to regulate https://doi.