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Bioisosteric replacement and scaffold hopping are powerful strategies in drug design useful for rationally modifying a hit compound towards novel lead therapeutic agents. Recently, we reported a series of thienopyrimidinones that compromise dynamics at the p66/p51 HIV-1 reverse transcriptase (RT)-associated Ribonuclease H (RNase H) dimer interface, thereby allosterically interrupting catalysis by altering the active site geometry. Although they exhibited good submicromolar activity, the isosteric replacement of the thiophene ring, a potential toxicophore, is warranted. Thus, in this article, the most active 2-(3,4-dihydroxyphenyl)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one 1 was selected as the hit scaffold and several isosteric substitutions of the thiophene ring were performed. A novel series of highly active RNase H allosteric quinazolinone inhibitors was thus obtained. To determine their target selectivity, they were tested against RT-associated RNA-dependent DNA polymerase (RDDP) and integrase (IN). Interestingly, none of the compounds were particularly active on (RDDP) but many displayed micromolar to submicromolar activity against IN.

Converting the single-stranded retroviral RNA into integration-competent double-stranded DNA is achieved through a multi-step process mediated by the virus-coded reverse transcriptase (RT). With the exception that it is restricted to an intracellular life cycle, replication of the Saccharomyces cerevisiae long terminal repeat (LTR)-retrotransposon Ty3 genome is guided by equivalent events that, while generally similar, show many unique and subtle differences relative to the retroviral counterparts. Until only recently, our knowledge of RT structure and function was guided by a vast body of literature on the human immunodeficiency virus (HIV) enzyme. Although the recently-solved structure of Ty3 RT in the presence of an RNA/DNA hybrid adds little in terms of novelty to the mechanistic basis underlying DNA polymerase and ribonuclease H activity, it highlights quite remarkable topological differences between retroviral and LTR-retrotransposon RTs. The theme of overall similarity but distinct differences extends to the priming mechanisms used by Ty3 RT to initiate (−) and (+) strand DNA synthesis. The unique structural organization of the retrotransposon enzyme and interaction with its nucleic acid substrates, with emphasis on polypurine tract (PPT)-primed initiation of (+) strand synthesis, is the subject of this review.

In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to genome-wide mapping of roX2, a well-studied ncRNA involved in dosage compensation in DROSOPHILA: CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the male-specific lethal complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to chromatin immunoprecipitation for proteins.

Antisense inhibition of gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that promote mRNA degradation through RNase H or RNase P, or by steric hindrance of translation. Bridge nucleic acids (BNAs) are nucleotide analogs available in a few chemical variants. We evaluated gapmers composed of an oligodeoxynucleotide flanked by BNA residues in a BNA -DNA -BNA configuration, using the available variants: the original locked nucleic acid (LNA; 2'-O,4'-methylene bridge), cET (2'-O,4'-ethyl bridge), cMOE (2'-O,4'-methoxyethyl bridge), and BNANC (2'-O,4'-aminomethylene bridge). These gapmers were tested in vitro for their ability to induce cleavage of the model mRNA. All gapmers complementary to a previously identified region suitable for interaction with antisense oligomers induced RNase H-mediated degradation. Instead, only the LNA-containing gapmer also elicited RNase P-dependent cleavage, demonstrating dual RNA- and DNA-mimicking capability. In vitro coupled transcription-translation assays using cell lysates or reconstituted systems confirmed inhibition of expression and ruled out steric hindrance as the mechanism. In contrast, gapmers targeting the ribosome-binding site strongly inhibited expression by steric hindrance. These findings demonstrate that LNA-containing gapmers can exert their effects through multiple mechanisms, depending on the targeted mRNA region, thereby supporting their potential for synergistic inhibition of gene expression.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I.

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication. Cellular energy production is a function of the abundance of the small circular DNA molecules in mitochondria. Mitochondrial DNA is replicated in both dividing and nondividing cells, and encoding ribonuclease H1 (RNase H1) is essential to this process. Here, we define its mechanistic role: the removal of the RNA primers used for mitochondrial DNA replication. In the absence of RNase H1, primers are fixed in both template strands of mitochondrial DNA. The retained primers are a major impediment to mitochondrial DNA polymerase γ, leading to the formation of persistent DNA gaps that are catastrophic for subsequent rounds of replication. Moreover, primer retention provides unambiguous identification of RNA-DNA transition sites in the control region of mitochondrial DNA, thereby defining two major origins of replication.

The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80–90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2′-O-methylated, the optimized sequence coverage makes it a viable method. Graphical Abstract