Gapmer antisense oligonucleotides (ASOs) hold therapeutic promise for allele-specific silencing, but face challenges in distinguishing between mutant and wild-type transcripts. This study explores new design strategies to enhance ASO specificity, focusing on a common dominant mutation in COL6A3 gene associated with Ullrich congenital muscular dystrophy. Initial gapmer ASO design exhibited high efficiency but poor specificity for the mutant allele. We then adopted a mixmer design, incorporating additional RNA bases based on computational predictions of secondary structures for both mutant and wild-type alleles, aiming to enhance ASO accessibility to mutant transcripts. The mixmer ASO design demonstrated up to a 3-fold increase in specificity compared with the classical gapmer design. Further refinement involved introducing a nucleotide mismatch as a structural modification, resulting in a 10-fold enhancement in specificity compared with the gapmer design and a 3-fold over the mixmer design. Additionally, we identified for the first time a potential role of the RNA-induced silencing complex (RISC), alongside RNase H1, in gapmer-mediated silencing, in contrast with what was observed with mixmer ASOs, where only RNase H1 was involved. In conclusion, this study presents a novel design concept for allele-specific ASOs leveraging mRNA secondary structures and nucleotide mismatching and suggests a potential involvement of RISC in gapmer-mediated silencing.

Combined therapies play a key role in the fight against complex pathologies, such as cancer and related drug-resistance issues. This is particularly relevant in targeted therapies where inhibition of the drug target can be overcome by cross-activating complementary pathways. Unfortunately, the drug combinations approved to date -mostly based on small molecules- face several problems such as toxicity effects, which limit their clinical use. To address these issues, we have designed a new class of RNase H-sensitive construct (3ASO) that can be disassembled intracellularly upon cell entry, leading to the simultaneous release of three different therapeutic oligonucleotides (ONs), tackling each of them the mRNA of a different protein. Here, we used Escherichia coli RNase H1 as a model to study an unprecedented mode of recognition and cleavage, that is mainly dictated by the topology of our RNA·DNA-based hybrid construct. As a model system for our technology we have created 3ASO constructs designed to specifically inhibit the expression of HER2, Akt and Hsp27 in HER2+ breast cancer cells. These trifunctional ON tools displayed very low toxicity and good levels of antiproliferative activity in HER2+ breast cancer cells. The present study will be of great potential in the fight against complex pathologies involving multiple mRNA targets, as the proposed cleavable designs will allow the efficient single-dose administration of different ON drugs simultaneously.

Members of the ribonuclease H (RNase H) family of enzymes (EC 3.1.26.4), which are found in nearly all organisms, are endoribonucleases that specifically hydrolyze the phosphodiester bond of RNA in a RNA-DNA hybrid. In retroviruses such as HIV-1, the RNase H activity is part of reverse transcriptase, the enzyme that converts the viral ssRNA into dsDNA suitable for integration into the host cell genome. In HIV-1, RNase H plays an essential role in various stages of reverse transcription, and it has been known for 20 years that inhibiting RNase H activity renders HIV noninfectious. However, the development of potent and selective antagonists of HIV RNase H has made surprisingly slow progress, and so far no RNase H inhibitor is in clinical trial, rendering this enzyme an important, but as yet underexplored, drug target. The recently described crystal structure of human RNase H in complex with a RNA-DNA hybrid provides new insight into the mechanism of HIV RNase H activity, with the potential to unveil new niches for therapeutic intervention. The current status of RNase H screening efforts is reviewed here.

Targeting Ribonuclease H (RNase H) has been considered a viable strategy for HIV therapy. In this study, a series of novel thiazolo[3, 2-a]pyrimidine derivatives were firstly designed and synthesized as potential inhibitors of HIV-1 RNase H. Among these compounds, A28 exhibited the most potent inhibition against HIV-1 RNase H with an IC50 value of 4.14 μM, which was about 5-fold increase in potency than the hit compound A1 (IC50 = 21.49 μM). To gain deeper insights into the structure-activity relationship (SAR), a CoMFA model was constructed to yield reasonable statistical results (q2 = 0.658 and R2 = 0.969). Results from magnesium ion chelation experiments and molecular docking studies revealed that these thiazolopyrimidine inhibitors may exert their inhibitory activity by binding to an allosteric site on RNase H at the interface between subunits p51 and p66. Furthermore, this analog demonstrated favorable physicochemical properties. Our findings provide valuable groundwork for further development of allosteric inhibitors targeting HIV-1 RNase H.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered \"Crab\"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. \"Crab\"-like configuration of two catalytic peptides (\"pincers\") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for \"single-pincer\" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled \"Crabs\" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5\'-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 μM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

We herein present a novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction. The detection probe employed as a key component in this technique serves as a substrate for RNase H and triggers the PS-THSP reaction upon the RNase H-mediated degradation of the probe. As a consequence, a large number of long concatemeric amplification products could be produced and used to identify the RNase H activity through the fluorescence signals produced by the nucleic acid-specific fluorescent dye, SYTO 9. Importantly, the use of the gp32 protein allowed the PS-THSP reaction to be performed at 37 °C, ultimately enabling an isothermal one-step RNase H assay. Based on this sophisticated design principle, the RNase H activity was very sensitively detected, down to 0.000237 U mL-1 with high specificity. We further verified its practical applicability through its successful application to the screening of RNase H inhibitors. With its operational convenience and excellent analytical performance, this technique could serve as a new platform for RNase H assay in a wide range of biological applications.

m6A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m6A is difficult to measure. Here, we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying m6A at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5\' of an adenosine site in an mRNA. This site is radiolabeled with 32P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and m6A at this site. Quantification of these spots reveals the m6A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m6A stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of m6A stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact m6A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m6A at previously mapped sites. SCARPET will be useful for testing specific sites for their m6A stoichiometry and to assess how m6A stoichiometry changes in different conditions and cellular contexts.

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.

t(8;14) translocation is the hallmark of Burkitt\'s lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt\'s lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt\'s lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.