Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

The molecular mechanism for TERRA recruitment and annealing to telomeres.

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere.

RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA-DNA hybrid cleavage.

RNase H1 has been acknowledged as an endoribonuclease specializing in the internal degradation of the RNA moiety within RNA-DNA hybrids, and its ribonuclease activity is indispensable in multifaceted aspects of nucleic acid metabolism. However, the molecular mechanism underlying RNase H1-mediated hybrid cleavage remains inadequately elucidated. Herein, using single-molecule approaches, we probe the dynamics of the hybrid cleavage by Saccharomyces cerevisiae RNase H1. Remarkably, a single RNase H1 enzyme displays 3'-to-5' exoribonuclease activity. The directional RNA degradation proceeds processively and yet discretely, wherein unwinding approximately 6-bp hybrids as a prerequisite for two consecutive 3-nt RNA excisions limits the overall rate within each catalytic cycle. Moreover, Replication Protein A (RPA) reinforces RNase H1's 3'-to-5' nucleolytic rate and processivity and stimulates its 5'-to-3' exoribonuclease activity. This stimulation is primarily realized through the pre-separation of the hybrids and consequently transfers RNase H1 to a bidirectional exoribonuclease, further potentiating its cleavage efficiency. These findings unveil unprecedented characteristics of an RNase and provide a dynamic view of RPA-enhanced processive hybrid cleavage by RNase H1.

Visual Evidence for the Recruitment of Four Enzymes with RNase Activity to the Bacillus subtilis Replication Forks.

Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5'->3' exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal.

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

In Vitro Enzymatic and Cell Culture-Based Assays for Measuring Activity of HBV Ribonuclease H Inhibitors.

HBV is a small, enveloped DNA virus that replicates by reverse transcription of an RNA intermediate. Current anti-HBV treatment regiments employ interferon α or nucleos(t)ide analogs, but they are not curative, are of long duration, and can be accompanied by systemic side-effects. The HBV ribonuclease H (RNaseH) is essential for viral replication; however, it is unexploited as a drug target. RNaseH inhibitors that actively block viral replication would represent an important addition to the potential new drugs for treating HBV infection. Here, we describe two methods to measure the activity of RNaseH inhibitors. The DNA oligonucleotide-directed RNA cleavage assay allows mechanistic analysis of compounds for anti-HBV RNaseH activity. Analysis of preferential inhibition of plus-polarity DNA strand synthesis by HBV RNaseH inhibitors in a cell culture model of HBV replication can be used to measure the ability of RNaseH inhibitors to block viral replication.

RNase H-sensitive multifunctional ASO-based constructs as promising tools for the treatment of multifactorial complex pathologies.

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.

Discovery of Benzisothiazolone Derivatives as Bifunctional Inhibitors of HIV-1 Reverse Transcriptase DNA Polymerase and Ribonuclease H Activities.

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure-activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.

Parsing Dynamics of Protein Backbone NH and Side-Chain Methyl Groups using Molecular Dynamics Simulations.

Experimental NMR spectroscopy and theoretical molecular dynamics (MD) simulations provide complementary insights into protein conformational dynamics and hence into biological function. The present work describes an extensive set of backbone NH and side-chain methyl group generalized order parameters for the Escherichia coli ribonuclease HI (RNH) enzyme derived from 2-µs microsecond MD simulations using the OPLS4 and AMBER-FF19SB force fields. The simulated generalized order parameters are compared with values derived from NMR 15N and 13CH2D spin relaxation measurements. The squares of the generalized order parameters, S2 for the N-H bond vector and Saxis2 for the methyl group symmetry axis, characterize the equilibrium distribution of vector orientations in a molecular frame of reference. Optimal agreement between simulated and experimental results was obtained by averaging S2 or Saxis2 calculated by dividing the simulated trajectories into 50 ns blocks (∼five times the rotational diffusion correlation time for RNH). With this procedure, the median absolute deviations (MAD) between experimental and simulated values of S2 and Saxis2 are 0.030 (NH) and 0.061 (CH3) for OPLS4 and 0.041 (NH) and 0.078 (CH3) for AMBER-FF19SB. The MAD between OPLS4 and AMBER-FF19SB are 0.021 (NH) and 0.072 (CH3). The generalized order parameters for the methyl group symmetry axis can be decomposed into contributions from backbone fluctuations, between-rotamer dihedral angle transitions, and within-rotamer dihedral angle fluctuations. Analysis of the simulation trajectories shows that (i) backbone and side chain conformational fluctuations exhibit little correlation and that (ii) fluctuations within rotamers are limited and highly uniform with values that depend on the number of dihedral angles considered. Low values of Saxis2, indicative of enhanced side-chain flexibility, result from between-rotamer transitions that can be enhanced by increased local backbone flexibility.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

N-Hydroxypiridinedione: A Privileged Heterocycle for Targeting the HBV RNase H.

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low µM range (1.1-7.7 µM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure-activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Design, synthesis and biological evaluation of Thiazolo[3, 2-a]Pyrimidine derivatives as novel RNase H inhibitors.

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.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

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.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

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.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Multivalent DNAzyme agents for cleaving folded RNA.

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.

Complete genome sequence of a novel badnavirus infecting Fatsia japonica in China.

The complete genome sequence of a novel badnavirus, tentatively named "fatsia badnavirus 1" (FaBV1, OM540428), was identified in Fatsia japonica. The infected plant displayed virus-like symptoms on leaves, including yellowing and chlorosis. The genome of FaBV1 is 7313 bp in length and similar in size and organization to other members of the genus Badnavirus (family Caulimoviridae), containing four open reading frames (ORFs), three of which are found in all known badnaviruses, and the other of which is only present in some badnaviruses. The virus has the genome characteristics of badnaviruses, including a tRNAMet binding site (5'-TCTGAATTTATAGCGCTA-3') and two cysteine-rich domains (C-X-C-2X-C-4X-H-4X-C and C-2X-C-11X-C-2X-C-4X-C-2X-C). Pairwise sequence comparisons of the RT+RNase H region indicated that FaBV1 shares 61.4-71.2% nucleotide (nt) sequence identity with other known badnaviruses, which is below the threshold (80% nt sequence identity in the RT+RNase H region) used for species demarcation in the genus Badnavirus. Phylogenetic analysis revealed that FaBV1, ivy ringspot-associated virus (IRSaV, MN850490.1), and cacao mild mosaic virus (CMMV, KX276640.1) together form a separate clade within the genus Badnavirus, suggesting that FaBV1 is a new member of the genus Badnavirus in the family Caulimoviridae. To our knowledge, this is the first report of a badnavirus infecting F. japonica.

Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR-targeted treatments. Therefore, we designed a protein- and genomic-based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS; 30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared with ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR-related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n = 12) versus Non-HomDel (n = 37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNA-SEH2B HomDels in U-LMS was 76%, 93%, and 71%, respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.

Approaches for Mapping and Analysis of R-loops.

R-loops are nucleic acid structures composed of a DNA:RNA hybrid with a displaced non-template single-stranded DNA. Current approaches to identify and map R-loop formation across the genome employ either an antibody targeted against R-loops (S9.6) or a catalytically inactivated form of RNase H1 (dRNH1), a nuclease that can bind and resolve DNA:RNA hybrids via RNA exonuclease activity. This overview article outlines several ways to map R-loops using either methodology, explaining the differences and similarities among the approaches. Bioinformatic analysis of R-loops involves several layers of quality control and processing before visualizing the data. This article provides resources and tools that can be used to accurately process R-loop mapping data and explains the advantages and disadvantages of the resources as compared to one another. © 2024 Wiley Periodicals LLC.