RNase H Research Articles

Cleavable molecular beacon-based loop-mediated isothermal amplification assay for the detection of adulterated chicken in meat.

A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for chicken authentication detection. LAMP and CMB were used for DNA amplification and amplicon analysis, respectively. Targeting the mitochondrial cytochrome b gene of chickens, five primers and one CMB probe were designed, and their specificity was validated against nine other animal species. The structure of CMB and concentrations of dNTPs, MgSO4, betaine, RNase H2, primers and CMB were optimized. The CMB-LAMP assay was completed within 17 min, and its limit of detection for chicken DNA was 1.5 pg μL-1. Chicken adulteration as low as 0.5% was detected in beef, and no cross-reactivity was observed. Finally, this assay was successfully applied to 20 commercial meat products. When combined with our developed DNA extraction method (the extraction time was 1 min: lysis for 10 s, washing for 20 s and elution for 30 s), the entire process (from DNA extraction to results analysis) was able to be completed within 20 min, which is at least 10 min shorter than other LAMP-based methods. Our method showed great potential for the on-site detection of chicken adulteration in meat.

Ribonuclease H2 Subunit A Preserves Genomic Integrity and Promotes Prostate Cancer Progression.

RNASEH2A was demonstrated to be highly upregulated in aggressive prostate cancer to degrade R-loop accumulation and preserve genomic stability for tumor growth, suggesting that RNase H2 activity could be a promising therapeutic target.

Regulation of Heat Stress in Physcomitrium (Physcomitrella) patens Provides Novel Insight into the Functions of Plant RNase H1s

RNase H1s are associated with growth and development in both plants and animals, while the roles of RNase H1s in bryophytes have been rarely reported. Our previous data found that PpRNH1A, a member of the RNase H1 family, could regulate the development of Physcomitrium (Physcomitrella) patens by regulating the auxin. In this study, we further investigated the biological functions of PpRNH1A and found PpRNH1A may participate in response to heat stress by affecting the numbers and the mobilization of lipid droplets and regulating the expression of heat-related genes. The expression level of PpRNH1A was induced by heat stress (HS), and we found that the PpRNH1A overexpression plants (A-OE) were more sensitive to HS. At the same time, A-OE plants have a higher number of lipid droplets but with less mobility in cells. Consistent with the HS sensitivity phenotype in A-OE plants, transcriptomic analysis results indicated that PpRNH1A is involved in the regulation of expression of heat-related genes such as DNAJ and DNAJC. Taken together, these results provide novel insight into the functions of RNase H1s.

Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion.

The in vivo role for RNase H1 in mammalian mitochondria has been much debated. Loss of RNase H1 is embryonic lethal and to further study its role in mtDNA expression we characterized a conditional knockout of Rnaseh1 in mouse heart. We report that RNase H1 is essential for processing of RNA primers to allow site-specific initiation of mtDNA replication. Without RNase H1, the RNA:DNA hybrids at the replication origins are not processed and mtDNA replication is initiated at non-canonical sites and becomes impaired. Importantly, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed accompanied by mtDNA depletion. The steady-state levels of mitochondrial transcripts follow the levels of mtDNA, and RNA processing is not altered in the absence of RNase H1. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain of RNase H1 causing impaired mtDNA replication. In contrast to catalytically inactive variants of RNase H1, this mutant version has enhanced enzyme activity but shows impaired primer formation. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.

A multi-locus genome-wide association study reveals the genetics underlying muscadine antioxidant in berry skin.

Muscadine berries display enhanced nutraceutical value due to the accumulation of distinctive phytochemical constituents with great potential antioxidant activity. Such nutritional and health merits are not only restricted to muscadine, but muscadine berries accumulate higher amounts of bioactive polyphenolics compared with other grape species. For the genetic study of the antioxidant trait in muscadine, a multi-locus genome-wide association study (GWAS) with 350 muscadine genotypes and 1,283 RNase H2 enzyme-dependent amplicon sequencing (rhAmpSeq) markers was performed. Phenotyping was conducted with several antioxidant-related traits, including total phenolic content (TPC), total flavonoid content (TFC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, and FRAP antioxidant assay in muscadine berry skin. The correlation coefficient analysis revealed that the TPC, and DPPH/FRAP activities were significantly correlated. Through the GWAS analysis, 12 QTNs were identified from the four traits, of which six were pleiotropic QTNs. Two pleiotropic QTNs, chr2_14464718 and chr4_16491374, were commonly identified from the TPC and DPPH/FRAP activities. Co-located genes with the two pleiotropic QTNs were isolated, and two candidate genes were identified with transcriptome analysis. UDP-glycosyltransferase and 4-hydroxy-4-methyl-2-oxoglutarate aldolase were the candidate genes that are positively and negatively correlated to the quantitative property of traits, respectively. These results are the first genetic evidence of the quantitative property of antioxidants in muscadine and provide genetic resources for breeding antioxidant-rich cultivars for both Muscadinia and Euvitis species.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2′-O-methyl (2′-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2′-methoxyethyl, 2′ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2′-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5′ end of the gap or in the 3′-wing of a Gap 2′-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2′-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

NAT10 and DDX21 Proteins Interact with RNase H1 and Affect the Performance of Phosphorothioate Oligonucleotides.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing.

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5′-3′ exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.

Analysis of ribonucleotide content in the genomic DNA of ribonuclease H2 A subunit (RH2A)-knockout NIH3T3 cells after transient expression of wild-type RH2A or RH2A variants with an Aicardi-Goutières syndrome-causing mutation.

Ribonuclease (RNase) H2 is involved in the removal of ribonucleotides embedded in genomic DNA. Eukaryotic RNase H2 is a heterotrimer consisting of the catalytic A subunit (RH2A) and the accessory B and C subunits. This study aimed to compare the cellular activities of wild-type ribonuclease (RNase) H2 and its variants with a mutation causing neuroinflammatory autoimmune disease, Aicardi-Goutières syndrome (AGS). We first analyzed cellular RNase H2 activity and ribonucleotide content in the genomic DNA of RH2A-knockout (KO) mouse fibroblast NIH3T3 cells after transfection with a transient expression plasmid encoding mouse wild-type RH2A. From 4h after transfection, the RNase H2 activity increased and the amount of ribonucleotides decreased, as compared with the corresponding non-transfected RH2A-KO cells. This demonstrated the rapidness of ribonucleotide turnover in mammalian genomic DNA and the importance of continuous expression of RNase H2 to maintain the ribonucleotide amount low. Next, we expressed mouse RH2A variants with a mutation corresponding to a human AGS-causing mutation in RH2A-KO NIH3T3 cells. Neither increase in RNase H2 activity nor decrease in ribonucleotide amount was observed for G37S; however, both conditions were observed for N213I and R293H. This corresponded with our previous results on the activity of recombinant human RNase H2 variants.

Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.

Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable invivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

Ribodysgenesis: sudden genome instability in the yeast Saccharomyces cerevisiae arising from RNase H2 cleavage at genomic-embedded ribonucleotides.

Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term ‘ribodysgenesis’. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30–40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/μL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.

Translin facilitates RNA polymerase II dissociation and suppresses genome instability during RNase H2- and Dicer-deficiency

The conserved nucleic acid binding protein Translin contributes to numerous facets of mammalian biology and genetic diseases. It was first identified as a binder of cancer-associated chromosomal translocation breakpoint junctions leading to the suggestion that it was involved in genetic recombination. With a paralogous partner protein, Trax, Translin has subsequently been found to form a hetero-octomeric RNase complex that drives some of its functions, including passenger strand removal in RNA interference (RNAi). The Translin-Trax complex also degrades the precursors to tumour suppressing microRNAs in cancers deficient for the RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax complex being explored as a therapeutic target. Additionally, Translin and Trax have been implicated in a wider range of biological functions ranging from sleep regulation to telomere transcript control. Here we reveal a Trax- and RNAi-independent function for Translin in dissociating RNA polymerase II from its genomic template, with loss of Translin function resulting in increased transcription-associated recombination and elevated genome instability. This provides genetic insight into the longstanding question of how Translin might influence chromosomal rearrangements in human genetic diseases and provides important functional understanding of an oncological therapeutic target.

RNase H2, mutated in Aicardi‐Goutières syndrome, resolves co-transcriptional R-loops to prevent DNA breaks and inflammation

RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus.

African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.

Analysis of clinical characteristics of children with Aicardi-Goutieres syndrome in China

BackgroundAicardi-Goutieres syndrome (AGS) is an inflammatory disorder belonging to the type I interferonopathy group. The clinical diagnosis of AGS is difficult, which can lead to a high mortality rate. Overall, there is a lack of large-sample research data on AGS in China. We aim to summarize the clinical characteristics of Chinese patients with AGS and provide clues for clinical diagnostic.MethodsThe genetic and clinical features of Chinese patients with AGS were collected. Real-time polymerase chain reaction was used to detect expression of interferon-stimulated genes (ISGs).ResultsA total of 23 cases were included, consisting of 7 cases of AGS1 with three prime repair exonuclease 1 mutations, 3 of AGS2 with ribonuclease H2 subunit B (RNASEH2B) mutations, 3 of ASG3 with RNASEH2C, 1 of AGS4 with RNASEH2A mutations, 2 of AGS6 with adenosine deaminase acting on RNA 1 mutations, and 7 of AGS7 with interferon induced with helicase C domain 1 mutations. Onset before the age of 3 years occurred in 82.6%. Neurologic involvement was most common (100%), including signs of intracranial calcification which mainly distributed in the bilateral basal ganglia, leukodystrophy, dystonia, epilepsy, brain atrophy and dysphagia. Intellectual disability, language disability and motor skill impairment were also observed. Skin manifestations (60.87%) were dominated by a chilblain-like rash. Features such as microcephaly (47.62%), short stature (52.38%), liver dysfunction (42.11%), thyroid dysfunction (46.15%), positive autoimmune antibodies (66.67%), and elevated erythrocyte sedimentation rate (53.85%) were also found. The phenotypes of 2 cases fulfilled the diagnostic criteria for systemic lupus erythaematosus (SLE). One death was recorded. ISGs expression were elevated.ConclusionsAGS is a systemic disease that causes sequelae and mortality. A diagnosis of AGS should be considered for patients who have an early onset of chilblain-like rash, intracranial calcification, leukodystrophy, dystonia, developmental delay, positive autoimmune antibodies, and elevated ISGs, and for those diagnosed with SLE with atypical presentation who are nonresponsive to conventional treatments. Comprehensive assessment of vital organ function and symptomatic treatment are important.

RNA modifications can affect RNase H1-mediated PS-ASO activity

Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection.

α‐hydroxytropolones Inhibit RNase H1 Noncompetitively Despite Binding in the Active Site

Human immunodeficiency virus (HIV) and Hepatitis B virus (HBV) ribonucleases H (RNase H) are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. RNases H cleave RNA in RNA/DNA hybrids. Eukaryotic RNase H1 is an essential protein and probable off‐target enzyme for viral RNase H inhibitors. α‐hydroxytropolones (αHTs) comprise an anti‐RNase H inhibitor class that can inhibit the HIV, HBV, and human RNases H1. These compounds work by binding the RNase H active site by chelating the catalytic divalent metal cofactors. We hypothesized that a better understanding of RNase H1 inhibition will help development of compounds selective for the viral RNases H. To this end, we expressed and purified recombinant human RNase H1 and determined its inhibition mechanism(s) in steady‐state kinetics by two αHTs, 110 and 404 (Fig. 1). Inhibition was not competitive with a 12‐mer RNA/DNA substrate, but the turnover rate was reduced despite inhibitor binding to the active site (Fig. 2). 110 and 404displayed inhibition constants of 9 μM and 3 μM in saturating substrate concentrations, respectively, and these values were elevated 2‐3‐fold in very low substrate. Saturating 110and 404concentrations modestly reduced the apparent substrate binding constant (KM) from 90 nM to ~30 nM, while reducing the turnover rate (kcat = 0.17 s‐1) ~20‐fold. We found that 110enhanced affinity of RNase H1 for substrate by 4‐fold using a fluorescence polarization (FP) substrate binding assay with Ca2+ instead of Mg2+ to prevent RNA cleavage. 404, on the other hand, competed with substrate in binding assays, raising the substrate's KD~7‐fold from 24 nM without compound to ~150 nM. Induced fit docking studies in the Schrödinger suite suggest 110 binds to the active site metals as expected, while the substrate is still capable of binding via RNase H1’s high‐affinity auxiliary RNA/DNA hybrid binding domain (HBD) and substrate binding groove within the RNase H domain. 110 made favorable contacts with both enzyme and substrate, stabilizing an ESI complex. 404, on the other hand, occupies much of the substrate binding groove as well as the active site due to its larger structure. This would explain why 404 competes with substrate binding, while 110enhances substrate binding. The reason the KM decreased with 404 despite its competitive behavior in substrate binding assays is not clear. However, we hypothesize that 404locally competes with the substrate for the RNase H1 active site and the substrate binding groove within the RNase H domain without interfering with the HBD:substrate interface. This could lower the overall ES affinity. However, we speculate that if substrate release is slow relative to RNA hydrolysis and product release, the compound could behave uncompetitively in kinetics assays by inhibiting the breakdown of the ES complex through catalysis, only permitting enzyme‐substrate dissociation via the putatively slow substrate release pathway. Thus, these results illustrate that non‐competitive steady‐state kinetics may be observed without an allosteric inhibitor binding site. This may be common to active‐site inhibitors of enzymes with auxiliary substrate binding domains.