RNASEH1 Antibody

What is RNASEH1 and why is it important in cellular processes?

RNASEH1 (Ribonuclease H1) is an enzyme that cleaves the RNA portion of RNA/DNA hybrid structures formed during replication and repair processes. These hybrids could lead to DNA instability if not properly processed. Eukaryotic RNases H are larger and more complex than their prokaryotic counterparts . RNASEH1 has acquired a Hybrid Binding Domain that confers processivity and affinity for its substrate . The protein plays crucial roles in:

• Maintaining genomic stability

• Mitochondrial DNA replication

• Processing R-loops during transcription

• Facilitating antisense oligonucleotide (ASO) activity

Knockout studies demonstrate that RNASEH1 is essential for viable mammalian development, with its absence leading to mitochondrial dysfunction and ultimately apoptosis .

What are the basic structural and biochemical characteristics of RNASEH1?

RNASEH1 has the following key characteristics:

• Calculated and observed molecular weight: 32 kDa

• Gene location: Human chromosome 2p25.3

• Protein length: 286 amino acids

• Cellular localization: Both nucleoplasmic and nucleolar, with dynamic localization patterns

RNASEH1 contains both catalytic and hybrid binding domains, with the catalytic domain responsible for the endonuclease activity and the hybrid binding domain providing specificity for RNA-DNA hybrid structures .

What are the recommended protocols for using RNASEH1 antibodies in Western blot experiments?

For optimal Western blot results with RNASEH1 antibodies:

Sample preparation and controls:

• Positive controls: HeLa cells, U2OS cells, A549 cells, HEK-293 cells, Jurkat cells

• Protein lysate preparation: Standard cell lysis protocols with protease inhibitors

• Expected band size: 32 kDa

Protocol recommendations:

• Load 20-40 μg total protein per lane

• Use recommended antibody dilutions:

  • For antibody 15606-1-AP: 1:1000-1:5000

  • For antibody 82771-1-RR: 1:5000-1:50000 (higher sensitivity)

• Incubate primary antibody overnight at 4°C

• Detect using standard secondary antibody and visualization systems

Troubleshooting tips:

• If multiple bands appear, optimize blocking conditions (5% BSA often works better than milk for nuclear proteins)

• Verify specificity using RNASEH1 knockdown/knockout controls

• For weaker signals, longer exposure times or signal enhancement systems may be required

How should immunofluorescence experiments be optimized when using RNASEH1 antibodies?

For immunofluorescence with RNASEH1 antibodies:

Sample preparation:

• Validated cell types: HeLa cells show consistent positive staining

• Fixation methods: Both paraformaldehyde (4%) and methanol fixation work, but epitope accessibility may differ

Protocol optimization:

• Recommended dilutions: 1:50-1:500 for antibody 15606-1-AP

• Include permeabilization step (0.2% Triton X-100) if using paraformaldehyde fixation

• Block thoroughly (1 hour at room temperature) to reduce background

• Incubate primary antibody overnight at 4°C

• Use appropriate fluorophore-conjugated secondary antibodies

• Counterstain nuclei with DAPI

Expected results:

• Mixed nucleoplasmic and nucleolar staining pattern in most cell types

• Dynamic localization may depend on cell cycle stage and transcriptional activity

• Co-staining with nucleolar markers can help confirm partial nucleolar localization

What methodology should be used for immunoprecipitation with RNASEH1 antibodies?

For immunoprecipitation using RNASEH1 antibodies:

Sample preparation:

• Validated cell types: HeLa cells show consistent positive results

• Recommended lysis buffer: Non-denaturing buffer containing 150 mM NaCl, 50 mM Tris pH 7.5, 1% NP-40, with protease inhibitors

Protocol recommendations:

• Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Pre-clear lysate with protein A/G beads

• Incubate with RNASEH1 antibody overnight at 4°C

• Add protein A/G beads and incubate 1-2 hours

• Wash thoroughly (at least 4 times)

• Elute and analyze by Western blot

Validation methods:

• Include IgG control IP

• Verify precipitated protein size by Western blot (32 kDa)

• For stringent validation, use RNASEH1 knockout/knockdown samples as negative controls

How can RNASEH1 antibodies be used to study R-loop dynamics in genomic research?

RNASEH1 antibodies can be valuable tools for investigating R-loop formation and dynamics:

Chromatin immunoprecipitation (ChIP) applications:

• RNASEH1 ChIP can identify genomic regions where the enzyme is recruited, indicating potential R-loop formation sites

• ChIP-seq can map genome-wide RNASEH1 binding profiles

• Research shows RNASEH1 distribution along rDNA coincides with sites where R-loops accumulate

Co-localization studies:

• Combined IF with the S9.6 antibody (RNA-DNA hybrid marker) can identify co-localization patterns

• Triple staining with RNASEH1, S9.6, and topoisomerase 1 can reveal functional relationships in R-loop processing

Experimental approaches:

• Perform ChIP using validated RNASEH1 antibodies at 1:100 dilution

• Compare RNASEH1 binding profiles in wild-type versus cells under replication stress

• Validate findings using RNASEH1 knockout/knockdown models

• Examine how RNASEH1 and Top1 cooperatively suppress RNAP I transcription-associated R-loops

Research has shown that loss of either RNASEH1 or Top1 causes R-loop accumulation, with exacerbated effects when both proteins are depleted, suggesting partial functional redundancy in mammalian cells .

What is the role of RNASEH1 in antisense oligonucleotide (ASO) mechanisms, and how can antibodies help study this?

RNASEH1 is essential for the activity of antisense oligonucleotides designed to work via an RNase H1-dependent mechanism:

Key experimental findings:

• Studies with viable RNASEH1 knockout mice have unequivocally demonstrated that RNase H1 is necessary for ASO activity

• In both constitutive and inducible RNASEH1 knockout mice, ASOs targeting various mRNAs showed no activity, while they demonstrated potent activity in control mice

• This effect was observed both in isolated primary hepatocytes and in vivo following ASO administration

Research methodologies:

• Verify RNASEH1 expression levels using immunoblotting with specific antibodies

• Compare ASO efficacy in cells with normal versus reduced RNASEH1 levels

• Use immunofluorescence to track RNASEH1 localization changes upon ASO treatment

• Perform RNA immunoprecipitation to examine RNASEH1 interaction with ASO-targeted RNAs

Experimental design considerations:

• Include dose-dependent ASO treatments (100-300 mg/kg range for in vivo studies)

• Measure target mRNA levels 48 hours post-ASO treatment

• Include both wild-type and RNASEH1-depleted models for comparison

• Monitor both RNASEH1 levels and target mRNA reduction

This research definitively shows that despite earlier speculations about potential contributions from RNase H2 or Flap endonuclease 1 (FEN1), RNASEH1 is the essential enzyme for the activity of RNase H1-dependent ASOs .

How can the catalytically inactive form of RNASEH1 be used as a detection tool for RNA-DNA hybrids?

Catalytically inactive RNASEH1 (with D210N mutation) tagged with GFP (GFP-dRNH1) serves as a superior reagent for imaging RNA-DNA hybrids compared to the conventional S9.6 antibody:

Advantages over S9.6 antibody:

• GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides

• S9.6 antibody readily binds to double-stranded RNA in vitro and in vivo, creating nonspecific signals

• GFP-dRNH1 is not susceptible to binding endogenous RNA, providing cleaner results

Methodology for using purified GFP-dRNH1:

• Fix cells using methanol fixation

• Apply purified GFP-dRNH1 protein directly to fixed cells

• Wash thoroughly to remove unbound protein

• Visualize directly via GFP fluorescence (no secondary antibody needed)

• For validation, create control samples by treating with E. coli RNase H1 prior to staining

Experimental validation approach:

• Transfect fluorescently labeled 60-mer oligonucleotides (ssDNA, ssRNA, dsRNA, or RNA-DNA hybrids) into cells

• After fixation, compare binding patterns of GFP-dRNH1 versus S9.6 antibody

• Analyze co-localization of GFP-dRNH1 with the hybrid oligonucleotides but not with other nucleic acid types

This method bypasses the need for cell line engineering with transgenic constructs, making it applicable to a wide range of cell types and experimental systems .

What is known about RNASEH1 in mitochondrial function and associated diseases?

RNASEH1 plays a critical role in mitochondrial function and homeostasis:

Mitochondrial functions of RNASEH1:

• Required for effective removal of mitochondrial S7 R-loops

• Essential for transcription of mitochondrial DNA

• Prevents accumulation of potentially harmful RNA-DNA hybrids in mitochondria

Experimental evidence from knockout models:

• In RNASEH1 knockout mice, there is significant accumulation of S7 R-loops in the mitochondria

• This leads to failure to transcribe essential mitochondrial DNA

• The consequence is progressive mitochondrial dysfunction, loss of mitochondria, and ultimately apoptosis

Research methodologies:

• Measure S7 R-loop levels using PCR primers targeting CSB III or OH regions of mitochondrial genome

• Confirm specificity by treating samples with E. coli RNase H1 prior to immunoprecipitation

• Track mitochondrial function in RNASEH1-depleted versus normal cells

• Compare R-loop levels at different time points post-RNASEH1 depletion

Elevated levels of S7 R-loops were observed in hepatocytes from both tamoxifen-treated inducible knockout mice and constitutive knockout mice, confirming RNASEH1's essential role in mitochondrial R-loop processing .

How has RNASEH1-AS1 been implicated in cancer research?

RNASEH1-AS1 (antisense RNA 1) is a long non-coding RNA that has shown abnormal expression patterns in multiple cancer types:

Cancer associations of RNASEH1-AS1:

• Abnormally expressed in lung squamous cell carcinoma, ovarian cancer, and non-small cell lung cancer

• Significantly elevated in hepatocellular carcinoma (HCC) tissues and cell lines

• Its knockdown suppresses proliferation, migration, and invasion of HCC cells

Immune relationship findings:

• RNASEH1-AS1 expression is inversely associated with infiltration of most immune cell types

• Specifically shows negative correlation with plasmacytoid dendritic cells, B cells, and neutrophils

Research approaches and findings:

• Expression of RNASEH1-AS1 can be analyzed using RT-qPCR in patient tumor samples

• In silico analysis identified 1109 positively co-expressed genes of RNASEH1-AS1 in HCC

• Gene Ontology and KEGG analysis revealed these genes relate to RNA processing, ribosome biogenesis, transcription, and histone acetylation

• A risk prediction model based on four RNASEH1-AS1-related hub genes (EIF4A3, WDR12, DKC1, NAT10) showed good prognostic potential

While RNASEH1-AS1 research primarily focuses on the RNA rather than the protein, understanding its relationship with RNASEH1 protein expression and function represents an important avenue for future investigation.

What are common technical challenges when using RNASEH1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with RNASEH1 antibodies:

Western blot issues:

• Multiple bands: Optimize antibody dilution (try 1:5000 instead of 1:1000) and use freshly prepared samples with protease inhibitors

• Weak signal: Try more sensitive detection systems or concentrate protein samples

• High background: Increase washing steps and optimize blocking (5% BSA often works better than milk)

Immunofluorescence challenges:

• Weak nuclear staining: Try different fixation methods; methanol fixation may preserve epitopes better for some antibodies

• High cytoplasmic background: Increase washing duration and stringency

• Inconsistent results: Standardize cell culture conditions as RNASEH1 expression may vary with cell cycle phase

Immunoprecipitation difficulties:

• Low IP efficiency: Increase antibody amount (up to 4.0 μg for 1-3 mg lysate)

• Non-specific bands: Use more stringent washing conditions and pre-clear lysates thoroughly

• Degradation products: Add protease inhibitors and keep samples cold throughout the procedure

Validation approaches:

• Compare results with multiple antibodies targeting different RNASEH1 epitopes

• Include appropriate positive controls (HeLa, U2OS cells show reliable expression)

• Use RNASEH1 knockdown/knockout samples as negative controls when possible

• For critical experiments, confirm antibody specificity by peptide competition assay

How can I compare the specificity and reliability of S9.6 antibody versus RNASEH1-based methods for detecting RNA-DNA hybrids?

Comparative analysis between S9.6 antibody and catalytically inactive RNASEH1 (GFP-dRNH1) reveals important differences in specificity:

Experimental design for comparison:

• Transfect cells with fluorescently labeled nucleic acids (ssDNA, ssRNA, dsRNA, RNA-DNA hybrids)

• Fix cells using methanol fixation

• Apply both detection methods to parallel samples:

  • S9.6 antibody with fluorescent secondary antibody

  • Purified GFP-dRNH1 (direct detection via GFP)

• Compare co-localization patterns with the transfected nucleic acids

Key findings from comparative studies:

• S9.6 signal can be detected with both RNA-DNA hybrids and dsRNA oligonucleotides

• GFP-dRNH1 shows selective binding to RNA-DNA hybrids but not to dsRNA

• S9.6 background can arise from endogenous RNA binding

Validation methodology:

• Treat samples with E. coli RNase H1 prior to immunoprecipitation to digest RNA-DNA hybrids

• Compare signal before and after treatment to confirm specificity

• For S9.6, include RNase A treatment controls to eliminate dsRNA signal

Research demonstrates that while S9.6 has been the standard tool for R-loop detection, GFP-dRNH1 offers superior specificity, particularly in RNA-rich cellular environments .

How do RNASEH1 and topoisomerase 1 (Top1) cooperate in R-loop regulation?

Recent research has revealed an important functional relationship between RNASEH1 and topoisomerase 1 (Top1) in R-loop regulation:

Key experimental findings:

• Distribution of RNASEH1 and Top1 along ribosomal DNA (rDNA) coincides at sites where R-loops accumulate

• Loss of either RNASEH1 or Top1 individually causes R-loop accumulation

• The accumulation of R-loops is significantly exacerbated when both proteins are depleted

• Protein levels of Top1 are negatively correlated with RNASEH1 abundance, suggesting a compensatory mechanism

Research implications:

• RNASEH1 and Top1 are partially functionally redundant in suppressing RNA polymerase I transcription-associated R-loops

• Cells may maintain R-loop homeostasis through a balance between these two proteins

• Studying either protein in isolation may not provide complete understanding of R-loop regulation

Suggested experimental approaches:

• Double knockdown/knockout experiments to further explore the cooperative relationship

• ChIP-seq of both proteins to map genomic co-occupancy

• R-loop mapping in cells with different ratios of RNASEH1 and Top1 expression

• Investigation of potential physical interaction or complex formation between the proteins

This research highlights the importance of considering multiple R-loop processing factors when studying genomic stability mechanisms .

What are the emerging applications of RNASEH1 antibodies in therapeutic development research?

RNASEH1 antibodies have growing importance in therapeutic research, particularly in antisense oligonucleotide (ASO) development:

Key research applications:

• ASO mechanism validation:

  • RNASEH1 antibodies can confirm the mechanism of action for RNase H1-dependent ASOs

  • Research has definitively demonstrated that RNASEH1 is necessary for ASO activity

  • No ASO activity was observed in RNASEH1 knockout models, even at high doses (300 mg/kg)

• Therapeutic resistance investigation:

  • RNASEH1 antibodies can help assess whether altered RNASEH1 expression contributes to ASO resistance

  • Immunohistochemistry on patient samples can correlate RNASEH1 levels with treatment response

• Combinatorial therapy research:

  • Understanding RNASEH1 expression in relation to drug targets can inform combination strategies

  • RNASEH1 antibodies can monitor effects of treatment combinations on RNASEH1 levels

• Predictive biomarker development:

  • RNASEH1 expression levels could potentially serve as biomarkers for ASO therapy response

  • Standardized immunohistochemistry protocols using validated antibodies (e.g., 82771-1-RR at 1:1500-1:6000 dilution) could be developed for clinical use

Future research directions:

• Investigation of RNASEH1 polymorphisms and their impact on ASO efficacy

• Development of methods to enhance RNASEH1 activity in targeted tissues

• Exploration of novel ASO chemical modifications optimized for human RNASEH1 recognition