Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes

Summary

This protocol enables the isolation of DNA:RNA hybrids and their associated RNA (including TERRA) across multiple tissues. While our approach captures a subset of hybrid interactions, additional genomic mapping would be required for full genome-wide characterization.

Abstract

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from all telomeres in higher eukaryotes, and only a subset of these molecules forms stable DNA/RNA hybrids at telomeres. We have outlined a detailed molecular protocol to identify and purify these hybrid estimates. This streamlined method enables the direct extraction of DNA/RNA hybrid interactions naturally formed in vivo and can be applied to detect all such regions. By minimizing procedural steps, this technique is highly efficient in isolating RNA attached to DNA, estimating the reliability and reproducibility of downstream sequencing. Guanidine isothiocyanate reagent is used for total cell lysis and homogenization, preserving the integrity of nucleic acids (DNA and RNA), particularly DNA/RNA hybrids. Adding chloroform initiates phase separation via centrifugation (three distinct layers): a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA, released from both the nucleus and cytoplasm, is retained in the aqueous phase, while high-molecular-weight DNA, including the DNA/RNA hybrid regions, is confined to the interphase. With isopropyl alcohol, followed by centrifugation, the aqueous phase (free RNA), interphase, and DNA, particularly DNA/RNA hybrids, are recovered. To remove the remaining traces of proteins, the sample is incubated with Proteinase K. Treatment with DNase, phenol/chloroform extraction, isopropyl alcohol, and centrifugation is used to release RNA hybridized to DNA.

Introduction

Transcription of the genome results in the formation of temporary hybrid molecules, known as R-loops, which consist of three-stranded structures. These structures include the non-coding and coding strands of DNA, with the non-coding strand displaced by the formation of hybrids with the complementary RNA¹. During genome transcription, a large number of non-coding RNAs are produced at varying rates from both DNA strands. Most of these RNAs serve as short-lived intermediates, but some play critical roles in the regulatory networks governing genome biology, particularly those originating from non-coding regions such as telomeres and centromeres¹. The distribution of DNA/RNA hybrids in the genome may also reflect cells' transcriptional activity at any given time.

As interest grows in the regulatory functions of non-coding RNAs, there is increasing curiosity to track their transcription and distribution across the genome, particularly in the form of temporary DNA/RNA hybrids. To address this, we developed an independent assay for the detection of stable DNA/RNA hybrids, applicable to any biological cell or tissue. This method enables the isolation of complementary RNAs that co-purify with the DNA, followed by analysis via PCR precipitation or sequencing.

Research from cell culture models established that during DNA replication and transcription, replication stress, DNA damage, replication fork collisions, and RNA polymerase II pausing can occur due to topological, structural, and hybridization events. These processes, particularly the unwinding of the DNA double helix, introduce torsional stress, which can lead to the formation of abnormal structures, such as R-loops, during transcription¹.

Most R-loop data have been generated using yeast or cultured cancer cells, relying on the DNA-RNA hybrid-specific monoclonal antibody S9.6. Structural studies have shown that the antigen-binding fragment (Fab) of S9.6 binds specifically to a 13 bp RNA-DNA hybrid duplex¹. Antibody-based detection of R-loops has primarily identified hybrids that form in guanine-rich regions during transcription, where enhanced RNA-DNA hybridization is likely to occur. In vitro studies using the S9.6 antibody suggest that RNAs containing four or more consecutive guanines near their 5′ end are more readily detected, correlating with increased R-loop formation¹. Various enzymes, such as helicases, topoisomerases, RNase H1, and RNase H2, help maintain R-loop balance during transcription. Disruption of R-loop homeostasis is linked to increased genomic instability, contributing to diseases like cancer and neurodegenerative disorders².

Telomeres, once believed to be transcriptionally silent, have been found to transcribe long non-coding RNAs known as Telomeric Repeat-Containing RNA (TERRA)³. These transcripts originate from subtelomeric regions and contain telomeric repeats, playing essential roles in maintaining telomere homeostasis⁴. In this manuscript, we refer to 'TERRA-hybrid RNA' as the RNA molecules released from DNA/RNA hybrid complexes via DNase I treatment, presumed to include TERRA sequences based on extraction properties and primer specificity.

TERRA transcription occurs in the centromere-to-telomere direction and is variable in length, ranging from 100 bp to 9 kb, with the majority of TERRA being recovered in the aqueous phase as a free RNA molecule. A small fraction remains attached to the DNA and is associated with key biological processes, such as cancer and aging^4,5. TERRA levels vary depending on the subtelomeric loci and are regulated by the heterochromatic state of telomeres⁶. Cell culture studies also indicate that TERRA transcripts from various subtelomeric regions exhibit heterogeneity in length. In some tumor-derived cell culture studies, researchers have found that the 20q subtelomeric region is the primary source of TERRA transcripts, and this region is often methylated at its CpG islands^7,8,9. In mice, the subtelomeric region of chromosome 18 is a major site of TERRA production¹⁰,¹¹. Additionally, PAR-TERRAs, which are TERRA transcripts originating from pseudoautosomal subtelomeric regions in embryonic cells, were shown to have up to 200x more TERRA compared to chromosome 18^11,12.

The transcription of TERRA is controlled by DNA methylation and histone modifications such as H3K9 and H4K20 trimethylation, which suppress TERRA, while histone acetylation positively correlates with TERRA transcription^6,13,14. TERRA also binds to extratelomeric regions, including intergenic sites and introns, indicating a broader role in genome regulation².

Additionally, the depletion of TERRA leads to dysregulation of numerous genes, particularly those near its binding sites, suggesting its role in epigenetic regulation of chromatin and gene expression². TERRA accumulation at shortened telomeres promotes homologous recombination (HR)-based telomere extension, contributing to telomere length homeostasis¹⁵,¹⁶.

To address the role of TERRA and other DNA/RNA hybrids, we employed an assay specifically designed to isolate RNA molecules stably hybridized to DNA. Briefly, nuclei were isolated and treated with DNase-free RNase to remove unprotected RNA. DNA/RNA hybrids were then extracted using selective DNase digestion, which releases RNA originally hybridized to DNA. This fraction, referred to as "DNA/RNA hybrid-associated RNA," was subsequently analyzed by RNA-seq and compared to total soluble RNA obtained by standard phenol-chloroform extraction¹⁷. This approach allowed us to identify both telomeric and non-telomeric sites of RNA-DNA hybrid formation, and to compare the transcriptomic profiles of DNA-bound versus soluble RNA pools. Once considered merely a byproduct of telomere transcription, TERRA is now recognized for its dual role in telomere maintenance, influencing telomerase activity and homologous recombination, which are crucial for telomere length homeostasis. Moreover, advancements in detection assays have enabled a more detailed understanding of the dynamic interactions between RNA and DNA across the genome. This knowledge paves the way for further exploration of the implications of DNA/RNA hybrids, such as TERRA, in cellular processes and their potential links to diseases such as cancer and neurodegenerative disorders. The ongoing research in this field will undoubtedly deepen our understanding of genomic regulation and its relevance to health and disease.

The method used to identify TERRA molecules and DNA/RNA hybrids in this study is the same optimized guanidinium thiocyanate-phenol-chloroform extraction protocol detailed herein. This protocol was developed to allow the concurrent isolation of RNA-DNA/RNA hybrids, total RNA, and genomic DNA within a unified workflow. By strategically modifying the classical three-phase separation method, this protocol facilitates the selective retention of DNA/RNA hybrids at the phenol interphase, thereby minimizing their loss-an inherent limitation of conventional extraction techniques.

Traditionally, the detection and isolation of DNA/RNA hybrids have relied on immunoprecipitation with the S9.6 monoclonal antibody, which specifically recognizes DNA/RNA hybrid structures^18,19. This S9.6-based approach, although widely used, can be limited by antibody accessibility, cost, and potential cross-reactivity. In contrast, our protocol offers a cost-efficient, antibody-independent alternative that is readily applicable to both cellular and tissue-derived samples.

To further enhance the purity and structural integrity of the isolated DNA/RNA hybrid fraction, we incorporated proteinase K-mediated deproteinization, followed by DNase I treatment. The DNase I step selectively digests the DNA strand of the DNA/RNA hybrids, thereby releasing the hybridized RNA for subsequent isolation and downstream molecular analysis²⁰. This approach ensures that the RNA species recovered are specifically those that were stably hybridized to DNA in vivo. Owing to its modular configuration and scalability, our protocol is broadly adaptable to diverse biological contexts, providing researchers with a robust tool for interrogating DNA/RNA hybrid biology in a wide array of experimental systems.

Protocol

All experimental protocols involving animals were approved by the Erciyes University Animal Ethics Committee (license number 19-07-2019, decision no. 19/127). Balb/c mice were used in this study: five healthy females, 6 weeks of age (25 g), and five males, 8 weeks of age (30 g). Mice were cared for and treated according to the Principles of Laboratory Animal Care (European rules). The study presented here utilized blood samples from 13 healthy control subjects, derived from a previous research project that received ethical approval from the Erciyes University Human Ethics Committee (Decision No: 17/002). A graphical abstract for this protocol is provided in Figure 1. Use RNase-free materials and aseptic techniques throughout all steps to minimize RNA degradation.

1. Mouse husbandry

1. To determine the appropriate anesthetic dosage needed for euthanasia, weigh each mouse and administer ketamine at a dose of 60 mg/kg and xylazine at 10 mg/kg via intraperitoneal injection.
2. After administering anesthesia, confirm full anesthesia by the absence of the pedal withdrawal reflex (toe pinch) and lack of response to tactile stimulation. Once anesthesia is confirmed, perform euthanasia by cervical dislocation.

2. Human samples

1. Select participants as healthy controls after screening them to exclude any history of chronic diseases (such as diabetes, cardiovascular disease, hypertension, or cancer), acute infections, autoimmune or inflammatory disorders, neurological or psychiatric conditions, and recent use of prescription medication¹⁷.
2. Collect skin biopsy samples from the area behind the knee (popliteal fossa) of each volunteer using a 2 mm punch tool. Administer local anesthesia subcutaneously prior to the procedure, and perform all biopsies under sterile conditions.

3. DNA/RNA hybrid extraction from all cell types

1. Tissue collection and lysis
   1. For each extraction, use 500 µL of heart blood, whole hypothalamic-pituitary complex, whole adrenal gland, or collected sperm sample from a single mouse.
   2. Transfer all collected samples into sterile 1.5 mL tubes. Add 500 µL of phenol to each tube. Vortex blood samples thoroughly to mix.
      1. For tissue samples, homogenize completely using a 2 mm syringe until no visible fragments remain.
   3. Place the tubes on a cooling block to maintain sample integrity.
      NOTE: Always follow laboratory safety guidelines when handling hazardous reagents such as phenol, chloroform, and ammonium acetate.
2. Phase separation
   1. Add 180 µL of chloroform to each tube and vortex briefly to mix. Incubate tubes on ice for 15 min to allow phase separation.
   2. Inspect the samples-ensure the aqueous phase appears clear. If the phase is cloudy, vortex again and return to ice for an additional 5 min.
   3. Centrifuge samples at 12,000 × g, 4 °C for 15 min.
3. RNA precipitation
   1. Carefully transfer the clear aqueous phase to a new sterile 1.5 mL microcentrifuge tube placed on a cooling block.
   2. Add an equal volume (1:1) of isopropanol to the aqueous phase, vortex briefly to mix, and incubate on ice for 15 min.
   3. Centrifuge at 12,000 × g, 4 °C for 15 min.
      NOTE: The RNA pellet should be visible at the bottom of the tube.
4. Washing the RNA pellet
   1. Carefully invert the tube to discard the supernatant without disturbing the pellet.
   2. Add 1.5 mL of 70% ethanol to the pellet. Gently tap the tube for 5 s to wash.
   3. Centrifuge at 7,500 × g, 4 °C for 5 min.
   4. Invert the tube to discard the supernatant. Repeat the ethanol wash 2x (for a total of three washes).
5. RNA purification
   1. After the final ethanol wash, invert the tube and allow any residual ethanol to air dry or carefully remove with a pipette.
   2. Add 100 µL of RNase-free water to the RNA pellet and gently pipette up and down or flick the tube to fully resuspend the RNA.
      NOTE: Always keep RNA samples on ice or a chilled rack/cooling block to prevent degradation.
6. RNA quantification and storage
   1. Place the tube containing resuspended RNA on ice.
   2. Measure RNA concentration and purity using a spectrophotometer. Record the absorbance at 260 nm (A260) to determine RNA concentration. Assess purity by calculating A260/A280 and A260/A230 ratios.
   3. Use only RNA samples with A260/A280 ratios between 1.9 and 2.1 for downstream applications.
      NOTE: In this study, typical RNA concentrations ranged from 100 to 180 ng/µL.
   4. Store RNA samples at −80 °C until further use.
7. Protein removal and isolation of DNA
   1. After RNA isolation, retain the interphase from the phenol extraction step.
      NOTE: This step is necessary because the interphase contains DNA, which can be further processed for DNA isolation following phenol addition and centrifugation.
   2. Proceed with DNA extraction by following steps 3.2.1-3.4.1, beginning with the interphase as the starting material. Carefully transfer the entire interphase (containing DNA) from the phenol extraction to a sterile 1.5 mL microcentrifuge tube kept on a cooling rack.
   3. Protein digestion: Add 0.5 mL of lysis buffer (20 mM Tris-HCl, pH 8.0; 100 mM EDTA; 0.1% SDS; 400 µg/mL Proteinase K) to the tube. Incubate overnight at 56 °C to digest proteins.
   4. Salt precipitation of DNA
      1. The following day, add 100 µL of 5 M ammonium acetate to each sample and mix briefly. Centrifuge at 1,788 × g for 20 min at room temperature.
      2. Carefully transfer the clear aqueous phase to a new sterile 1.5 mL microcentrifuge tube. Add an equal volume of molecular biology-grade isopropanol (1:1 ratio) to the aqueous phase, mix by gentle inversion, and centrifuge at 1,788 × g for 10 min at room temperature. Discard the supernatant without disturbing the DNA pellet.
      3. Wash the DNA pellet with 1 mL of 70% ethanol, then centrifuge at 1,788 × g for 10 min.
      4. Remove and discard the ethanol wash. Allow the DNA pellet to dry air briefly.
      5. Resuspend the dried DNA pellet in 50 µL of Nuclease-free water.
8. Remove free nucleic acid to clean up DNA:
   1. Load the dissolved DNA onto a DNA spin column. Centrifuge at 15,000 × g for 15 s, and discard the flowthrough.
   2. Wash the column 2x with 70% ethanol, centrifuging each time for 1 min at 15,000 × g. Discard the flowthrough after each wash.
   3. Transfer the spin column to a clean 1.5 mL collection tube. Add 30 µL of nuclease-free water to the column matrix, incubate at room temperature for 5 min, then centrifuge for 1 min at 15,000 × g to elute DNA.
   4. Place the eluted DNA on ice. Divide the sample into two tubes: one for DNA storage at 4 °C, the second aliquot for subsequent DNA/RNA hybrid extraction.
9. Extraction of RNA attached to DNA (DNA/RNA Hybrids)
   1. DNase treatment
      1. Prepare a DNase digestion mixture by combining 5 µL (15 U) of DNase I and 75 µL of 10x DNase digestion buffer.
      2. Add 80 µL of the DNase mixture to the dried DNA pellet. Incubate the sample at room temperature for 15 min.
   2. Phenol-chloroform extraction
      1. Add 300 µL of phenol to the sample and incubate at room temperature for 5 min.
      2. Add 180 µL of chloroform to the mixture and incubate for an additional 5 min at room temperature.
      3. Centrifuge at maximum speed (e.g., 15,000 × g) for 10 min at 4 °C to separate the phases. Carefully transfer the upper aqueous phase (containing RNA:DNA hybrids) to a new tube.
10. DNA/RNA hybrid isolation
    1. Complete the isolation of DNA/RNA hybrids from steps 3.3 to 3.5.2.
    2. Add an equal volume (1:1) of isopropanol to the aqueous phase, vortex briefly to mix, and incubate on ice for 15 min.
    3. Centrifuge at 12,000 × g, 4 °C for 15 min. Look for the RNA pellet at the bottom of the tube. Carefully invert the tube to discard the supernatant without disturbing the pellet.
    4. Add 1.5 mL of 70% ethanol to the pellet. Gently tap the tube for 5 s to wash. Centrifuge at 7,500 × g, 4 °C for 5 min. Invert the tube to discard the supernatant. Repeat the ethanol wash 2x (for a total of three washes).
11. RNA purification:
    1. After the final ethanol wash, invert the tube and allow any residual ethanol to air dry or carefully remove with a pipette.
    2. Add 30 µL of RNase-free water to the DNA/RNA hybrid pellet and gently pipette up and down or flick the tube to fully resuspend the RNA.
    3. Store DNA/RNA hybrid samples at −80 °C until further use.

4. cDNA synthesis

NOTE: Ensure RNA, cDNA, and DNA integrity by using RNase/DNase-free materials and keeping samples on ice throughout the procedure.

1. For each sample, use 6 µL (20 ng of RNA; as isolated in Step 3.6.5 for free RNA or Step 3.13.3 for DNA/RNA hybrid RNA). Transfer the required volume of RNA into labeled PCR tubes.
2. Prepare the cDNA synthesis master mix according to the kit manufacturer's instructions. For each reaction, ensure that the master mix contains reverse transcriptase, dNTPs, primers, buffer, and RNase inhibitors. For each reaction, combine 14 µL of master mix with 6 µL of the RNA sample in a PCR tube (total volume: 20 µL per reaction). Include at least one no-reverse transcriptase (-RT) control by preparing a parallel reaction without adding reverse transcriptase enzyme, using the same amount of RNA.
3. Perform cDNA synthesis in a thermal cycler using the temperature program specified in Table 1.
4. Dilute the resulting cDNA 1:5 with nuclease-free water and store at −80 °C until qPCR analysis.

5. Determination of TERRA expression levels via Q-PCR

1. Prepare the qPCR reaction mixture using a SYBR Green-based qPCR kit, following the manufacturer's instructions. For each reaction, combine 1 µL of SYBR Green Master Mix, 0. µL of forward primer for TERRA (see Table 2), 0. µL of reverse primer for TERRA (see b), and  µL of nuclease-free water.
2. Dispense 16 µL of the reaction mix into each well of a 96-well qPCR plate.
3. Add 4 µL of diluted cDNA (from Step 4.6) to each well, for a final reaction volume of 20 µL.
   NOTE: For each sample, include at least two technical replicates and two biological replicates. Include no-template controls (NTCs) to check for contamination and no-reverse transcriptase controls to ensure specificity for RNA-derived signals.
4. Perform qPCR using the temperature program specified in Table 3.
5. Normalize TERRA expression to β-Actin (housekeeping gene) using the 2^-ΔΔCt method.

6. Determination of telomere length

1. Prepare the qPCR mixture for telomere length measurement ²¹.For each reaction, combine 1 µL of SYBR Green Master Mix, 0. µL of telomere forward primer (see Table 2]), 0. µL of telomere reverse primer (see Table 2), and  µL of nuclease-free water.
2. Dispense 16 µL of the telomere reaction mix into each well of a 96-well qPCR plate.
3. Add 4 µL of genomic DNA (from Step 3.10.5) to each well, for a final reaction volume of 20 µL.
4. Perform qPCR according to the appropriate program parameters specified by the manufacturer (see Table 3).
5. In parallel, set up reactions for a single-copy reference gene (36B4) using the same concentrations and final volume.
6. Standard curves were generated using serial dilutions of a synthetic telomeric oligonucleotide (Table 2) to estimate relative abundance and amplification efficiency. Absolute quantification was not performed. The relative telomere length is calculated as the T/S ratio (telomere repeat copy number to single-copy gene number) using the ΔCt method²¹.

Results

In this study, we quantified and compared the levels of free TERRA, DNA/RNA hybrid TERRA, and telomere length across multiple mouse tissues (blood, sperm, skin, hypothalamus, pituitary, adrenal) as well as in human blood and skin samples, to investigate tissue-specific dynamics and the relationship to telomere biology. Free TERRA was isolated from the aqueous phase following phenol-chloroform extraction (protocol steps 3.6.1-3.6.5), enriching for cytoplasmic and nucleopla...

Discussion

A central strength of the protocol presented here lies in its stepwise extraction and discrimination of free TERRA and DNA/RNA hybrid TERRA from complex tissue samples. Critical steps include the careful separation of the aqueous and interphase fractions during phenol-chloroform extraction, as cross-contamination can confound the distinction between free and hybrid TERRA. The subsequent DNase I treatment of the interphase fraction is also pivotal: insufficient digestion may leave DNA-bound RNA inaccessible, while overdig...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	Greiner	671201
15 mL Falcon Tubes	Isolab	078.02.001
2 mL Syringe	Hayat	N/A
96- Multiwell plate	Greiner	50-720-3203
Absolute Ethanol	Merck	1070172511
Amonnium Acetate	Sigma	238074-500G
cDNA Synhesis Kit	Roche	07 912 439 001
Chloroform	Merck	1024451000
DNAse I Kit	Zymo	E1010
EDTA	Sigma	E9884-1KG
Glass bottle	Isolab	LB.IS.061.01.901
Graduated cylinder	Isolab	015.01.901
Guanidine isothiocyanate	Sigma	G9277-100G
Isoproponal	Merck	1096342511
Light Cycler 480 II	Roche	5015278001
Microcentrifuge Tubes (1.5 mL)	Greiner	616201
Micropipettes (10 µL)	Gilson	FD10001
Micropipettes (100 µL)	Gilson	FD10004
Micropipettes (1000 µL)	Gilson	FD10003
Micropipettes (2 µL)	Gilson	GFAM00064
Nanodrop	Shimadzu	C101-E112
PBS Tablet	Sigma	P4417-50TAB
PCR Device	Sensoquest	T3-0140
PGL3 Basic Vector	Promega	#212936
Pipette tips (10 µL, 200 µL, 1000 µL)	Greiner	772352
Plastic Petri dishes	Greiner	627102
Proteinase K	Sigma	P6556-10MG
SDS	Sigma	8170342500
Serological Pipette	Greiner	760107
Spin	Gilson	PMC880
Sybr Green Master	Roche	4707516001
Thermomixer	Eppendorf	#5355
Trizma base	Sigma	1503-1KG
Trizol	Biorad	7326890
Unimax 1010 DT Shaker	Heidolph	543-12310-00-6
Vortex	Heidolph	541-10000-00-1
Zymo-Spin I Columns	Zymo	C1003-50