A Proximity Ligation-Based Method to Detect RNA-DNA Association

Abstract

We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the “RNA-DNA interaction assay” (RDIA), involves differentially labeling DNA and RNA with EdU and BrU, respectively. Once labeled, PLA is performed to assess if the labeled molecules are in close proximity. Here we provide a detailed description of the modified RDIA protocol utilizing currently commercially available BrdU antibodies. As an example, we show its ability to detect nascent transcripts on recently synthesized DNA in both cultured H1299 cells and mouse embryonic stem cells.

1. Introduction

Here we describe a protocol that utilizes two different molecular techniques: copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reactions and proximity ligation assays (PLA). CuAAC click reactions are a rapid, highly efficient method to conjugate terminal azide and alkyne moieties using a copper (I) catalyst. Because of its reproducibility and efficiency, this method has been widely adopted for bioconjugation experiments [1]. An example of the biological utilization of click chemistry is the method of assessing DNA replication by incubating cycling cells with 5-ethynyl-2′-deoxyuridine (EdU), which is incorporated into replicating regions of the genome. Since EdU contains an alkyne group, click chemistry can be used to conjugate other compounds containing azide functional groups, including fluorophores such as Alexa Fluor 488 azide, which allows for rapid fluorescent detection of replicating DNA [2].

Proximity ligation assays are an antibody technology that were initially developed to study protein-protein interactions [3, 4] These in situ assays utilize two primary antibodies from different species to detect two proteins in fixed cells or tissues. The primary antibodies are detected by secondary antibodies that have been conjugated with oligonucleotides (termed “PLA probes”). If the two proteins are in close proximity (less than or equal to 40 nm), then these probes can be bridged by linker oligonucleotides and ligated into a closed circle. This closed circle then undergoes rolling circle amplification (RCA) by incubation with a polymerase enzyme and the amplified, concatemeric region of DNA is subsequently detected by a fluorescent oligonucleotide. The power of these assays lies in their high sensitivity (10⁻⁴⁰ M), technical simplicity, and quantifiability.

Our lab combined PLA and click chemistry to track protein recruitment to nascent chromatin in a technique called the chromatin assembly assay (CAA; [5–9]) and more recently to understand nascent transcription on labeled DNA and transcript stability during mitosis using the RNA-DNA interaction assay (RDIA; [7, 10]). In these experiments, we utilized CuAAC to conjugate biotin (in the form of biotin azide) to EdU-labeled DNA and then performed PLA between biotin and proteins of interest (CAA) or between biotin and BrU-labeled RNA (RDIA). Here we describe in detail the RNA-DNA interaction assay (scheme in Fig. 1). This method labels DNA with EdU and then RNA with BrU. Following fixation, biotin is covalently attached to EdU using click chemistry and PLA is performed between anti-biotin antibody (to detect EdU) and anti-BrdU antibody (to detect BrU). Following PLA, biotinylated EdU is immunostained in order to identify replicating cells. In addition, cells can be immunostained to mark their identity or differentiation status. The RDIA allows the identification of the timing of transcriptional resumption following DNA replication, or assessment of different RNAs associated with DNA at other stages of the cell cycle including mitosis.

Fig. 1.

Scheme of RDIA. DNA is labeled with EdU (red) and RNA is labeled with BrU (blue). Biotin (purple) is covalently attached to EdU by two click reactions. PLA is then performed with an anti-BrdU antibody (to detect BrU) and an anti-biotin antibody (to detect EdU)

Since the initial iteration of this protocol we have made several adjustments to improve both its sensitivity and specificity. These alterations are mainly due to issues in specificity of anti-BrdU antibodies, which are notoriously promiscuous for other nucleotide analogs, including EdU [11]. While the experiments in Black et al. [7] utilized a robust antibody that happened to also be highly specific for BrU and not for EdU, we later switched to the MoBu-1 monoclonal antibody that was reported to be selective for BrdU by Liboska et al. [10, 11]. Recently we have encountered issues with this antibody and have thus further adapted the protocol to use a different anti-BrdU antibody by making several changes to ensure specificity of this antibody for BrU-labeled RNA. The most important of these changes was altering the timing of the click reaction from 30 min to two tandem 15-min click reactions. This modification improved the efficiency of EdU conjugation to biotin, thereby blocking it from detection by most commercially available anti-BrdU antibodies.

2. Materials

All solutions should be prepared with deionized water unless otherwise noted.

2.1. Tissue Culture and DNA and RNA labeling

1. Cell lines: H1299 (ATCC) and mouse embryonic stem cells (mESC; line E14TG2a, ATCC).

2. Cell culture medium: For H1299 cells supplement high-glucose DMEM with 12% fetal bovine serum (FBS) and 2 mM l-glutamine. For mES cells supplement high-glucose DMEM with 15% FBS, 1× nonessential amino acids, 2 mM l-glutamine, 10 mM HEPES, and 0.1 mM 2-mercaptoethanol. Additionally, freshly add 1000 U/mL LIF, 1 µM MEK inhibitor PD0325901, and 3 µM GSK3 inhibitor CHIR99021 to maintain mESC pluripotency.

3. 0.25% Trypsin-EDTA (with phenol red).

4. Chamber slides: Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber Slide™ System.

5. 5-Ethynyl-2′-deoxyuridine (EdU): Prepare a 1 mM stock in water. Aliquot and store at −20 °C in the dark.

6. 5-Bromouridine (BrU).

7. 37% Formaldehyde with 10–15% methanol for stabilization, molecular biology grade.

8. 20% Triton X-100 solution in water.

2.2. Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) (See Note 1)

1. Phosphate-buffered saline (PBS; 10×): Dissolve 80 g NaCl, 2 g KCl, 11.5 g Na₂HPO₄·7H₂O, and 2 g KH₂PO₄ in 800 mL of water. Adjust pH to 7.4 with 12 M HCl, bring the final volume to 1 L by adding water, and filter through a 0.22 µm filter. Store at room temperature (see Note 2).

2. 10% Bovine serum albumin (BSA) solution in water: Store at −20 ° C.

3. 0.1 M Copper (II) sulfate solution in water: Store at 4 °C in the dark for up to 4 months.

4. 1 mM Biotin azide: Dissolve 1 mg biotin azide (also known as PEG4 carboxamide-6-azidohexanyl biotin) in 1.62 mL of DMSO. Aliquot and store at −20 °C, protected from light. Avoid repeated freeze–thaw cycles.

5. 0.1 M l-Ascorbic acid sodium salt in water: Freshly prepare it, immediately prior to use in click reaction.

2.3. Immunofluorescence and Proximity Ligation Assay

1. ImmEdge pen (Vector Laboratories).

2. Western blocking reagent (10×; Roche).

3. Normal rabbit serum.

4. Primary antibodies: Rabbit anti-biotin (Abcam) and mouse anti-BrdU (clone: B44; BD Biosciences).

5. PLA probes: Duolink In Situ PLA Probe anti-rabbit minus and Duolink In Situ PLA Probe anti-mouse plus (Sigma Aldrich).

6. Duolink In Situ Detection Kit Reagent Red (Sigma Aldrich).

7. PLA buffer A (10×): Dissolve 88 g NaCl, 12 g Tris base, and 5 mL Tween 20 in 800 mL of water. Adjust the pH to 7.4 with 12 M HCl, and then add water to the final volume of 1 L. Store at 4 °C. For use, dilute to 1× with water (final concentrations 0.01 M Tris, 0.15 M NaCl, and 0.05% Tween 20), and bring to room temperature.

8. Secondary antibodies: Donkey anti-rabbit IgG Alexa Fluor 488 and mouse anti-biotin Alexa Fluor 488 (Jackson ImmunoResearch).

9. Vectashield Mounting Media with DAPI.

10. Coverslips: 24 × 50 mm, No. 1 thickness.

3. Methods

Both EdU and BrU are light sensitive, so keep the stock solutions protected from light as much as possible, and once samples have been labeled keep them covered for all incubation steps. Additionally, make sure to quickly change solutions between steps to prevent samples from overdrying because this will increase the number of nonspecific PLA signals. Please follow appropriate handling and disposal procedures for all chemicals.

Here we describe the protocol for adherent cells, using H1299 and mES cells as examples. We have also performed these experiments with suspension cells [10] and provided the appropriate variations for this type of culture in Subheading 4.

1. Seed chamber slides with cells so that in 1–2 days the slides will be 60–70% confluent (see Note 3). Make sure to have enough wells including two control samples: EdU-only and BrU-only.

2. Once cells have reached 60–70% confluency, label with EdU by collecting the cultured media from the wells and using it to dilute the stock EdU 1:100 (final concentration 10 µM). Add medium containing 10 µM EdU to chambers and place in cell culture incubator (37 °C with 5% CO₂) for 15 min (see Note 4).

3. Freshly prepare a 125 mM stock of BrU in 1× PBS (see Note 5).

4. Remove EdU-containing medium and add medium that has been supplemented with 1–5 mM BrU. Place in the incubator for 25 min (see Notes 5–7).

5. Prepare fixation solution by diluting 37% formaldehyde 1:10 in 1× PBS (final concentration 3.7%).

6. Remove medium, add 1× PBS to samples, immediately remove it, and add fixation solution. Cover samples and incubate for 12–15 min (see Note 8).

7. Remove fixation solution and wash samples with 1× PBS (three times, 5 min each).

8. Prepare permeabilization solution by diluting 20% Triton X-100 1:80 in 1× PBS (final concentration 0.25%). Remove PBS, add permeabilization solution to samples, cover, and incubate for 10 min at room temperature.

9. Remove permeabilization solution and wash with 1× PBS (three times, 5 min each).

10. Prepare 1% BSA by diluting 10% BSA 1:10 in 1× PBS. Remove PBS and incubate samples in 1% BSA for 5 min at room temperature.

11. Freshly prepare 0.1 M sodium ascorbate solution in water.

12. Prepare solution with click reaction in 1 mL aliquots; be sure to use solution with click reaction immediately after preparation. Combine the following reagents in the listed order: 10 µL 10× PBS, 865 µL water, 20 µL of 0.1 M copper (II) sulfate, 5 µL of 1 mM biotin azide, and 100 µL of 0.1 M sodium ascorbate (final concentrations 0.1 × PBS, 2 mM copper (II) sulfate, 5 µM biotin azide, 10 mM sodium ascorbate). Remove 1% BSA, add solution with click reaction, cover, and incubate for 15 min at room temperature (see Notes 9 and 10).

13. Prepare a second, fresh solution with click reaction. Remove first solution with click reaction and add fresh solution to samples for an additional 15 min at room temperature (see Note 11).

14. Remove solution with click reaction and wash with 1× PBS (three times, 5 min each).

15. Prepare blocking solution by diluting 10× Western blocking reagent 1:10 in 1× PBS and supplement with 1.5% normal donkey serum and 0.01% Triton X-100.

16. Remove chambers from the chamber slide and circumscribe each spot with the ImmEdge pen in order to sequester reagents on each individual sample. Incubate samples in blocking solution for 30 min at room temperature.

17. Prepare antibody diluent solution by diluting 10× Western blocking 1:10 in 1× PBS and supplementing with 0.01% Triton X-100. Dilute the following antibodies in antibody diluent: rabbit anti-biotin 1:1000 and anti-BrdU (clone B44) 1:10. Cover each spot on the slide with 30–35 µL.

18. Place in a humidity chamber and incubate overnight at 4 ° C in the dark.

19. The following morning thoroughly wash samples with 1× PBS (three times, 8 min each).

20. Prepare antibody diluent by diluting 10× Western blocking 1:10 in 1× PBS, and dilute Duolink In Situ PLA Probe anti-rabbit minus and Duolink In Situ PLA Probe anti-mouse plus each 1:5. Prepare enough solution to incubate each spot with 20 µL. Incubate in humidity chamber at 37 °C for 1 h.

21. Wash with 1× PLA Buffer A (three times, 5 min each).

22. Prepare ligation reaction by diluting 5× ligation buffer 1:5 in water and supplementing with 1 µL ligase per 40 µL of reaction. Prepare enough solution to incubate each spot with 20 µL. Incubate in humidity chamber at 37 °C for 30 min.

23. Wash with 1× PLA Buffer A (three times, 5 min each).

24. Prepare amplification reaction by diluting 5× amplification buffer 1:5 in water and supplementing with 1 µL polymerase per 80 µL of reaction. Prepare enough solution to incubate each spot with 20 µL. Incubate in humidity chamber at 37 °C for 100 min (see Note 12).

25. Wash with 1× PBS.

26. Prepare secondary antibodies by diluting donkey anti-rabbit IgG Alexa Fluor 488 1:1000 and mouse anti-biotin Alexa Fluor 488 1:1000 in 1× Western blocking in PBS. Incubate samples with secondary antibodies for 1 hour at room temperature in the dark.

27. Wash samples with 1× PBS (three times, 5 min each). Remove wash, add Vectashield mounting media, and place a coverslip on top of samples.

28. Visualize with a fluorescence microscope. DAPI can be visualized by a DAPI filter, EdU staining with Alexa Fluor 488 can be visualized by a FITC filter, and the PLA (red, 594 dye) can be visualized by a Texas Red filter (see Note 13). All PLA signals should be visualized in EdU-labeled nuclei. See Fig. 2 for sample images of RDIA in H1299 and mES cells.

Fig. 2.

RDIA in H1299 cells (a) and mES cells (b). PLA was performed following 15-min labeling with EdU followed by 25-min labeling with BrU (rightmost panels). To show specificity of PLA for labeled molecules additional controls included (from left to right) treatment with neither EdU nor BrU, 25-min BrU-only, and 15-min EdU-only. PLA (red), EdU (green), DAPI (blue). PLA only is shown in black and white below each composite image