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. Author manuscript; available in PMC: 2021 Jun 28.
Published in final edited form as: Methods Mol Biol. 2020 Jan 1;2166:473–486. doi: 10.1007/978-1-0716-0712-1_27

Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging

Manisha B Walunj, Seergazhi G Srivatsan
PMCID: PMC7611080  EMSID: EMS128228  PMID: 32710426

Abstract

Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which works under mild conditions and enables the direct installation of various biophysical reporters and tags. This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine-5′-triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/ ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.

Keywords: Nucleotide analog, Suzuki-Miyaura reaction, Bio-orthogonal reaction, Posttranscriptional chemical modification, RNA labeling, RNA imaging, Environment-sensitive probe

1. Introduction

Several chemoselective transformations that can be carried out under biologically benign conditions serve as valuable tools to label and study biomacromolecular structure and function in both cell-free and cellular environments [1–4]. Some of the prominent examples of chemical labeling approaches, which have been categorized as bio-orthogonal reactions, include azide-alkyne cycloaddition [5], Staudinger ligation [6], and inverse electron demand Diels-Alder reactions [7]. While these reactions have been conveniently implemented in labeling proteins, sugars, and DNA, their use in labeling RNA has not been very straightforward due to inherent low stability of RNA [8]. In this regard, we and other groups have designed posttranscriptional and chemo-enzymatic labeling approaches employing above-said bio-orthogonal reactions to functionalize [9–13] and image RNA in cells [14–19]. Nevertheless, new chemoselective reactions that can be tuned to work in aqueous and under milder conditions are constantly needed to expand the repertoire of functionalized RNA. In this context, palladium (Pd)-catalyzed C-C bond formation, which is a powerful reaction in synthetic organic chemistry, is proving useful as a chemoselective transformation for post synthetic labeling of biomolecules [20–23].

Like other methods, Pd-mediated cross-coupling reactions were first established on DNA and protein [20, 21]. Bromo- or iodo-modified DNA oligonucleotides (ONs) and proteins, prepared by chemical or enzymatic methods, are reacted with a cognate reactive handle tagged with a reporter in the presence of a water-soluble Pd-ligand catalytic system. The Manderville and Jaschke groups developed aqueous-phase Suzuki-Miyaura coupling reaction to functionalize DNA by reacting halogen-modified DNA ONs with boronic acid/ester substrates in the presence of Pd(OAc)2 and a water-soluble triphenylphosphane-3,3′, 3′-trisulfonate ligand [24, 25]. However, this catalyst-ligand combination requires elevated temperature (>70 °C), basic pH buffer conditions, and long reaction times to produce coupled ON products. Understandably, RNA will not survive these high temperature and basic pH buffer conditions. Meanwhile, Davis and Lin group formulated a Pd-ligand catalytic system to execute Suzuki and Sonogashira reactions, respectively, on proteins under very mild conditions [26, 27]. The catalytic system was made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), which enabled the direct incorporation of functional labels onto DNA ONs at room temperature and at a pH (8.5) slightly higher than physiological pH [28]. Encouraged by this report, we established a milder and efficient Suzuki-based labeling method to generate RNA functionalized with different biophysical reporters [29]. In this strategy, 5-iodouridine-5′-triphosphate (IUTP 1) was incorporated into RNA transcripts by in vitro transcription reaction catalyzed by T7 RNA polymerase. Iodo-labeled transcripts were then posttranscriptionally reacted with various cognate reactive partners (boronic acid/ester substrates) labeled with a desired biophysical reporter or tag in the presence of Pd(OAc)2 and ADHP or DMADHP complex. The reaction proceeds under benign conditions (37 °C and pH 8.5) and produces coupled RNA products in moderate to good yields. Taken together, this method is modular, and provides access to RNA labeled with (a) fluorogenic environment-sensitive nucleoside for probing nucleic acid structure and recognition, (b) fluorescent probes for microscopy, and (c) affinity tags for pull-down and immunoassays (Fig. 1). Further, metabolic labeling of nascent RNA with IU/IUTP followed by Pd-mediated coupling chemistry could be potentially used to image as well as profile cellular RNA. This notion is supported by the recent development of a Pd nanoparticle-based catalytic system, which enables the Suzuki reaction in the cellular environment [30].

Fig. 1.

Fig. 1

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Schematic diagram illustrating the incorporation of IUTP 1 into RNA transcripts by in vitro transcription followed by posttranscriptional functionalization using a Suzuki-Miyaura cross-coupling reaction to generate RNA labeled with different probes. This figure has been reproduced by permission of Nucleic Acids Research: Oxford Journals [29]

Here, we describe a detailed stepwise protocol to incorporate 5-iodouridine-5′-triphosphate into RNA transcripts during in vitro transcription using T7 RNA polymerase. Following this section, the preparation of water-soluble Pd-ligand catalysts, the setting up of posttranscriptional Suzuki reactions and the purification of coupled RNA products are illustrated in detail. Although we have performed coupling reactions with several substrates, emphasis is laid on preparing RNA labeled with fluorogenic environmentsensitive nucleoside probes (4 and 7), a fluorescent probe (9) suitable for imaging, and an affinity tag (10) suitable for pulldown and immunoassays (Fig. 2).

Fig. 2. (a) Chemical structure of boronic acid/ester substrates used in the posttranscriptional Suzuki-Miyaura coupling reactions. (b) Ligand L1 and L2.

Fig. 2

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2. Materials

Prepare all reagents, substrates, and buffer stocks in autoclaved water unless indicated otherwise. Store enzymes, DNA ONs, NTPs, and reagents in a deep freezer (–20 or –40 °C).

2.1. In Vitro Transcription

  1. 5x Transcription buffer: 200 mM Tris–HCl (pH 7.9), 30 mM MgCl2, 50 mM DTT, 50 mM NaCl, and 10 mM spermidine. This buffer is usually supplied along with T7 RNA polymerase.

  2. Annealing buffer: 10 mM Tris–HCl, 1 mM EDTA, 100 mM NaCl, pH 7.8. Weigh 12.11 mg of Tris base and 58.44 mg of NaCl in a 15 mL centrifuge tube. Add 8–9 mL of water and 20 μL of 0.5 M EDTA. Adjust the pH to 7.8 by adding 1 M HCl. Make the final volume to 10 mL using autoclaved water and check the pH again.

  3. 20 U/μL T7 RNA polymerase stock (see Note 1).

  4. NTPs minus UTP mix: 20 mM Stock solution in autoclaved water. Mix 20 μL of 100 mM stock solutions of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and adenosine triphosphate (ATP) in a 1.5 mL microcentrifuge tube. Make it to 100 μL by adding 40 μL autoclaved water. Vortex, centrifuge, and store the NTP-UTP mix at –40 °C.

  5. 20 mM 5-Iodouridine-5′-triphosphate (IUTP 1, ε260 = 3100 M–1 cm–1, can be purchased from a supplier): Prepare this stock solution in autoclaved water. Store the stock solution at –40 °C.

  6. 200 mM MgCl2: Weigh 40.66 mg of MgCl2 in a 1.5 mL microcentrifuge tube. Dissolve the solid in 1 mL of autoclaved water. Store the solution at –40 °C.

  7. 40 U/μL RiboLock (see Note 1).

  8. 1 M Dithiothreitol (DTT): Weigh 7.71 mg in 0.5 mL microcentrifuge tube and dissolve in 50 μL of autoclaved water (see Note 2).

  9. 5 μM Promoter-template DNA duplex: Transfer 5 μL of promoter DNA ON 11 (100 μM) and template DNA ON 12 (100 μM) into a 1.5 mL microcentrifuge tube containing 90 μL of the annealing buffer. Vortex the solution and centrifuge for 10 s. Anneal the ON mix by placing the centrifuge tube on the heating block at 90 °C for 3 min. Remove the heating block from the dry-block heater and allow it to cool to room temperature (RT). Place the annealed promotertemplate DNA duplex (final conc. 5 μM) in an ice bath for 30 min. Store the promoter-template duplex at –40 °C.

2.2. PAGE Equipment

  1. Gel electrophoresis equipment.

  2. Glass plates (45 cm x 20 cm, 4.8 mm thick).

  3. Power supply.

  4. 1.5 mm Spacer.

  5. 1.5 mm Comb.

  6. Plastic wrap (UV transparent).

  7. Sterile scalpel.

  8. Sterile glass rod.

  9. Poly-Prep columns (Bio-Rad Laboratories).

  10. Sep-Pak C18 cartridges (Waters Corporation).

  11. UV lamp.

  12. Rotary mixture.

2.3. PAGE Purification of IU-Labeled RNA Transcript

  1. 10 x TBE resolving buffer: 0.89 M Tris, 0.89 M boric acid, and 20 mM EDTA. Dissolve 108 g Tris base and 55 g boric acid in 800 mL autoclaved water. To this solution add 40 mL of 0.5 M EDTA (pH 8.0). Adjust the volume to 1 L with autoclaved water. Filter the buffer through 0.45 μm filter paper. Store the buffer at RT.

  2. Denaturing acrylamide/bis-acrylamide solution (19:1): Weigh 190 g of acrylamide and 10 g of bis-acrylamide in 1 L reagent bottle. Add autoclaved water to a volume of 300 mL. Further add 420 g of urea followed by addition of 100 mL 10 x TBE buffer. Stir the mixture vigorously using a magnetic stir bar. Make up the volume to 1000 mL with autoclaved water and filter using 0.45 μm filter paper. Store this solution at 4 °C protected from light (see Note 3).

  3. 10% (wt/vol) Ammonium persulfate (APS): Weigh 1 g of APS in 15 mL centrifuge tube and make the volume up to 10 mL by adding autoclaved water (see Note 4).

  4. N,N,N′,N′-tetramethylethylenediamine (TEMED).

  5. Denaturing loading buffer: 7 M Urea, 10 mM Tris–HCl, 100 mM EDTA, pH 8.0. Dissolve 4.2 g urea and 15.76 mg Tris–HCl in 5 mL of autoclaved water. Add 2 mL of 0.5 M EDTA (pH 8.0). Adjust the volume to 10 mL. Store the buffer at room temperature.

  6. 0.3 M Sodium acetate solution: Prepare 20 mL of 0.3 M sodium acetate solution in autoclaved water.

2.4. Posttranscriptional Suzukh-Miyaura Coupling Reactions

  1. 100 mM Stock solution of boronic acids/esters: Prepare 50 μL solution of each substrate by dissolving respective amount of boronic acid/ester substrates in BioUltra-grade DMSO (see Note 5). Vortex the solution, spin, and store at –40 °C (see Note 5).

  2. Pd-ligand catalyst (see Note 6): Add ADHP (65 mg, 0.5 mmol, 2 equiv.) or DMADHP (78 mg, 0.5 mmol, 2 equiv.) to a 5 mL volumetric flask containing 3 mL of autoclaved water. Add 100 μL of 10 mM NaOH (4 mole equivalents) to above solution. Stir the solution for 5 min at RT until all solids dissolve completely. Add 56 mg of Pd(OAc)2 (0.25 mmol, 1 mol equivalent) to the flask. Stir the mixture at 65 °C for 60 min. Allow the solution to attain RT. Remove the stir bar and adjust the volume to 5 mL with autoclaved water to make a 50 mM stock solution of the Pd-ligand catalyst. Store the catalyst at –40 °C. For cross-coupling reactions use 10 mM solution of the catalyst diluted using autoclaved water. Store 10 mM stock solution at –40 °C (see Note 6).

  3. 250 mM Tris–HCl reaction buffer (5x), pH 8.5: Weigh 303 mg of Tris base in 15 mL centrifuge tube. Dissolve Tris base in 8 mL of autoclaved water. Adjust the pH of the solution to 8.5 by adding 1 M HCl. Adjust the volume to 10 mL by adding autoclaved water and filter the buffer using 0.22 μm syringe filter. Store the buffer at room temperature.

2.5. HPLC Purification of Cross-Coupled RNA Products

  1. 1 M Triethylammonium acetate buffer (TEAA), pH 7.0: Take 139.4 mL of HPLC-grade triethylamine in 1 L reagent bottle. Add 700 mL of autoclaved water and stir the mixture on an ice bath. Add dropwise 57.2 mL HPLC-grade acetic acid with stirring. Adjust the pH of the solution to 7.0 and make the total volume to 1 L by adding autoclaved water. Check the pH again and store the buffer at 4 ° C. From this stock make 1 L of 50 mM solution of TEAA in autoclaved water. Filter the buffer through 0.22 μm filter paper prior to HPLC purification.

  2. Phenomenex-Luna C18 column (250 x 4.6 mm, 5 μm) or an equivalent column.

  3. Use spin filters (0.45 μm) to filter the samples before injecting on to the HPLC column.

  4. Mobile phase A: 50 mM TEAA buffer, pH 7.0.

  5. Mobile phase B: Acetonitrile. Filter the HPLC-grade acetonitrile through 0.22 μm filter paper.

3. Methods

3.1. Incorporation of IUTP into RNA Transcript by In Vitro Transcription Reaction (Fig. 3)

Fig. 3.

Fig. 3

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Incorporation of IUTP 1 by in vitro transcription reaction using T7 RNA polymerase, template DNA ON 12, and T7 promoter 11. The sequence of IU-modified RNA transcript 13 is shown, which upon posttranscriptional Suzuki coupling with boronic acid/ester substrates 4, 7, 9, and 10 gives coupled RNA ON products 14, 15, 16, and 17, respectively. Part of this figure has been reproduced by permission of Nucleic Acids Research: Oxford Journals [29]

  1. Thaw transcription buffer, reagents, and enzymes necessary for transcription reaction on an ice bath.

  2. Spin, vortex, and spin the buffer and reagents, and keep them on ice bath (see Note 1).

  3. Perform large-scale transcription reaction in 250 μL reaction volume. Pipette 72.50 μL autoclaved water and 50 μL transcription buffer (5x) into a 1.5 mL microcentrifuge tube.

  4. Add 25 μL of 20 mM NTPs-UTP mix, 25 μL of 20 mM IUTP, 17.5 μL of 200 mM MgCl2, 2.5 μL of 1 M DTT, and 15 μL of 5 μM promoter-template DNA duplex.

  5. Mix the above reaction mixture well with pipette and add 2.5 μL of 40 U/μL RiboLock.

  6. Initiate the transcription reaction by adding 40 μL T7 RNA polymerase (20 U/μL). Mix the reaction mixture by pipetting up and down few times.

  7. Incubate the reaction mixture at 37 °C for 6 h (see Note 7).

3.2. Preparative-Scale PAGE Purification of IU-Labeled RNA Transcript

  1. Mix 150 mL of 20% denaturing acrylamide/bis-acrylamide solution with 1.4 mL of 10% aqueous APS in a 250 mL Erlenmeyer flask and add 70 μL of TEMED.

  2. Pour the solution between the glass plates and insert a comb (1.5 mm thick with 4 wells). Polymerize for ~1 h.

  3. Rinse the wells thoroughly with Millipore or autoclaved water to remove unpolymerized acrylamide and place it on the electrophoresis apparatus.

  4. Fill the upper and lower chambers with 1x TBE buffer; pre-run the gel for at least 60 min at 25 W.

  5. In the meantime, reduce the reaction volume (see Subheading 3.1) to nearly 1/3 in a speed vac (e.g., Labconco). Add 50 μL denaturing loading buffer (see Note 8).

  6. After pre-run, turn off the power supply and wash the wells again thoroughly with 1 x TBE buffer.

  7. Load the samples on the preparative 20% denaturing polyacrylamide gel using pipette.

  8. Run the gel at constant 25 W for ~6 h.

  9. Turn off the power supply and carefully separate the gel from the plates. Cover the gel using plastic wrap.

  10. Mark the band corresponding to the full-length transcript by UV shadowing (254 nm). This is usually a slower migrating intense band. Cut this band using a sterile scalpel.

  11. Carefully transfer the gel pieces to a Poly-Prep column (BioRad) and crush the gel with a sterile glass rod into fine pieces.

  12. Add 3 mL of 0.3 M aqueous sodium acetate solution and cap the Poly-Prep column.

  13. Place the Poly-Prep column on a rotary mixer and allow the contents to mix well for ~12 h at RT.

  14. Centrifuge the Poly-Prep column for 10–15 min and collect the solution in a 15 mL centrifuge tube.

  15. Meanwhile condition the reversed-phase Sep-Pak column (Waters) by washing with 5 mL of acetonitrile and 15 mL of autoclaved water. The Sep-Pak column can be easily connected to a plastic syringe, which can be used for washing and loading the sample.

  16. Load the filtrate from step 14 onto the conditioned reversed-phase Sep-Pak column.

  17. Wash the column with 12 mL of autoclaved water and then elute the transcript with 4 mL of 40% (v/v) acetonitrile in autoclaved water. Collect ~1 mL fractions in 1.5 mL microcentrifuge tubes.

  18. Record the absorbance of the fractions at 260 nm to confirm the presence of the transcript.

  19. Evaporate the fractions containing the transcript to dryness. Dissolve the residue in a known volume of autoclaved water and determine the concentration using the molar extinction coefficient at 260 nm (ε260 = 84,300/M/cm) [29].

  20. Confirm the purity and identity of IU-labeled RNA transcript by HPLC and mass analysis. For details refer supporting information of reported literature [29].

3.3. Posttran-scriptional Suzuki-Miyaura Cross-Coupling (Fig. 3)

  1. Pipette 10 μL of 5 x Tris–HCl buffer and 7.5 μL DMSO into a 0.5 mL microcentrifuge tube. Add IU-labeled RNA ON 13 (9.1 μL of 550 μM stock, 5 nmol, 1 mol equivalent).

  2. To the above reaction mixture add boronic acid/ester (2.5 μL of 100 mM stock in DMSO, 50 equiv.). Add 19.9 μL of autoclaved water.

  3. Initiate the reaction by adding Pd(OAc)2(L1)2 or Pd (OAc)2(L2)2 catalyst (1 μL of 10 mM stock, 2 equiv.). The final reaction volume is 50 μL. The final concentration of RNA 13, boronic acid/ester substrate, catalyst, and DMSO% is 100 μM, 5 mM, 200 μM, and 20%, respectively (see Note 9).

  4. Incubate the reaction mixture at 37 °C for 6 h (see Note 10).

  5. Quench the reaction by freezing the sample at –40 °C.

3.4. HPLC Purification of Functionalized RNA Products

  1. Filter the reaction mixture and wash the spin filter with 40 μL of autoclaved water (see Note 11).

  2. Analyze the filtrate by RP-HPLC (see Note 12).

    Separate the cross-coupled product from the reaction mixture using following mobile phases and gradient: mobile phase A: 50 mM TEAA buffer (pH 7.0); mobile phase B: acetonitrile. Flow rate of 1 mL/min gradient: 0–30% B (100–70% A) in 35 min, 30–100% B (70–0% A) in 10 min, 100% B for 5 min, and 100% A for 5 min (see Note 13).

  3. Monitor the HPLC run at 260 nm and at the respective wavelength of each fluorophore (see Note 14).

  4. Collect the fraction corresponding to the coupled RNA product (see Note 14) and lyophilize the buffer. Add water to the residue and lyophilize again. This step removes the volatile TEAA buffer efficiently.

  5. Dissolve the residue in a known volume of autoclaved water and quantify the coupled RNA product by measuring the absorbance. Refer reported literature for the molar extinction coefficient of each cross-coupled product [29] (see Note 15).

  6. Confirm the identity of coupled RNA products by mass spectroscopy (see Note 16). If the product is fluorescent, then analyze its emission properties. For example, see Figs. 4 and 5 for the fluorescence profile of coupled RNA products, which are labeled with a fluorogenic dye, environment-sensitive fluorescent nucleoside probe [31, 32], and fluorescence microscopy-compatible NBD dye [33].

Fig. 4.

Fig. 4

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Top: Image showing the fluorogenic Suzuki coupling of IU-labeled transcript 13 with boronic ester 7 and the reaction product 15. The samples were irradiated using 365 nm light source. Bottom: Emission spectrum of boronic ester 7 and coupled RNA product 15. CPS = counts per second. Part of this figure has been reproduced by permission of Nucleic Acids Research: Oxford Journals [29]

Fig. 5.

Fig. 5

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(a) Suzuki reaction on IU-labeled RNA ON 13 using 2 equiv. of Pd catalyst and 50 equiv. of NBD boronic ester 9. The reaction mixture was resolved by PAGE under denaturing conditions and UV shadowed. UV shadow of the gel at 254 nm (left) and at 365 nm (right) shows the formation of dye-labeled RNA product 16. (b) Emission spectrum of the NBD-modified RNA product 16. The sample was excited at 480 nm with an excitation and emission slit width of 8 nm and 10 nm, respectively. Part of this figure has been reproduced by permission of Nucleic Acids Research: Oxford Journals [29]

Acknowledgments

This work was supported by Wellcome Trust-DBT India Alliance (IA/S/16/1/502360) grant to S.G.S. M.B.W. thanks CSIR, India, for a graduate research fellowship. The authors wish to thank Arun Tanpure for discussion and help with work that has led to the optimization of this protocol.

Footnotes

1

Thaw T7 RNA polymerase and RiboLock on an ice bath. Centrifuge the enzymes for few seconds (3–4 s). Do not vortex the enzyme solutions as they may denature.

2

Make aliquots of 20 μL of 1 M DTT solution and store at –20 or –40 °C. Do not use DTT that is freeze-thawed more than two times.

3

Acrylamide is highly toxic. Always wear protective gloves while handling acrylamide. The acrylamide solution can be stored at 4 °C for a month; however, it hydrolyzes to acrylic acid and ammonia if kept for longer period of time.

4

Use freshly prepared APS for efficient polymerization. If APS solution is old, the efficiency of polymerization will dramatically reduce. Store APS solution at 4 °C.

5

Boronic acid substrate 4 can be purchased from a supplier, and substrates 7, 9, and 10 can be prepared as has been reported [29]. Avoid prolonged exposure to light, in particular fluorescent NBD boronic ester 9. Store the stock solutions in aluminum-wrapped centrifuge tube or in amber-colored plastic/glass vials.

6

ADHP (L1) can be purchased from a supplier. DMADHP (L2) can be synthesized as described [34]. The catalyst is stable when stored at –20 or –40 °C. Precipitation was observed when stored at RT for more than a month. We preferred to use freshly prepared 10 mM solution from 50 mM stock solution stored at –40 °C.

7

We have observed deiodination of the IU-labeled transcripts if the transcription reaction is performed for longer time. 6 h of incubation time gave 10–12 nmol of the IU-labeled transcript without detectable deiodination of the RNA transcript. So we recommended shorter reaction time (e.g., 6 h) and wrapping the microcentrifuge tube with aluminum foil.

8

We preferred not to add bromophenol blue (BPB) in the loading buffer so as to avoid any adventitious contamination of BPB in the transcript. To track the electrophoresis, we loaded an extra lane with BPB in the same denaturing buffer.

9

After optimization, we found that this concentration and ratio of IU-labeled RNA 13, boronic acid/ester substrate, and catalytic system gave the best results.

10

Depending on the boronic acid/ester substrate the reaction time and yield could differ.

11

Filter the reaction mixture before injecting into the HPLC column. Otherwise fine particles of Pd formed in the reaction could affect the performance of the HPLC column.

12

We preferred HPLC purification of Suzuki-coupled RNA products as opposed to gel electrophoretic purification because it was observed that Pd chelated to the RNA was not easily removable by PAGE. However, with TEAA buffer system we were able to successfully isolate the pure coupled products by HPLC.

13

In our experiments, using the substrates listed in Fig. 2, we used the conditions given in Subheading 3.4. These conditions provide good separation between the coupled RNA product, IU-labeled RNA substrate, deiodinated RNA, and boronic acid/ester substrates. However, the HPLC purification method could vary depending on the substrate and product, and hence the readers are recommended to optimize the purification conditions.

14

Apart from monitoring the run at 260 nm (λmax of RNA), it would be useful to monitor HPLC chromatogram at the characteristic absorption wavelength of the probe attached to boronic acid/ester. A peak corresponding to the retention time that absorbs at 260 nm and also at the characteristic wavelength of the probe indicates that the fraction corresponds to the coupled RNA product.

15

Coupling of boronic esters 6 and 7 with RNA transcript 13 using catalyst Pd(OAc)2(L1)2 gave two regioisomers (major trans and minor cis) of the RNA product [29]. When Pd (OAc)2(L2)2 was used a single isomer (trans) was formed. However, the overall yield of the coupled RNA product was better when the reaction was performed using Pd(OAc)2(L1)2. Therefore, depending on the boronic acid/ester substrate, we recommend the users of this protocol to optimize the conditions in terms of reaction time and type of catalyst that can be used.

16

Mass of oligonucleotides can be measured using Applied Biosystems 4800 Plus MALDI TOF/TOF analyzer.

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