Synthesis of DNA and RNA Oligonucleotides Containing a Dual-Purpose Selenium-Modified Fluorescent Nucleoside Probe

Abstract

Development of efficient tools that would enable direct correlation of nucleic acid structure and recognition in solution and in solid state at atomic resolution is highly desired. In this context, we recently developed dual-purpose nucleoside probes made of a 5-selenophene-modified uracil core, which serves both as a conformation-sensitive fluorophore and as an X-ray crystallography phasing agent. In this article, we provide a detailed synthetic procedure to synthesize the phosphoramidites of 5-selenophene-modified 2’-deoxyuridine and 5-selenophene-modified uridine analogs. We also describe their site-specific incorporation into therapeutically relevant DNA and RNA oligonucleotide motifs by an automated solid support synthesis protocol. The dual-purpose and minimally invasive nature of the probes enables efficient analysis of the conformation and ligand binding abilities of bacterial decoding site RNA (A-site) and G-quadruplex structures of the human telomeric overhang in real time by fluorescence and in 3D by X-ray crystallography.

Introduction

Correlative understanding of nucleic acid structure and its ability to bind to ligands in solution and in solid state is necessary and could provide opportunities to develop new experimental strategies to modulate the function of the target nucleic acid motif. In this regard, we used an innovative probe design approach, wherein we developed dualpurpose nucleoside analog probes containing a conformation-sensitive fluorophore and a very good anomalous X-ray scattering atom (Se). The nucleoside probes, 5-selenophene- modified 2'-deoxyuridine (SedU) and 5-selenophene-modified uridine (SeU), were synthesized by attaching selenophene at the C5 position of the uracil ring and were then incorporated into DNA and RNA oligonucleotides (ONs) by phosphoramidite chemistry (Fig. 1). The present protocols are based on our recently published work (Nuthanakanti, Boerneke, Hermann, & Srivatsan, 2017; Nuthanakanti, Ahmed, Khatik, Saikrishnan, & Srivatsan, 2019), wherein we demonstrated the utility of the probes in (1) monitoring RNA–antibiotic interaction and (2) detecting various G-quadruplex (GQ) structures of the human telomeric DNA repeat.

Figure 1.

Workflow showing the synthesis of oligonucleotides (ONs) labeled with 5-selenophene-2'-deoxyuridine (SedU) or 5-selenophene uridine (SeU) probes. The nucleoside analogs are made of a conformation-sensitive fluorophore component and a selenium atom, which serves as an anomalous X-ray scattering label. The nucleoside analogs incorporated into target DNA and RNA ON motifs, here bacterial ribosomal decoding site (A-site) RNA (PDB: 5T3K; Nuthanakanti et al., 2017), can be used to study the target's structure and recognition by fluorescence and X-ray crystallography.

Basic Protocol 1 describes the synthesis of an SedU 2 analog and its phosphoramidite substrate 5 suitable for incorporation into DNA ONs (Fig. 2). Basic Protocol 2 illustrates the stepwise synthesis of an SeU 7 analog and its phosphoramidite substrate 11 suitable for incorporation into RNA ONs (Fig. 3). Finally, in Basic Protocols 3 and 4, we describe site-specific incorporation of phosphoramidite substrates 5 and 11 into DNA and RNA ONs, respectively, on solid support; cleavage from the solid support; and purification of the ONs by polyacrylamide gel electrophoresis (PAGE).

Figure 2. Synthesis of 5-selenophene-2'-deoxyuridine (SedU) 2 and its phosphoramidite substrate 5.

Figure 3. Synthesis of 5-selenophene uridine (SeU) 7 and its phosphoramidite substrate 11.

CAUTION: Perform all reactions in a well-ventilated fume hood. Wear safety glasses and a laboratory apron. Wear sterile gloves during the course of DNA and RNA ON synthesis.

NOTE: Several steps in these protocols require anhydrous reaction conditions; hence, use oven-dried glassware and dry solvents. Perform reactions under nitrogen or argon atmosphere.

Synthesis Of 5-Selenophene-2'-Deoxyuridine 2 And Its Phosphoramidite 5

SedU 2 and its phosphoramidite substrate 5 are synthesized according to the steps illustrated in Figure 2. 5-Iodo-2'-deoxyuridine is reacted with 2-(tri-n-butylstannyl) selenophene (Pawar, Nuthanakanti, & Srivatsan, 2013; Yang et al., 2005) under Stille cross-coupling reaction conditions to prepare the nucleoside analog 2. Synthesis of phosphoramidite substrate 5 is accomplished in two steps: (1) 5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 (Shah, Wu, & Rana, 1994) is reacted with 2-(tri-n-butylstannyl) selenophene to obtain compound 4, and (2) compound 4 is reacted with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce the desired SedU phosphoramidite substrate 5. The phosphoramidite can be stored under nitrogen or argon atmosphere at –20°C or –40°C for long-term storage.

Materials

5-iodo-2'-deoxyuridine (e.g., Sigma-Aldrich, cat. no. I7125)

2-(tri-n-butylstannyl) selenophene (see Support Protocol 1; Pawar et al., 2013) ŵis(Triphenylphosphine)-palladium(II) chloride (e.g., Sigma-Aldrich, cat. no.8.04174)

1,4-dioxane, anhydrous

Methylene chloride (dichloromethane [CH₂Cl₂])

Methanol (MeOH)

Celite

5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 (see Support Protocol 2; Shah et al., 1994)

Triethylamine (TEA; e.g., Sigma-Aldrich, cat. no. 471283) A,A-diisopropylethylamine (e.g., Sigma-Aldrich, cat. no. 387649) 2-cyanoethyl A,A-diisopropylchlorophosphoramidite (e.g., Alfa Aesar, cat. no.L18575)

Ethyl acetate

15% (w/v) sodium bicarbonate

Brine

Sodium sulfate

Petroleum ether

Silica gel, 100 to 200 mesh

25- and 50-ml round-bottom flask

Magnetic stir plate and stir bar

Reflux condenser

Rotary evaporator

Vacuum concentrator

Gas-tight glass syringe (e.g., Hamilton)

100-ml separatory funnel

Cotton plug

2 × 40–cm glass chromatography column

Test tubes

Additional reagents and equipment for thin-layer chromatography (TLC; see

Current Protocols article: Meyers & Meyers, 2008) and silica gel chromatography (see Current Protocols article: Chakravarti, Mallik, & Chakravarti, 2016)

Synthesis of 5-selenophene-2'-deoxyuridine (SedU) 2

• 1Weigh out 0.2 g (0.56 mmol) of 5-iodo-2'-deoxyuridine, 0.47 g (1.24 mmol) of 2-(tri-n-butylstannyl) selenophene (Pawar et al., 2013), and 0.02 g (0.03 mmol) bis(triphenylphosphine)-palladium(II) chloride in a 25-ml round-bottom flask containing a stir bar.
• 2Add 6 ml anhydrous dioxane. Fit a reflux condenser, and heat reaction mixture at 95°C for 2.5 hr under nitrogen atmosphere. Monitor reaction for completion by TLC using 8:2 (v/v) CH₂Cl₂:MeOH as mobile phase. Expected R_f of the product is ~0.70.
• 3Filter reaction mixture using Celite pad, and wash Celite pad two times with 10 ml CH₂Cl₂.
• 4Evaporate filtrate to dryness using a rotary evaporator.
• 5Purify residue by silica gel column chromatography using CH₂Cl₂:MeOH. Rotary evaporate appropriate fractions, and dry product under high vacuum.

Product 2 is obtained as an off-white solid in ~60% yield. TLC (CH₂Cl₂:MeOH = 8:2); R_f = 0.71; ¹HNMR (400 MHz, DMSO-d₆): δ (ppm) 11.78 (s, 1H), 8.73 (s, 1H), 8.10 (dd, J₁ = 6.0 Hz, J₂ = 0.8 Hz, 1H), 7.59-7.58 (m, 1H), 7.30–7.27 (m, 1H), 6.22 (t, J = 6.4 Hz, 1H), 5.36 (br, 1H), 5.29 (br, 1H), 4.33-4.30 (m, 1H), 3.86-3.84 (m, 1H), 3.733.71 (m, 1H), 3.67-3.64 (m, 1H), 2.29-2.16 (m, 2H); ¹³CNMR (100 MHz, DMSO-d₆): δ (ppm) 161.8,149.2,137.7,134.5,131.7,128.6,122.8,109.9, 87.6, 84.9, 69.9, 60.8, 40.5. HRMS: m/z calcd.for C₁₃H₁₃N₂O₅Se [M-H]^- = 356.9990, observed = 356.9991.

Synthesis of 5'-O-DMT-protected 5-selenophene-2'-deoxyuridine 4

• 6Prepare 5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 as described in Support Protocol 2 (Shah et al., 1994).
• 7Weigh 0.71 g (1.04 mmol) compound 3, 1.0 g (2.37 mmol) of 2-(tri-n-butylstannyl) selenophene (Pawar et al., 2013), and 0.05 g (0.06 mmol) bis(triphenylphosphine)- palladium(II) chloride in a 50-ml round-bottom flask containing a stir bar.
• 8Add 25 ml anhydrous dioxane, and heat reaction mixture at 95°C for 2.5 hr under nitrogen atmosphere. Monitor reaction for completion by TLC using 95:5 (v/v) CH₂Cl₂:MeOH containing 1% (v/v) TEA as mobile phase.

Expected R_f of the product is 0.62.

• 9Cool reaction mixture to room temperature, and filter using Celite pad. Wash Celite pad two times with 20 ml CH₂Cl₂.
• 10Evaporate filtrate to dryness using rotary evaporator.
• 11Purify residue by silica gel column chromatography using CH₂Cl₂:MeOH containing 1% (v/v) TEA. Rotary evaporate appropriate fractions, and further dry product under high vacuum.

Compound 4 is obtained as a foam in 80% to 90% yield: TLC (CH₂Cl₂:MeOH = 95:5, 1% TEA); Rf = 0.62; ¹HNMR (400 MHz, CDCl3): δ (ppm) 9.39 (s, 1H), 7.99 (s, 1H), 7.91-7.90 (m, 1H), 7.41-7.39 (m, 2H), 7.29-7.27 (m, 4H), 7.25-7.17 (m, 3H), 6.90-6.88 (m, 2H), 6.78-6.76 (m, 4H), 6.39 (dd, J₁ = 7.6Hz, J₂ = 6.0 Hz, 1H), 4.53-4.51 (m, 1H), 4.17-4.14 (m, 1H), 3.74 (s, 6H), 3.46-3.42 (m, 1H), 3.38 (dd, J₁ = 10.4 Hz, J₂ = 4.0 Hz, 1H), 2.57-2.51 (m, 1H), 2.33-2.26 (m, 1H); ¹³CNMR (100 MHz, CDCl₃): δ (ppm)161.5, 158.8, 149.5, 144.4, 136.8, 135.6, 135.5, 133.0, 132.1, 130.1, 130.1, 128.9, 128.2,128.1, 127.2, 124.7, 113.4, 112.3, 87.0, 86.4, 85.7, 72.5, 63.7, 55.4, 40.9; HRMS: m/z calcd.for C₃₄H₃₁N₂O₇Se [M-H]^- = 659.1296, observed = 659.1290.

Synthesis of 5-selenophene-modified 2'-deoxyuridine-3'-O-phosphoramidite 5

• 12Weigh 0.33 g (0.55 mmol) compound 4 in a 25-ml round-bottom flask containing a stir bar, and apply high vacuum for 6 to 8 hr (or overnight).
• 13Add 2.5 ml anhydrous CH₂Cl₂ and 0.44 ml (2.53 mmol) N,N-diisopropylethylamine. Stir reaction mixture under ice-cold conditions for 10 min.

N,N-diisopropylethylamine should be stored in molecular sieves of 4 Å for at least 12 hr.

• 14Add 0.17 ml (0.76 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to the reaction mixture drop-wise using a gas-tight syringe.
• 15Stir reaction mixture under nitrogen or argon atmosphere at room temperature for 1.5 hr.
• 16Evaporate reaction mixture to almost dryness, and redissolve residue by adding 20 ml ethyl acetate.
• 17Transfer solution from step 16 to a 100-ml separatory funnel, and add 15 ml of 5% (w/v) sodium bicarbonate. Extract organic phase, and add 15 ml brine.
• 18Extract organic phase again, dry over sodium sulfate, and filter through a cotton plug.
• 19Evaporate organic extract using rotary evaporator, and purify by silica gel column chromatography using petroleum ether:ethyl acetate containing 1 % (v/v) TEA. Use 2 × 40–cm glass column, and pack with silica gel slurry up to 15 cm. Collect 10-ml fractions and product eluents in ~20 test tubes. Rotary evaporate appropriate fractions, and dry product under high vacuum.

Store product 5 in nitrogen or argon atmosphere at –20°C or –40°C.

Compound 5 is obtained as a white solid in ~55% yield. TLC (CH₂Cl₂:MeOH = 95:5 with 1% of TEA); R_t = 0.58; ¹HNMR (400 MHz, CDCl₃): δ (ppm) 8.02 (br, 1H), 7.88– 7.87 (m, 1H), 7.40–7.38 (m, 2H), 7.28–7.17 (m, 8H), 6.82–6.75 (m, 6H), 6.82–6.75 (m, 6H), 6.39-6.35 (m, 1H), 4.61–4.58 (m, 1H), 4.24 (br, 1H), 3.73 (s, 6H), 3.66–3.54 (m, 4H), 3.50–3.47 (m, 1H), 3.33–3.30 (m, 1H), 2.57–2.54 (m, 1H), 2.40 (t, J = 6.4 Hz, 2H), 2.33–2.26 (m, 1H), 1.16–1.15 (m, 12H); ¹³CNMR (100 MHz, CDCl₃): δ (ppm) 161.4, 158.8, 149.2, 144.4, 136.6, 135.5, 135.5, 132.9, 132.0, 130.2, 130.2, 128.9, 128.3, 128.1, 127.2,124.7,117.5,113.3,112.3, 86.8, 86.0, 85.5, 73.7, 73.5, 63.2,58.4,58.2, 55.4, 43.5, 43.3,40.4,40.4,24.8,24.7,24.6,20.3,20.3; ³¹PNMR (162 MHz, CDCl₃): δ (ppm) 149.7; HRMS: m/z calcd.for C₄₃H₄₉N₄O₈PSeK [M+K]⁺ = 899.2090, observed = 899.2299.

Synthesis Of 2-(Tri-N-Butylstannyl) Selenophene

2-(Tri-n-butylstannyl) selenophene is prepared by activating selenophene with butyllithium and N,N,N',N'-tetramethylethylenediamine (TEMED) in anhydrous diethyl ether at –78°C and then reacting with tributyltin chloride (Pawar et al., 2013; Yang et al., 2005).

Materials

Selenophene (e.g., Sigma-Aldrich, cat. no. 367141)

Diethyl ether, anhydrous, or tetrahydrofuran (THF), anhydrous Dry ice/acetone mixture

TEMED (e.g., Sigma-Aldrich, cat. no. 8.08742)

2 M n-butyllithium (e.g., Sigma-Aldrich, cat. no. 302120) in cyclohexane Tributyltin chloride (e.g., Sigma-Aldrich, cat. no. T50201)

Ammonium chloride, saturated

Sodium sulfate

RediSep Rf reversed-phase C18 column, 43 g, packed with silica Petroleum ether

100-ml round-bottomed flask

Magnetic stir plate and stir bar

Separatory funnel

Filtration system

Rotary evaporator

• 1Weigh 2.0 g (15.2 mmol) selenophene in an oven-dried 100-ml round-bottom flask, and dissolve in freshly prepared 30 ml anhydrous diethyl ether (or anhydrous THF; Yang et al., 2005). Cool flask to –78°C using dry ice/acetone mixture.
• 2Add 2.7 ml (18.1 mmol) TEMED to the reaction mixture, and stir for 30 min at –78°C.
• 3Add 9.1 ml (18.2 mmol) of 2 M n-butyllithium (in cyclohexane) dropwise along the side of the flask over 20 min.
• 4Allow reaction mixture to come to room temperature, and stir for 3 hr. Cool flask again to –78°C.
• 5Add 4.9 ml (18.1 mmol) tributyltin chloride slowly (over 5 min), and continue stirring for 1.5 hr at –78°C.

CAUTION: Tributyltin chloride is toxic and foul-smelling. Perform reaction in a well- ventilated hood and wear appropriate laboratory safety equipment.

• 6Bring reaction mixture to room temperature, and dilute with 20 ml saturated ammonium chloride. Transfer mixture to a separatory funnel, and extract two times with 50 ml diethyl ether.
• 7Dry organic extract over sodium sulfate, filter, and evaporate to dryness using a rotary evaporator.
• 8Purify residue by flash column chromatography using a silica RediSep Rf 43 g column with petroleum ether as mobile phase (isocratic, 5 ml/min flow rate, 15-ml fractions).
• 9Evaporate fractions and store product at 4°C.

2-(Tri-n-butylstannyl) selenophene is obtained as a colorless oil in nearly 70% yield: TLC (petroleum ether:ethyl acetate = 9:1), R_f = 0.72; ¹HNMR (400 MHz, CDCl₃): δ (ppm) 8.36 (m, 1H), 7.51-7.48 (m, 2H), 1.61-1.51 (m, 6H), 1.37-1.29 (m, 6H), 1.12-1.08 (m, 6H), 0.92-0.88 (m, 9H); ¹³CNMR (100MHz, CDCl₃): δ (ppm) 143.7,138.0, 135.4,130.7, 29.1, 27.4,13.8, 11.2. HRMS: m/z calcd. for C₁₆H₃₁SeSn [M+H]⁺ = 423.0613, observed = 423.0074.

Synthesis Of 5'-O-Dmt-Protected 5-Iodo-2'-Deoxyuridine 3

5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 is synthesized by reacting 5-iodo- 2'-deoxyuridine with 4,4'-dimethoxytrityl chloride (DMT-Cl) in the presence of 4-dimethylamino pyridine (DMAP; Shah et al., 1994).

Materials

5-iodo-2'-deoxyuridine (e.g., Sigma-Aldrich, cat. no. I7125)

DMT-Cl (e.g., Sigma-Aldrich, cat. no. 38827)

DMAP (e.g., Avra Synthesis, cat. no. ASD1298)

Pyridine, anhydrous (e.g., Sigma-Aldrich, cat. no. 270970)

CH₂Cl₂

MeOH

TEA (e.g., Sigma-Aldrich, cat. no. 471283)

5% (w/v) sodium bicarbonate

Sodium sulfate

50-ml round-bottom flask

Magnetic stir plate and stir bar

Rubber septum

High-vacuum line

Stainless steel needles

Vacuum pump

Separatory funnel

Filtration system

Rotary evaporator

Additional reagents and equipment for TLC (see Current Protocols article: Meyers & Meyers, 2008) and silica gel chromatography (see Current Protocols article: Chakravarti et al., 2016)

• 1Weigh 0.5 g (1.41 mmol) of 5-iodo-2'-deoxyuridine and 0.57 g (1.69 mmol) DMT-Cl in a 50-ml round-bottom flask containing a magnetic stir bar.
• 2Add 0.02 g (0.14 mmol) DMAP. Seal flask with a rubber septum, and apply vacuum using a needle fitted to the high-vacuum line. Apply vacuum for 30 to 60 min.
• 3Close vacuum line, and add 10 ml anhydrous pyridine to the reaction flask.
• 4Perform reaction under nitrogen atmosphere for 12 hr at room temperature.
• 5Monitor reaction using TLC (CH₂Cl₂:MeOH = 95:5 with few drops of TEA).

Expected R_f of the product is ~0.60.

• 6Evaporate pyridine under reduced pressure using a vacuum pump in a well- ventilated fume hood.
• 7To the residue, add 40 ml CH₂Cl₂, and wash two times with 20 ml of 5% (w/v) sodium bicarbonate using a separatory funnel. Repeat step one additional time.
• 8Dry organic extract over sodium sulfate, filter, and evaporate using a rotary evaporator.
• 9Purify residue by silica gel column chromatography using CH₂Cl₂:MeOH containing 1% (v/v) TEA.
• 10Collect appropriate fractions, and evaporate solvent under vacuum. Store product at 4°C.

Product 3 is obtained as an off-white solid in 79% yield: TLC (CH₂Cl₂:MeOH = 95:5 with a few drops of TEA); R_f = 0.61; ¹HNMR (400 MHz, CD?): δ (ppm) 8.85 (s, 1H), 8.14 (s, 1H), 7.42–7.40 (m, 2H), 7.34–7.30 (m, 4H), 7.29–7.28 (m, 1H), 7.25–7.21 (m, 1H), 7.18–7.16 (m, 1H), 6.86–6.82 (m, 4H), 6.31 (dd, J₁ = 7.6 Hz, J₂ = 5.6 Hz, 1H), 4.55–4.53 (m, 1H), 4.10–4.08 (m, 1H), 3.79 (s, 3H), 3.80 (s, 3H), 3.41 (dd, J₁ = 11.6Hz, J₂ = 3.2 Hz, 1H), 3.36 (dd, J₁ = 10.6 Hz, J₂ = 3.4 Hz, 1H), 2.51–2.46 (m, 1H), 2.32– 2.25 (m, 1H); ¹³CNMR (100 MHz, CDCl₃): δ (ppm) 160.0, 158.8, 150.0,1445.5, 135.6, 135.5, 130.2, 130.2, 129.3, 128.3, 128.2, 128.0, 127.9, 127.3, 127.2, 113.5, 113.3, 87.2, 86.6, 85.7, 72.6, 68.7, 63.6, 55.4, 55.4; HRMS: m/z calcd.for C30H30IN2O7 [M+H]⁺ = 657.1098, observed = 657.1030.

Synthesis Of 5-Selenophene-Modified Uridine 7 And Its Phosphoramidite 11

SeU 7 (Pawar et al., 2013) and its phosphoramidite 11 (Nuthanakanti et al., 2017) are synthesized as shown in Figure 3. The nucleoside analog 7 is synthesized in one step by a Stille cross-coupling reaction between 5-iodouridine and 2-(tri-n-butylstannyl) selenophene. Phosphoramidite 11 is synthesized in four steps starting from 5-iodouridine: (1) protection of 5-iodouridine using an acid-labile DMT group to obtain 5'-O- DMT-protected 5-iodouridine 8 (Shah et al., 1994); (2) Stille cross-coupling reaction between 8 and 2-(tri-n-butylstannyl) selenophene to give compound 9; (3) subsequent reaction with tert-butyldimethylsilyl chloride (TBDMS-Cl) in the presence of AgNO₃ to give 2'-O-TBDMS-protected intermediate 10 (Hakimelahi, Proba, & Ogilvie, 1982; Somoza, 2008); and (4) treatment of compound 10 with 2-cyanoethyl N,N- diisopropylchlorophosphoramidite to obtain SeU phosphoramidite 11. The phospho- ramidite can be stored under nitrogen or argon atmosphere at –20°C or –40°C for longterm storage.

Materials

5-iodouridine (e.g., Sigma-Aldrich, cat. no. 852597) bis(Triphenylphosphine)-palladium(II) chloride (e.g., Sigma-Aldrich, cat. no.

8.04174)

1,4-dioxane, anhydrous

2-tri-n-butylstannyl selenophene (see Support Protocol 1; Pawar et al., 2013) Celite

Petroleum ether

RediSep Rf reversed-phase C18 column, 43 g, packed with silica

Acetonitrile

5'-O-DMT-protected 5-iodouridine 8 (see Shah et al., 1994)

CH₂Cl₂

MeOH

TEA (e.g., Sigma-Aldrich, cat. no. 471283)

Silver nitrate (e.g., Sigma-Aldrich, cat. no. 209139)

Pyridine, anhydrous (e.g., Sigma-Aldrich, cat. no. 270970)

THF, anhydrous

TBDMS-Cl (e.g., Sigma-Aldrich, cat. no. 190500)

Ethyl acetate

5% (w/v) sodium bicarbonate

Brine

Sodium sulfate

N,N-diisopropylethylamine (e.g., Sigma-Aldrich, cat. no. 387649) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (e.g., Alfa Aesar, cat. no. L18575)

Silica gel, 100 to 200 mesh

10-, 25-, and 50-ml round-bottom flask

Magnetic stir plate and stir bar

Reflux condenser

90°C silicone oil bath

Rotary evaporator

Vacuum concentrator

100- and 150-ml separatory funnel

Gas-tight glass syringe (e.g., Hamilton)

Cotton plug

2 × 40-cm glass chromatography column

Test tubes

Synthesis of 5-selenophene-modifed uridine (SeU) 7

• 1Dissolve 0.25 g (0.68 mmol) of 5-iodouridine and 0.024 g (0.034 mmol) bis(triphenylphosphine)-palladium(II) chloride in 7.5 ml degassed anhydrous dioxane in a 25-ml round-bottom flask.
• 2Add 0.425 g (1.01 mmol) of 2-tri-n-butylstannyl selenophene (Pawar et al., 2013). Fit a reflux condenser, and heat suspension at 90°C for 2 hr under nitrogen atmosphere.
• 3Filter reaction mixture through Celite pad. Wash pad three times with 10 ml dioxane.
• 4Evaporate filtrate using rotary evaporator until viscous. Precipitate crude product using ~25 ml petroleum ether.
• 5Purify precipitate by flash column chromatography using a C18 RP column (10% to 55% acetonitrile in water for 35 min, 10 ml/min flow rate). Collect 10-ml fractions.

The product starts to elute at around 25% acetonitrile in water (six test tube fractions).

• 6Collect fractions corresponding to product. Evaporate solvent using a rotary evaporator, and dry under high vacuum.

Product fraction will absorb at ~260 and ~324 nm.

Compound 7 is obtained as an off-white solid in ~45% yield: TLC (CHCl₃:MeOH = 8:2) Rf = ũ.63; ¹HNMR (400 MHz, DMSO-d6): δ = 11.81 (s, 1H), 8.80 (s, 1H), 8.08 (d, J = 5.6 Hz,1H), 7.60 (d, J = 3.6 Hz, 1H), 7.28 (dd, J = 5.6 Hz, J = 4.0 Hz, 1H), 5.84 (d, J = 3.6 Hz, 1H), 5.51-5.49 (m, 2H), 5.11 (d, J = 5.2 Hz, 1H), 4.13 (dd, J₁ = 9.2 Hz, J₂ = 4.8 Hz, 1H), 4.07 (dd, J₁ = 10.4 Hz, J₂ = 5.2 Hz, 1H), 3.93-3.92 (m, 1H), 3.81-3.76 (m, 1H), 3.68-3.63 (m, 1H)ppm; ¹³CNMR (100MHz, DMSO-d6): δ = 161.8, 149.4, 137.6, 134.5, 131.7, 128.6, 122.8, 109.9, 88.9, 84.6, 74.4, 69.2, 60.0 ppm; HRMS: m/z calcd. for C₁₃H₁₄N₂O₆SeNa [M+Na]⁺ = 396.9915; observed = 396.9873.

Synthesis of 5'-O-DMT-protected SeU 9

• 7Weight out 1.0 g (1.49 mmol) of 5'-O-DMT-protected 5-iodouridine 8 (Shah et al., 1994), 1.37 g (3.27 mmol) of 2-(tri-n-butylstannyl) selenophene, and 0.084 g (0.12 mmol) bis(triphenylphosphine)-palladium(II) chloride in a 50-ml roundbottom flask containing a stir bar.
• 8Add 25 ml anhydrous dioxane, and stir reaction mixture at 90°C for 2 hr under nitrogen atmosphere.
• 9Filter reaction mixture through Celite pad, and wash pad two times with 20 ml CH₂Cl₂.
• 10Evaporate filtrate using a rotary evaporator, and purify residue by silica gel column chromatography using CH₂Cl₂:MeOH containing 1% (v/v) TEA. Rotary evaporate appropriate fractions, and further dry product under high vacuum.

Compound 9 is obtained as an off-white foam in —70% yield: TLC (CH₂Cl₂:MeOH = 95:5, 1% TEA); R_f = 0.36. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.02 (s, 1H), 7.85 (dd, J₁ = 5.0 Hz, J₂ = 1.4 Hz, 1H), 7.40-7.38 (m, 2H), 7.28-7.26 (m, 4H), 7.23-7.15 (m, 3H), 6.85-6.83 (m, 2H), 6.77-6.75 (m, 4H), 5.99 (d, J = 4.8 Hz, 1H), 4.49 (t, J = 5.0 Hz, 1H), 4.35 (t, J = 4.6 Hz, 1H), 4.32-4.31 (m, 1H), 3.73 (s, 6H), 3.49-3.47 (m, 1H), 3.35 (dd, J₁ = 10.6 Hz, J₂ = 3.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl3): δ (ppm) 162.2, 158.7, 151.0, 144.4, 136.3, 135.6, 135.5, 132.0, 130.1, 130.1, 128.9, 128.2, 128.1, 127.2.124.7.113.4.112.2, 90.7, 86.9, 84.8, 75.6, 71.2, 63.4, 55.3; HRMS: m/z calcd. for C₃₄H₃₃N₂O₈Se [M+H]⁺ = 677.1402, observed = 677.1394.

Synthesis of 2'-O-silyl-protected nucleoside intermediate 10

• 11Weigh out 0.95 g (1.41 mmol) compound 9 and 0.62 g (3.65 mmol) silver nitrate in an oven-dried 25-ml round-bottom flask containing a stir bar.
• 12Transfer 4.8 ml anhydrous pyridine to flask.
• 13Add 14 ml anhydrous THF into reaction mixture, and stir for 10 min under nitrogen atmosphere at room temperature.
• 14Add 0.55 g (3.65 mmol) TBDMS-Cl to flask, and stir reaction mixture at room temperature for 30 min.
• 15Filter reaction mixture through Celite pad, and wash pad two times with 15 ml ethyl acetate.
• 16Transfer filtrate to a 150-ml separatory funnel, and add 20 ml of 5% (w/v) sodium bicarbonate. Extract organic phase, and wash with 20 ml brine.
• 17Dry organic extract over sodium sulfate, and evaporate solvent using a rotary evaporator.
• 18Purify residue by silica gel column chromatography using petroleum ether:ethyl acetate containing 1% (v/v) TEA. Rotary evaporate appropriate fractions, and further dry product under high vacuum.

The 2'-O-TBDMS-protected isomer elutes first, followed by the 3'-O-TBDMS-protected isomer (Davis & Bajji, 2005).

Since the isomers run close on the silica gel column, column chromatography may have to be performed again with fractions containing both isomers.

Compound 10 is obtained as an off-white solid in ~50% yield: TLC (petroleum ether:ethyl acetate = 6:4, 1% TEA); Rf = 0.42; ¹H NMR (400 MHz, CDCl3): δ (ppm) 8.62 (br, 1H), 7.98 (s, 1H), 7.88 (d, J = 5.6 Hz, 1H), 7.42-7.40 (m, 2H), 7.30-7.28 (m, 5H), 7.24–7.19 (m, 2H), 6.78-6.67 (m, 6H), 6.12 (d, J = 5.6Hz, 1H), 4.51 (t, J = 5.2 Hz, 1H), 4.26-4.22 (m, 2H), 3.74 (s, 6H), 3.54-3.52 (m, 1H), 3.40 (dd, J₁ = 11.0 Hz, J₂ = 3.0 Hz, 1H), 0.93-0.91 (s, 9H), 0.15 (s, 3H), 0.14 (s, 3H);¹³ C NMR (100 MHz, CDCl₃): δ (ppm) 161.3,158.8,149.4,144.4,136.1,135.4,135.3,132.6,132.2,130.2,130.1,128.9, 128.2,128.1, 127.3, 124.9, 123.9, 113.4, 88.0, 87.0, 84.0, 75.8, 71.3, 63.4, 55.4, 25.8, 18.1, –4.5, –4.9; HRMS: m/z calcd.for C4oH₄₆N₂OsSeSiNa [M+Na]⁺ = 813.2086, observed = 813.2123.

Synthesis of 5-selenophene-modified uridine-3'-O-phosphoramidite 11

• 19Weigh 0.41 g (0.52 mmol) compound 10 in a dried 10-ml round-bottom flask containing a stir bar.
• 20Dry compound under high vacuum for few hours (~3 to 4 hr).
• 21Transfer 4.1 ml anhydrous CH₂Cl₂, which should dissolve compound 10. Maintain reaction flask under nitrogen atmosphere.
• 22Add 0.23 ml (1.30 mmol) N,N-diisopropylethylamine, and stir reaction mixture at room temperature for 10 min.

N,N-diisopropylethylamine should be stored in molecular sieves of 4 Å for at least 12 hr.

• 23Add 0.14 ml (0.63 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to the reaction mixture in a drop-wise manner (over 20 s) using a gas-tight syringe, and stir reaction mixture for 12 hr at room temperature.
• 24Evaporate reaction mixture to dryness, and then add 20 ml ethyl acetate.
• 25Transfer solution to a 100-ml separatory funnel, and wash with 15 ml of 5% (w/v) sodium bicarbonate followed by 15 ml brine.
• 26Dry organic extract over sodium sulfate, and then filter through a cotton plug.
• 27Evaporate filtrate using a rotary evaporator, and purify residue by silica gel column chromatography using petroleum ether:ethyl acetate containing 1% (v/v) TEA. Use a 2.5 × 40-cm glass column, and pack with silica gel slurry up to 15 cm. Collect 10-ml fractions and product eluents in ~20 test tubes.
• 28Rotary evaporate appropriate fractions, and further dry product under high vacuum. Store phosphoramidite under nitrogen or argon atmosphere at -20°C or -40°C until further use.

Phosphoramidite 11 is obtained as an off-white solid in 60% yield: Analytical data were obtained using one of the diastereomers, which eluted first. TLC (petroleum ether:ethyl acetate = 6:4, 1% TEA); Rf = 0.40; ¹HNMR (400 MHz, CDCl₃): δ (ppm) 8.03 (s, 1H), 7.86-7.84 (m, 1H), 7.44-7.42 (m, 2H), 7.32-7.28 (m, 5H), 7.25-7.20 (m, 2H), 6.78-6.70 (m, 6H), 6.12 (d, J = 6.4 Hz, 1H), 4.49 (t, J = 5.8 Hz, 1H), 4.37 (br, 1H), 4.24-4.20 (m, 1H), 3.74 (s, 6H), 3.61-3.51 (m, 5H), 3.30 (dd, J₁ = 10.6Hz, J₂ = 2.6Hz, 1H), 2.28-2.24 (m, 2H), 1.18-1.13 (m, 12H), 0.88 (s, 9H), 0.11 (s, 3H), 0.07(s, 3H); ¹³CNMR (100MHz, CDCl₃): δ (ppm) 161.3, 158.8, 149.4, 144.4, 135.4, 132.8, 132.0, 130.3, 130.3, 130.2, 128.9, 128.4, 128.2, 128.1, 127.3, 124.8, 113.4, 87.6, 87.0, 84.2, 75.8, 74.9, 63.4, 55.4, 43.6, 43.5, 29.8, 25.8, 24.8, 22.7, 20.3, 18.1, –4.4, –4.7; ³¹P NMR (162 MHz, CDCl₃): δ (ppm) 151.6; HRMS: m/z calcd. for C₄₉H₆₃N₄O₉PSeSiNa [M+Na]⁺ = 1013.3165, observed = 1013.3191.

Synthesis Of Dna Oligonucleotides Containing 5-Selenophene-Modified 2'-Deoxyuridine 2

Selenophene-modified DNA ONs (12 to 15) corresponding to the human telomeric repeat are synthesized at a 1-μmol scale (1000-Å controlled pore glass [CPG] solid support) using an ABI-392 synthesizer (Table 1; Nuthanakanti et al., 2019; Tanpure & Srivatsan, 2012). Site-specific incorporation of modified phosphoramidite substrate 5 into DNA ONs is achieved on the solid support using standard phosphoramidites of natural nucleosides (see Current Protocols article: Beaucage & Caruthers, 2000). After deprotection of the DMT group using trichloroacetic acid in CH₂Cl₂, individual phosphoramidites are coupled according to the input sequence in the presence of 5-(ethylthio)-1H-tetrazole. The coupling reaction with modified phosphoramidite 5 (~10 equiv. with respect to the solid support) is performed two times with a reaction time of 4 min each. The coupling efficiency, as determined using trityl monitor, for the incorporation of 5 into ONs 12 to 15 is 55% to 70%. Cleavage from the solid support and global deprotection are performed in a one-shot reaction by treating the ONs with 30% aqueous ammonium hydroxide for 15 hr at 55°C. The ON products are resolved by PAGE (20% gel) under denaturing conditions. The gel is visualized with UV light, the appropriate band is cut, and the ON is extracted using ammonium acetate. The extract is passed through a C18 column to desalt the ON solution, which is then dried under vacuum. The ONs are reconstituted in sterile water, and purity is determined by reversed-phase high-performance liquid chromatography (RP-HPLC; Fig. 4A). The product is quantified using an extinction coefficient, as given in Table 1.

Table 1. Modified DNA 12 to 15 and RNA 16 Oligonucleotide Sequences Synthesized Using Basic Protocols 3 and 4.

Entry	ON	Calcd. mass (m/z)	Observed mass (m/z)	ε260 (M-1cm-1)a
12	d(5' AGGG2TAGGGTTAGGGTTAGGG 3')	7081.5	7082.4	230080
13	d(5' AGGGTTAGGG2TAGGGTTAGGG 3')	7081.5	7080.2	230080
14	d(5' AGGGTTAGGGT2AGGGTTAGGG 3')	7081.5	7081.6	230080
15	d(5' AGGGTTAGGGTTAGGG2TAGGG 3')	7081.5	7081.5	230080
16	5' CAGCG7CACACCACCC 3'	5127.1	5126.9	140776

^aε260 of modified oligonucleotides was determined using an OligoAnalyzer 3.1. The extinction coefficient of nucleoside 2 (ε260 = 96 80 M^-1cm^-1) and nucleoside 7 (ε260 = 8576 M^-1 cm^-1)was used in place of thymidine and uridine, respectively.

Figure 4.

Representative high-performance liquid chromatography (HPLC) profile of polyacrylamide gel electrophoresis-purified DNA oligonucleotide (ON) 13 (A) and RNA ON 16 (B) at 260 nm. Mobile phase A = 100 mM triethylammonium acetate (pH 75), mobile phase B = acetonitrile; flow rate = 1 ml/min; gradient = 0% to 10% B over 10 min and 10% to 100% B over 20 min. HPLC analysis was performed using a Luna C18 column (250 mm × 4.6 mm, 5 μm). Figure adapted and reprinted with permission from the authors (Nuthanakanti et al., 2017, 2019).

Materials

Reagents for DNA synthesis:

Deblock: 3% trichloroacetic acid in CH₂Cl₂

Cap A: 8:1:1 (v/v/v) 2,6-lutidine/acetic anhydride/THF Cap B: 8:1:1 (v/v/v) N-methylimidazole/THF/pyridine Oxidizer: 90.54:9.05:0.41:0.43 (v/v/v/w) THF/water/pyridine/iodine Activator: 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile DMT dG (dmf) phosphoramidite: N2-dimethylformamidine-5'-O-(4,4'- dimethoxytrityl)-2'-deoxyguanosine-3'-O-[O-(2-cyanoethyl)-N,N ^/- diisopropylphosphoramidite]

DMT dA (bz) phosphoramidite: N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'- deoxyadenosine-3' - O-[O-(2-cyanoethyl)-N,N -diisopropylphosphoramidite]

DMT dT phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine-3'- O-[O-(2-cyanoethyl)-N,N'-diisopropylphosphoramidite]

SedU phrophoramidite 5 (see Basic Protocol 1)

Acetonitrile, anhydrous

30% aqueous ammonium hydroxide (e.g., Sigma-Aldrich, cat. no. 338818) Denaturing loading buffer (see recipe)

20% preparative polyacrylamide gel (19:1 acrylamide:bisacrylamide, 7 M urea in 1 × TBE buffer)

0.5 M ammonium acetate (e.g., Sigma-Aldrich, cat. no. 431311)

40% (v/v) acetonitrile

V-shaped vial

DNA synthesizer (e.g., Applied Biosystems)

1-μmol column with CPG support

Vacuum line

Screw-cap vial

Teflon tape

55°C incubator

Microcentrifuge

2.0-ml microcentrifuge tube

Centrifuge vacuum concentrator (e.g., SpeedVac)

UV light

Scalpel, sterile

Poly-Prep column (e.g., Bio-Rad)

Glass rod, sterile

Rotary mixer (e.g., RotoSPIN)

Sep-Pak C18 classic cartridge (e.g., Water Corporation, cat. no. WAT051910)

Additional reagents and equipment for denaturing PAGE (see Current Protocols article: Albright & Slatko, 1994)

NOTE: Wear sterile gloves and use sterile plastic and glassware. Use sterile water for all steps detailed in this protocol.

Synthesis of DNA ONs 12 to 15 containing 5-selenophene-modified 2'-deoxyuridine

• 1Set up reagents for DNA synthesis. Dissolve SedU phosphoramidite 5 (~10 equiv. with respect to the solid support) in anhydrous acetonitrile at 0.11 M final concentration in a V-shaped vial, and attach vial to the synthesizer.
• 2Attach a 1-μmol column composed of CPG support corresponding to the first nucleotide at the 3'-end of the desired sequence.
• 3Perform automated solid-phase ON synthesis from the 3'-end with DMT-off as the end step. Perform coupling reaction two times with a reaction time of 4 min each.
• 4Upon completion of synthesis, detach column from synthesizer, and dry under high vacuum for ~20 min.

Deprotection and purification of DNA ONs

• 5Transfer solid support to a screw-cap vial, and add 1 ml of 30% aqueous ammonium hydroxide. Tighten vial and wrap with Teflon tape.
• 6Incubate sample at 55°C for 15 hr, and then bring to room temperature.
• 7Centrifuge sample 2 min at 2680 × g, room temperature, and transfer supernatant to a clean 2-ml microcentrifuge tube. Wash solid support with 200 μl sterile water, and transfer solution to 2-ml tube containing sample.
• 8Evaporate sample to dryness at 30°C using a centrifuge vacuum concentrator.

This step could take up to 3 to 4 hr.

• 9Dissolve sample in 200 μl sterile water, and add 200 μl denaturing loading buffer.
• 10Load 100 μl sample per well in four wells of a 20% preparative polyacrylamide gel.

A recommended size for the gel is 20 cm width × 45 cm length × 1.5 cm thick.

• 11Run gel at 25 W for 5 to 6 hr until bromophenol blue runs 80% of the gel. Using a 254-nm UV light, carefully cut band related to the full-length product using a sterile scalpel.

  The slowest running band is usually the full-length ON product.

• 12Transfer gel pieces to a poly-prep column, and crush gel using a sterile glass rod. Add 3 ml of 0.5 M ammonium acetate, and place column in a rotary mixer.
• 13Incubate overnight at room temperature, and then centrifuge sample 10 min at 1000 × g, room temperature.
• 14Load filtrate on a Sep-Pak C18 classic cartridge. Wash cartridge with ~10 ml water, and then elute ON with 5 ml of 40% acetonitrile in water. Collect 1-ml fractions in 2.0-ml microcentrifuge tubes.

Typically, the ON should elute in the first 3 ml of 40% acetonitrile in water. It is recommended that each fraction is analyzed for the presence of ON by measuring UV absorbance at 260 nm.

• 15Pool fractions and evaporate to dryness in a vacuum concentrator.
• 16Dissolve ON samples in sterile water (e.g., 200 μl), and measure absorbance at 260 nm. Calculate concentration of the ONs using the extinction coefficient given in Table 1. Store samples at -20°C or -40°C.

Characterization data—including HPLC profile, melting temperature (Tm), circular dichroism (CD), and matrix-assisted laser desorption/ionization (MALDI) mass spectra—of the modified ONs have been previously published (Nuthanakanti etal., 2019).

Synthesis Of An Rna Oligonucleotide Containing 5-Selenophene-Modified Uridine 7

Basic Protocol 4 describes the incorporation of SeU 7 into the bacterial ribosomal decoding site (A-site) RNA ON sequence (Nuthanakanti et al., 2017). The synthesis is performed on a 1-μmol scale using CPG solid support (1000 Å) and 2'-O-TBDMS- protected phosphoramidites (Sproat, 2005). While the coupling reaction with native phos- phoramidites is performed with a reaction time of 10 min (two times), incorporation of 5-selenophene-modified-2'-O-TBDMS-protected phosphoramidite 11 is performed with a reaction time of 15 min (two times). The coupling efficiency for the modified phosphoramidite 11, based on a trityl monitor assay, is around 40%. The RNA ON is cleaved from the solid support, and protecting groups on nucleobases are deprotected by treatment with a 1:1 solution of 10 M methylamine in ethanol and water for 12 hr at room temperature. Further, the silyl group is deprotected using TEA trihydrofluoride (TEA·3HF). The ON is purified by PAGE, and purity is determined by RP-HPLC and quantified as in Basic Protocol 3 (Fig. 4B; Table 1).

Materials

Reagents for RNA synthesis:

Deblock: 3% trichloroacetic acid in CH₂Cl₂

Cap A: 8:1:1 (v/v/v) 2,6-lutidine/acetic anhydride/THF Cap B: 8:1:1 (v/v/v) N-methylimidazole/THF/pyridine Oxidizer: 90.54:9.05:0.41:0.43 (v/v/v/w) THF/water/pyridine/iodine Activator: 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile 5'-dimethoxytrityl-N-benzoyl-adenosine-2'-O-TBDMS-3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite

5'-dimethoxytrityl-N-acetyl-cytidine-2'-O-TBDMS-3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite

5'-dimethoxytrityl-N-acetyl-guanosine-2'-O-TBDMS-3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite

5'-dimethoxytrityl-uridine-2'-O-TBDMS-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite

5-ethylthio-1H-tetrazole (e.g., Glen Research)

SeU phosphoramidite 11 (see Basic Protocol 2)

Acetonitrile, anhydrous

10 M methylamine in ethanol (e.g., Sigma-Aldrich, cat. no. 534102) 10 M methylamine in water (e.g., Sigma-Aldrich, cat. no. 426466) Dimethylsulfoxide (DMSO), anhydrous

TEA·3HF (e.g., Sigma-Aldrich, cat. no. 344648)

V-shaped vial

RNA synthesizer (e.g., Applied Biosystems)

1-μmol column with CPG support Vacuum line

Screw-cap vials Microcentrifuge 2.0-ml microcentrifuge tube Centrifuge vacuum concentrator (e.g., SpeedVac) 65°C incubator

Lyophilizer

Additional reagents and equipment for purification and quantification of ONs (see Basic Protocol 3)

NOTE: Wear sterile gloves and use sterile plastic and glassware. Use sterile water for all steps detailed in this protocol.

Synthesis of RNA ON 16 containing 5-selenophene-modified uridine

• 1Set up reagents for RNA synthesis. Dissolve SeU phosphoramidite 11 in anhydrous acetonitrile at 0.11 M final concentration in a V-shaped vial, and attach vial to the synthesizer.
• 2Attach a 1-μmol column composed of CPG support corresponding to the first nucleotide at 3'-end of the desired sequence.
• 3Perform automated solid-phase ON synthesis from the 3'-end with DMT-off as the end step.
• 4Upon completion of synthesis, detach column from synthesizer, and dry under high vacuum for ~20 min.

Deprotection and purification of RNA ONs

• 5Transfer solid support to a screw-cap vial, and add 750 μl of 10 M methylamine in ethanol and 750 μl of 10 M aqueous methylamine. Incubate sample overnight at room temperature.
• 6Centrifuge sample 2 min at 2680 × g, room temperature, and transfer supernatant to a clean 2-ml microcentrifuge tube. Wash solid support with 500 μl sterile water, and transfer solution to 2-ml microcentrifuge tube containing sample.
• 7Evaporate sample to dryness at 30°C using a centrifuge vacuum concentrator.
• 8Add 100 μl anhydrous DMSO to residue, and heat sample at 65°C for 5 min.
• 9Bring sample to room temperature, and add 150 μl TEA·3HF. Heat sample at 65°C for 2.5 hr, and then bring to room temperature.
• 10Dilute sample with 500 μl sterile water, and freeze-dry using a lyophilizer.
• 11Follow Basic Protocol 3 steps 9 to 16 to purify and quantify the labeled RNA ON. Store sample at -20°C or -40°C for long-term storage.

Characterization data—including HPLC profile, Tm, CD, and MALDI mass spectra—of the modified ON have been previously published (Nuthanakanti et al., 2017).

Reagents And Solutions

Denaturing loading buffer

7 M urea

10 mM Tris·HCl

100 mM EDTA

0.05% (w/v) bromophenol blue

Adjust pH to 8 with NaOH Store at 4°C for up to a few months

Commentary

Background Information

Probing nucleic acid structures and their interaction with proteins, metabolites, and synthetic ligands has been greatly aided by biophysical techniques, including fluorescence imaging, nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray crystallography (Nguyen & Qin, 2012; Salgado, Cazenave, Kerkour, & Mergny, 2015; Serganov & Patel, 2012; Sinkeldam, Greco, & Tor, 2010). These methods of analysis commonly use ONs labeled with appropriate fluo- rophores, isotope labels, spin labels, and heavy atoms, as natural nucleosides that make up nucleic acids are practically nonemissive or do not contain intrinsic labels. Over the years several nucleoside analogs have been developed that serve as efficient fluorescent probes (Phelps, Morris, & Beal, 2012). Similarly, chemistry has been developed to install isotope, spin, and heavy atom labels on the nucleoside, which when incorporated into ONs facilitate efficient probing of nucleic acid structure and recognition by the abovementioned techniques (Wachowius & Höbart-ner, 2010). However, the majority of these tools, which are based on the so called “one label–one technique,” cannot be implemented in multiple techniques. Hence, correlating the information obtained under equilibrium conditions, in solid state, and in cells becomes very difficult using ONs labeled with traditionally designed probes. We came up with an innovative probe design strategy and developed multifunctional nucleoside analogs equipped with two or more labels, which would serve as common probes in analyzing nucleic acids by fluorescence, NMR, and X-ray crystallography techniques.

We recently introduced dual-application nucleoside probes based on 5-selenophene- modified and 5-fluorobenzofuran-modified uracil cores (Manna, Sarkar, & Srivatsan, 2018; Pawar et al., 2013). Of particular interest to this article, synthesis and incorporation of SedU and SeU into DNA and RNA ONs is described in detail. These analogs are designed based on simple physical and organic chemistry concepts and literature precedence, wherein conjugation of a heterocyclic moiety onto nucleobases can generate environmentsensitive nucleoside analogs. In addition, we took advantage of the good anomalous scattering property of the Se atom that has been commonly used in protein X-crystallography, and in nucleic acids (Carrasco, Buzin, Tyson, Halpert, & Huang, 2004; Egli & Pallan, 2007), to build the dual-purpose probes SedU and SeU. These analogs, when incorporated into ONs, are minimally invasive and importantly report antibiotic binding to the A-site RNA motif and small molecule binding to the human telomeric DNA GQs via changes in fluorescence properties. Further, the crystals of both ON systems were grown and their structures solved at high resolution. By comparing fluorescence data and X-ray structure, we were able to understand the structural basis of how the probe senses nucleic acid conformation in the absence and presence of ligands. Taken together, when judiciously placed, the selenophene-modified nucleoside analogs can aid analysis of ON motifs of interest by using fluorescence and X-ray crystallography techniques.

Critical Parameters and Troubleshooting

Some of the steps in this protocol use flammable and toxic materials and involve air- and/or moisture-sensitive reactions. It is recommended to refer to material safety data sheets and safety information before using the reagents. Prior experience in organic synthesis, workup procedures, and chromatographic purification techniques is required. Basic knowledge of solid support ON synthesis and experience in operating a DNA/RNA synthesizer are necessary.

The Stille coupling reaction to produce SedU 2 gave moderate yields possibly due to deiodination of 5-iodo-2'-deoxyuridine substrate. 2'-O-TBDMS-protected nucleoside 10 is obtained in 50% yield, as the 3'-O- TBDMS-protected isomer is also formed in this silylation reaction. During silica gel column chromatography purification of any DMT-protected nucleoside, use a mobile phase containing 1% to 2% TEA to avoid detritylation on the column. It is worth noting that the phosphoramidite chemistry is highly moisture sensitive. Therefore, prepare all stocks of phosphoramidites and activator in anhydrous acetonitrile. While it is preferred to synthesize phosphoramidites 5 and 11 fresh before use, they can be stored under nitrogen or argon atmosphere at –20°C or –40°C for a month before incorporation. All other reagents for ON synthesis are commercially available in high purity and are usually configured for a given DNA/RNA synthesizer. When synthesizing and purifying DNA and RNA ONs, it is recommended to use sterile plastic and glassware and to wear sterile gloves to avoid degradation of ONs. Prepare solutions for PAGE purification in a well-ventilated fume hood as acrylamide is considered a neurotoxin. When synthesizing highly structured ON sequences, coupling efficiency could be poor and deprotection may not complete within the prescribed time. In the case of poor coupling efficiency, which could be an inherent problem for a given sequence, perform synthesis using multiple columns. In case of a deprotection problem, vary the reaction time in the deprotection step.

Understanding Results

SedU, SeU, and corresponding phospho- ramidites are synthesized in moderate to good yields. Phosphoramidites 5 and 11 can be efficiently incorporated into DNA and RNA ONs using a solid-phase synthesizer. Typically, a reaction performed using a 1-μmol scale solid support yields 200 to 250 nmol of the PAGE-purified 5-selenophene-modified DNA ON products (22-mer; Table 1). However, RNA ON synthesis performed using a 1-μmol scale solid support produces nearly 100 nmol of the modified RNA ON (16-mer) after PAGE purification. This procedure to synthesize SedU- and SeU-modified GQ-forming DNA and decoding site RNA ONs can be used to synthesize other site- specifically labeled ONs of interest.

Time Considerations

With basic experience in nucleoside chemistry and solid-phase synthesis, one could prepare the phosphoramidites in 1 week and then incorporate and obtain purified ONs within a week.