Attachment of Nitroxide Spin Labels to Nucleic Acids for EPR

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

In addition to X?ray, NMR, and FRET, electron paramagnetic resonance (EPR) can be applied to elucidate the structure of different macromolecular systems and determine local surroundings of paramagnetic centers in DNA and RNA. This technique permits structural characterization as well as dynamic structural changes of macromolecular systems. To do so, free radicals with good stability must be introduced. Here, the site?directed spin labeling of DNA and RNA based on the Sonogashira cross?coupling reaction is described. First, the appropriate building blocks, either 5?iodo?substituted pyrimidine deoxy? or ribo?nucleoside phosphoramidites are prepared and incorporated by solid?phase synthesis. Following this, the protected oligonucleotides are ?on column? reacted with the acetylenic nitroxide spin labels and subsequently purified. Applications of this technique for duplexes, hairpins, and riboswitches in vitro and in cell are described. Curr. Protoc. Nucleic Acid Chem. 49:7.17.1?7.17.40. © 2012 by John Wiley & Sons, Inc.

Keywords: site?directed spin labeling (SDSL); nitroxide; on?column synthesis; Sonogashira cross coupling

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Introduction
Basic Protocol 1: Synthesis of 15N‐Marked Spin Label TPA
Basic Protocol 2: Synthesis of DNA Building Blocks for Spin Label Attachment
Basic Protocol 3: Synthesis of RNA Building Blocks for Spin Label Attachment
Basic Protocol 4: DNA Synthesis without Interruption
Basic Protocol 5: RNA Synthesis without Interuption
Alternate Protocol 1: RNA Synthesis with Interruption
Basic Protocol 6: Sonogashira Cross‐Coupling Reaction on the Solid Phase
Basic Protocol 7: Probe Preparation and EPR Measurements
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Synthesis of 15N‐Marked Spin Label TPA

Materials

Sodium carbonate (Na₂ CO₃ )
Magnesium oxide (MgO)
Ammonium chloride ¹⁵ N (NH₄ Cl; 99%; Cambridge Isotope Laboratories)
Acetone
Silica gel (grade 60, 70 to 230 mesh, 60 Å; Sigma‐Aldrich)
Ethyl acetate (EtOAc; p.a.; Merck)
Methanol (MeOH)
n ‐Hexane (p.a.; Merck), 4°C
Acetic acid (AcOH)
Bromine (Br₂ )
Concentrated aqueous ammonia
KOH (sat. aqueous solution)
Ethanol (EtOH)
Methyltrioxorhenium (Sigma‐Aldrich)
Hydrogen peroxide (H₂ O₂ )
Chloroform
Anhydrous sodium sulfate (Na₂ SO₄ )
10% aqueous NaOH
2 M HCl
Dichloromethane (CH₂ Cl₂ or DCM)
Disiopropylethylamine (DIPEA)
Argon source
Hydroxybenzotriazole (HOBt)
Diisopropylcarbodiimide (DIC)
N ,O ‐Dimethylhydroxylamine hydrochloride (Sigma‐Aldrich)
Tetrahydrofurane (absolute, puriss., THF; over molecular sieve; Fluka)
Liquid nitrogen
DIBAL‐H solution (1 M in hexane)
Ammonium chloride
Celite
Heptane
(Chloromethyl)triphenylphosphonium chloride (Sigma‐Aldrich)
n ‐Butyllithium in n ‐hexane (n ‐BuLi, Merck)
Diethyl ether (Et₂ O; p.a.; Merck)
Potassium tert ‐butylate (KOtBu)

Autoclave
Büchner funnel with appropriate round filter papers
Vacuum/inert gas manifold
Rotary evaporator (Buchi)
250‐mL round‐bottomed flasks equipped
Reflux condensers
Hotplate and magnetic stirrer
500‐mL three‐necked round‐bottomed flasks
250‐mL, 500‐mL, and 1‐liter extraction funnels
Rubber septa
2 ³ /⁴ ‐in. needle and 10‐mL syringe
Water baths
Glass‐fritted funnels with medium porosity
Fluted filter paper

CAUTION: n ‐Butyllithium is sensitive to moisture and air, and reacts violently with water, liberating extremely flammable gases (butane, hydrogen). n ‐BuLi is pyrophoric (ignites in air). It is spontaneously flammable in air and causes burns. Wearing gloves during transfer of solutions containing n ‐butyllithium is highly recommended.

Basic Protocol 2: Synthesis of DNA Building Blocks for Spin Label Attachment

Materials

3′,5′‐Bis‐O ‐(tert ‐butyldimethylsilyl)‐2′‐O ‐deoxyguanosine (Hayakawa et al., )
Dichloromethane (CH₂ Cl₂ ; puriss.; absolute; over molecular sieve; Fluka)
Triethylamine, purum (NEt₃ ; Riedel deHaën)
N ,N ‐Dimethylaminopyridine (DMAP; puriss. >99% ; Fluka)
2,4,6‐Triisopropyl‐benzenesulfonic‐chloride (puriss. >99%; Fluka)
Sodium hydrogen carbonate (NaHCO₃ ; 5% w/w aqueous solution)
MgSO₄
Silica gel (grade 60, 70 to 230 mesh, 60 Å; Merck; Sigma‐Aldrich)
n ‐Hexane; (p.a.; Merck)
Ethyl acetate (EtOAc; p.a.; Merck)
Tetrahydrofurane (THF; puriss.; absolute; over molecular sieve; Fluka)
Iodine (I₂ ; puriss. >99.5%; Fluka)
Copper iodide (CuI)
CH₂ I₂ (puriss. >99%; Fluka)
Isopentyl nitrite (purum >97%; Fluka)
Anhydrous sodium sulfate (Na₂ SO₄ )
Ammonia‐saturated ethanol
N ,N ‐Dimethylformamide‐dimethylacetal (purum >95%; Fluka)
Dimethylformamide (DMF; puriss.; p.a. >99.8%; ACS Fluka)
Methanol (MeOH)
TEA⋅3HF (Fluka)
Pyridine (puriss.; absolute; over molecular sieve; Fluka)
4,4′‐Dimethoxy‐trityl chloride (DMTr‐Cl; 98%; Avocado)
Toluene
DCI
2‐Cyanethoxy‐di‐(di‐N ,N ‐iso ‐propylamino) phosphine (99%; Aldrich)
Brine
Acetone
5% TEA

Magnetic hotplate and stir bar
250‐mL extraction funnel
Rotary evaporator (Buchi)
Three‐necked flasks with condensers
Rubber septa (+15.9 mm)
500‐mL tight, high‐pressure tubes (schott)
Glass‐fritted funnels with medium porosity
Fluted filter paper

Basic Protocol 3: Synthesis of RNA Building Blocks for Spin Label Attachment

Materials

Cytidine
Pyridine (puriss., absolute; over molecular sieve; Fluka)
Markiewicz‐protecting group (C₁₂ H₂₈ OSi₂ Cl₂ )
Toluene
Silica gel (grade 60, 70 to 230 mesh, 60 Å; Merck or Sigma‐Aldrich)
Ethyl acetate (EtOAc; p.a.; Merck)
Methanol (MeOH)
N ,N ,N ′,N ′‐Tetramethylethylendiamine (TEMED; puriss. 98%; Fluka)
N ‐Iodosuccinimide (98%; Avocado)
Dimethylformamide (puriss.; p.a. >99.8%; ACS Fluka)
Dichloromethane (CH₂ Cl₂ ; puriss.; absolute; over molecular sieve; Fluka)
Saturated sodium thiosulfate solution
Anhydrous sodium sulfate (Na₂ SO₄ )
Isopropanol (i ‐Pro)
N ,N ‐Dimethylformamide‐dimethylacetal (purum >95%; Fluka)
n ‐Hexane (p.a.; Merck)
Acetone
Tris(2‐acetoxyethyl)orthoformate (Dharmacon)
Pyridinium p ‐toluenesulfonate (puriss. >99%; Fluka)
4‐tert ‐Butyl‐dimethyl‐siloxy‐3‐penten‐2‐one (Fluka)
Acetonitrile
Hydrofluoric acid
Diisopropylamine (DIPA; puriss.; p.a. >99.0%; Fluka)
Benzhydroxy‐bis(trimethylsilyloxy)chlorosilane (BzHCl; Dharmacon)
Sodium hydrogencarbonate (5% w/w NaHCO₃ aqueous solution)
Brine
Triethylamine (Et₃ N; purum; Riedel deHaeën)
Methyl‐N ,N ,N ′,N ′‐tetraisopropylphosphordiamidite (puriss.; p.a. >99.0%; Fluka)
1H ‐Tetrazole (0.45 M in ACN for DNA synthesis; Fluka)
Ethanol

Magnetic stir plate and stir bar
Rotary evaporator (Buchi)
Vacuum/inert gas manifold
50‐ and 100‐mL flask with reflux condenser
Rubber septa (+15.9 mm)
Glass‐fritted funnels with medium porosity
Fluted filter paper
Büchner funnel with appropriate round paper filter

Basic Protocol 4: DNA Synthesis without Interruption

Materials

Reagents for synthesis:
- DNA phosphoramidites of the natural bases and 5‐iodouridine (Applied Biosystems), 5‐iodo‐desoxycytidine, and 2‐iodo‐desoxyadenosine (synthesize in laboratory)
- Trichloroacetic acid (TCA) in CH₂ Cl₂ (Perseptive Biosystems)
- CAP A (N ‐methyl‐imidazole; Proligo Biochemie)
- CAP B (acetic anhydride; Proligo Biochemie)
- Activator: dicyanoimidazole (DCI), 0.25 M in acetonitrile (Proligo Biochemie) or tetrazole, 0.45 M in acetonitrile (Biosolve), store over molecular sieve
- Oxidizer: iodine, 4.3 g in THF/pyridine/water, 7:1:2 v/v/v (Biosolve)
- Prepacked columns: clear pore glass (CPG) with the first base attached via a succinyl‐linker, 1 mmol, 500 Å; (Applied Biosystems)
- Phosphate buffer (see recipe )
Ammonia (p.a. 32%; Grüssing) mixed with p.a. methanol in a ratio of 3:1
Sterile water (Millipore)
0.25 M Tris⋅Cl, pH 8, HPLC‐grade
1 M NaCl, HPLC‐grade
PD‐10 Sephadex columns (Amersham Biosciences)
0.1 M triethylammonium acetate (TEAA), pH 7
Acetonitrile, HPLC‐grade

Oligonucleotide synthesizer (Perseptive Biosystems)
4‐mL vials
Filter (Whatman Spartan 13/0.45 RC, 0.24‐µm)
Speed‐Vac rotary evaporator (model no. SC110, Savant)
Glass‐fritted funnels with medium porosity
Fluted filter paper
HPLC device (JASCO) in combination with an anion exchange column (DNAPack PA‐100, semi‐prep, 9 × 250–mm; Dionex (flow: 5 mL/min)
EXPEDITE synthesizer (Perseptive Biosystems)
Reversed‐phase HPLC column (Phenomenex, Jupiter 4µ‐Poter‐90A)

Basic Protocol 5: RNA Synthesis without Interuption

Materials

Solutions for RNA synthesizer:
- Acetonitrile for DNA synthesis (Biosolve)
- RNA phosphoramidites of the natural bases and 5‐iodouridine (Dharmacon), 5‐iodocytidine and 2‐iodoadenosine (synthesized in laboratory)
- Tetrahydrofurane (THF; puriss.; absolute; over molecular sieve; Fluka)
- CAP A: N ‐methylimidazole (redist. 99%; Aldrich), 10% in acetonitrile
- CAP B: acetic anhydride (puriss. p.a. ACS >99%; Riedel‐de Haën), 10% in acetonitrile
- 3% dichloroacetic acid (puriss. 99%; Fluka) in absolute CH₂ Cl₂ over molecular sieve (Fluka)
- 1H ‐Tetrazole (0.45 M in ACN; Fluka)
- t BuOOH (purum, 70% in water; Fluka) in toluene (ACS; Fluka) (see recipe )
- Prepacked columns (aminomethyl‐polystyrene with the first base protected with DMT attached via a succinyl linker, 0.2 mmol; Dharmacon)
Ammonia
Methanol (MeOH)
DEPC‐treated water (see recipe )
N ‐methylpyrrolidone
Triethylamine (Et₃ N or TEA, purum, Riedel de Haën; or puriss. p.a. >99.5%; Fluka), dried with calcium hydride (3 to 5 g per 250 mL) by heating to reflux for 3 days and distilled under inert gas before use
Triethylamine‐hydrofluoride (Et₃ N‐3HF or HF⋅TEA; see recipe )
n ‐Butanol
1 M LiCl: dissolve 42.4 g LiCl in 1 liter Millipore water
PD‐10 Sephadex columns (Amersham Biosciences)
0.4 M disodium‐2‐carbamoyl‐2‐ cyanoethylene‐1,1‐dithiolate‐trihydrate (S₂ Na₂ ; see recipe )
N ,N ‐dimethyl‐formamide (DMF, puriss. p.a. ACS >99.8%; Fluka)
40% methylamine in water (Aldrich)
TEMED/acetic acid (see recipe )

ABI 392 synthesizer (Applied Biosystems)
Magnetic hotplate and stir bar
Speed‐Vac (SC110, Savant)
Refrigerated centrifuge
Filters (0.45‐µm pore size)
Buchner funnel with appropriate round paper filter
Anion‐exchange HPLC device (JASCO) with an anion‐exchange column (DNAPack PA‐100, semi‐prep, 9 × 250–mm, Dionex), flow 5 mL/min
PD‐10 sepharose columns
MALDI‐TOF VOYAGER DE‐PRO mass spectrometer (Applied Biosystems)
Vacuum manifold
4‐mL vials

CAUTION: HF is extremely toxic and must not be used without prior knowledge. Upon adding HF, a little bit of smoke evolves and the reaction is slightly exothermic. Transfer this solution directly into a 500‐mL polyethylene glycol bottle (Nalgene) and install it immediately onto the synthesizer.

Alternate Protocol 1: RNA Synthesis with Interruption

Materials

RNA/DNA synthesized on column
Copper iodide (CuI; >99%; Riedel de Haën)
Dichloromethane (CH₂ Cl₂ ; puriss.; absolute; over molecular sieve; Fluka)
Triethylamine (Et₃ N; purum; Riedel deHaeën)
Pd(II)(PPh₃ )₂ Cl₂ (purum, >98%; Fluka)
2,2,5,5‐Tetramethyl‐pyrrolin‐1‐oxyl‐3‐acetylene (TPA)

Vacuum/inert gas manifold
10‐mL one‐necked flasks (pear‐shape or round‐bottom flasks)
1‐mL vials (4‐cm high, +8 mm, Carl Roth, cat. no. H‐300.1)
Hotplate
Teflon‐coated magnetic stir bars
Plastic polypropylene syringes
Reusable syringe needles (steel, 150‐mm length, +1.2 mm) and disposable syringe needles (120‐mm length,+0.8 mm)
Shaker

Basic Protocol 6: Sonogashira Cross‐Coupling Reaction on the Solid Phase

Materials

Oligonucleotides
10 mM sodium cacodylate
0.2 mM disodium EDTA, pH 7
2.5% formamide solution
Diethylpyrocarbonate (DEPC) water (see recipe )
Liquid nitrogen
20% aqueous ethylene glycol solution or 20% aqueous sucrose solution

Heating block
UV‐cuvettes
UV‐/VIS‐600 (Jasco) spectrophotometer equipped with a Peltier thermostat
JASCO J‐710 spectropolarimeter with a Peltier thermostat
Quartz EPR tubes
Sterile tips (Eppendorf)
80°C oven
Vivaspin concentrator (Sartorius Stedim Biotech)
Plastic polypropylene syringes
Reusable syringe needles (steel, 150‐mm length, +1.2 mm) and disposable syringe needles (120‐mm length, +0.8 mm)
Rotary evaporator (Buchi)

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Figures

Figure 7.17.1 Synthesis of the ¹⁵ N‐nitroxide TPA undefined.

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Figure 7.17.2 a. TiPBS‐Cl, 80%; b. I₂ , CuI, CH₂ I₂ , isopentyl nitrite, 84%; c. EtOH/NH₃ , 78%; d. TEA‐3HF, 86%.

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Figure 7.17.3 Synthesis of the 5‐iodo‐cytidine phosphoramidite: a. Pyridine, C₁₂ H₂₈ OSi₂ Cl₂ , 0°C, overnight, 99%; b. N ‐iodosuccinimide, DMF, 50°C, 4 hr, 73%; c. N , N ‐Dimethyl‐formamide‐dimethylacetal, DMF, 50°C, overnight, 58%; d. Tris(2‐acetoxyethyl)orthoformate, pyridinium p ‐toluene‐sulfonate, 4‐tert‐butyl‐dimethyl‐siloxy‐3‐penten‐2‐one, DCM, room temperature, 48 hr, 85%; e. TEMED⋅HF, 0°C, 2 hr, quantitative; f. diisopropylamine, BzHCl, CH₂ Cl₂ , 0°C, 83%; g. 1 H ‐Tetrazole, methyl‐ N , N , N ′, N ′‐tetraisopropylphosphordiamidite, 0°C to room temperature, 76%.

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Figure 7.17.4 Sonogashira cross‐coupling reaction.

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Figure 7.17.5 Photo of the equipment for Sonogashira cross‐coupling reaction.

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Figure 7.17.6 Available nitroxides. #, deuterated compound; *, ¹⁵ N‐labeled compound.

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Figure 7.17.7 Synthesis of the nitroxide TEMPA 2.

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Figure 7.17.8 DNA building blocks. These are the iodo‐modified phosphoramidites for the DNA synthesis for Sonogashira cross‐coupling chemistry.

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Figure 7.17.9 RNA building blocks. These are the different phosphoramidite building blocks for RNA synthesis by ACE or TBDMS chemistry that can be used for Sonogashira cross‐coupling chemistry.

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Figure 7.17.10 Correlation of PELDOR and MD distances for RNAs1‐6 (open squares) and DNAs 1‐5 (open circles).

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Figure 7.17.11 Schematic secondary structure diagram of the cUUCGg tetraloop showing nucleotide conformations and critical interactions determined by NMR and MD‐simulation.

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Figure 7.17.12 (A ) Free; (B ) bound secondary structure of aptamer; (C ) neomycin‐B.

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Figure 7.17.13 CD‐spectrum of double‐labeled oligonucleotides.

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Figure 7.17.14 2‐D‐NMR data before and after the reduction of the spin label (Wurm et al., ).

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Figure 7.17.15 Secondary structures (with spin‐labeled nucleotides in red), baseline‐corrected PELDOR time traces, and distance distributions for double‐labeled 14‐mer cUUCGg tetraloop hairpin RNA (upper panel) and the 27‐mer neomycin‐sensing riboswitch (lower panel). In‐cell data after different incubation times are compared with in vitro data (green).

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Figure 7.17.16 Secondary structure (with spin‐labeled nucleotides in red), baseline corrected PELDOR time traces and distance distribution for 12‐bp double‐labeled DNA in vitro (green), and in‐cell (incubation time indicated in legend). Data were fitted with two Gaussians (Krstic et al., ).

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Literature Cited

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