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Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis

来源:生物谷  2010/10/12   访问量:5445
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Methods for fluorescent probing at a defined position of RNA provide powerful tools for analyzing the local structural conformation of functional RNA molecules by tracking fluorescence changes. In this article, we describe the site-specific fluorescent probing of RNA by transcription with an expanded genetic alphabet, using an extra, unnatural base pair between 2-amino-6-(2-thienyl)purine (s) and pyrrole-2-carbaldehyde (Pa). The protocol comprises template DNA preparation containing Pa, transcription involving fluorescent s incorporation and structural analysis of transcripts. The s base is strongly fluorescent, and its nucleoside 5′-triphosphate is site-specifically incorporated into RNA transcripts, opposite Pa in DNA templates, by conventional T7 transcription. The fluorescent intensity of s changes depending on its environment around the probe site, providing clues about the local structural features of RNA molecules. This is the first protocol for RNA transcript preparation with fluorescent labeling at a desired position. The procedure for s-containing RNA preparation takes about 2–3 d.

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Introduction

The tertiary folding and structural changes of nucleic acids are closely related to their biological functions. Thus, analytical methods for the physical properties of DNA and RNA molecules in solution will provide important information. One of the methods is the fluorescent probing of a specific region in functional nucleic acid molecules by introducing a base-analogue fluorophore into RNA, in which the fluorescent intensity changes depending on the local environment. Many reports have described the development and application of fluorescent base analogues, such as formycin, 2-aminopurine, 1,N6-ethenoadenosine, 1,3-diaza-2-oxo-phenoxazine (tCo), 4-amino-1H-benzo[g]quinazoline-2-one, 5-methoxyquinazoline-2,4-(1H,3H)-dione and size-expanded base analogues1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. For example, a fluorescent adenine analogue, 2-aminopurine, has been widely used for analyzing the local structures of DNA and RNA molecules1, 5, as well as for detecting their interactions with other molecules2, 6. Their fluorescent intensity changes reflect the conformational changes of the local nucleic acid structures, because fluorescence is strongly quenched when the base is stacked with neighboring bases or other aromatic components. However, a limitation of using such fluorescent base analogues is the difficulty encountered in the preparation of labeled nucleic acids. In general, nucleic acids containing these fluorescent base analogues are chemically synthesized, and it is difficult to generate large nucleic acids, especially RNA molecules. The enzymatic ligation of two or more short RNA fragments is an alternative method14, but it is still laborious. Although the enzymatic incorporation of the triphosphate substrates of fluorescent base analogues into nucleic acids can be accomplished by polymerase reactions3, 4, these analogues are incorporated in place of one of the four natural bases, and thus, the site-specific incorporation at a desired position is difficult.

Another new method for site-specific, fluorescent labeling is the use of an expanded genetic alphabet with an extra, unnatural base pair. Many types of unnatural base pairs have been designed, and their enzymatic incorporation into nucleic acids was examined in polymerase reactions15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. If we create a fluorescent unnatural base that can be directly incorporated into RNA by transcription mediated by the complementarity of the unnatural base pair, we could provide site-specific fluorescent labeling at a desired position in RNA molecules22, 23, 25.

We present an unnatural base pair system that enables the site-specific incorporation of a fluorescent base analogue into RNA by transcription. We have developed several unnatural base pairs that function in replication and/or transcription16, 17, 19, 21, 24, 25, 27. Among them, the unnatural base pair between a fluorescent purine analogue, 2-amino-6-(2-thienyl)purine (denoted by s), and its pairing partner, pyrrole-2-carbaldehyde (denoted by Pa) (Fig. 1a), enables the efficient site-specific incorporation of the fluorescent s base into RNA, opposite Pa in DNA templates, by T7 RNA polymerase25 (Fig. 1b). The fluorescence of the s base is characterized by excitation at 299 or 352 nm and emission at 434 nm (Fig. 1c), and is also quenched by stacking with neighboring bases30. Relative to those of 2-aminopurine, both the excitation and emission centers of s are shifted to longer wavelengths, and the fluorescent s probe is not strongly quenched by stacking with neighboring bases in nucleic acid strands. These properties of the s base are suitable for observing fluorescent intensity changes and provide a useful tool for studying the local structural conformations of functional RNA molecules, even with longer sizes. Pa-containing DNA templates are prepared by a DNA synthesizer using the amidite reagent of Pa, and the triphosphate of s (sTP), commercially available from Glen Research. The DNA templates can be amplified by PCR using another unnatural DsPa pair system24. In this article, we describe the fluorescent s labeling of specific positions of tRNA molecules (76-mer) by T7 transcription using Pa-containing DNA templates, and show the structural analysis of the L-shaped formation of tRNA molecules, depending on the temperature and magnesium concentration. The analysis revealed that one magnesium ion is important to form the tertiary structure around position 47 in the extra loop of the tRNA.

Figure 1: The sPa transcription system.
Figure 1 : The s|[ndash]|Pa transcription system.

(a) The structure of the sPa pair. (b) Site-specific fluorescent s incorporation by T7 transcription mediated by the sPa pairing. (c) Excitation and emission scans of the s ribonucleoside (1 μM), in 50-mM sodium cacodylate (pH 7.2), 50-mM KCl and 0.1-mM EDTA, at 20 °C.

Full size image (42 KB)

Although the efficiency of transcription involving s incorporation is as high as that of the usual transcription comprising natural bases, the incorporation of s into the aborting region (+1 to +8) in transcripts or into the 3′ terminus is less efficient, and thus these positions should be avoided. In addition, this protocol is for in vitro transcription using the unnatural sPa pair and not for in vivo transcription. Although the s-labeled RNA molecules prepared by this method could be used for a wide variety of experiments, we do not recommend their application in in vivo experiments because of the uncertain toxicity and biological activity of s-nucleoside derivatives31.

Experimental design

The protocol comprises four steps: (i) design and preparation of DNA templates (Fig. 2), (ii) T7 transcription, (iii) purification of transcripts and (iv) structural analysis suited for various purposes.


Design and preparation of DNA templates. The DNA templates for T7 transcription are designed by replacing the natural base at the desired position by Pa. The promoter region requires its nontemplate strand to form a duplex, but for other template regions, either single or double strands are acceptable. Partially single-stranded DNA templates with base pairs only in the −17 to +1 promoter region are as active in in vitro transcription as fully double-stranded DNA templates32, 33. In the PROCEDURE reported below, both template and nontemplate strands are synthesized and then annealed before T7 transcription. Synthetic DNA fragments containing Pa are prepared by chemical DNA synthesis34, 35, using the amidite reagent of Pa (Glen Research), or are available commercially. There is no protecting group in the Pa amidite, and standard solid-phase DNA synthesis can be applied without any changes. The protecting groups for the natural bases in the chemically synthesized DNA fragments are removed by the conventional method (deprotection)34, 35, by heating the DNA solution at 55 °C for 6 h after the fragment release from the CPG column by an incubation at room temperature (RT, 18–25 °C) for an hour in concentrated NH4OH. After deprotection, the solvent is evaporated to dryness with a centrifugal evaporator.

There are several options for preparing Pa-containing DNA templates, depending on the size of the transcript and the labeled position. Figure 2 shows the flowchart to choose the appropriate method for template preparation. For RNA transcripts shorter than 70–80 nucleotides, Pa-containing DNA templates (less than 100 nucleotides containing the T7 promoter region) are directly prepared by chemical synthesis. However, the direct chemical synthesis of DNA fragments longer than 100 nucleotides is not recommended, because of the accumulation of sequence errors with the increase in solid-phase synthesis cycles36, 37. The direct syntheses of 90-mer DNA fragments increased the error ratio to 10–11 random errors per kb, relative to 4–5 random errors per kb in 60-mer DNA syntheses37. For 80- to 150-nucleotide RNA transcription, primer extension using two DNA fragments is applicable24, 38. For the generation of RNAs longer than 150 nucleotides, templates can be prepared by enzymatic ligation using a set of short DNA fragments24. Recently reported chemical gene synthesis methods will also be useful for DNA template construction for much longer (>200-mer) transcripts39, 40, 41. If the fluorescent-labeling position is located near the 3′ terminus of the transcript, PCR amplification using 3′ primers containing Pa is a convenient method19, 21. For these enzymatic preparations, any natural bases can be used as the complementary base of Pa. Thus, any PCR method without unnatural base substrates can be used. However, as the base pairings between Pa and natural bases cause duplex destabilization during the annealing process for ligation and PCR priming, localization of Pa in the middle of the DNA fragment is recommended.

T7 transcription. Conventional transcription methods using T7 RNA polymerase can be used32, 33 with a Pa-containing DNA template and the ribonucleoside triphosphate of s (sTP). In general, equal amounts of sTP and natural base triphosphates (NTPs), at final concentrations of 1 to 2 mM each, are added to the reaction. There is no need to extend the incubation period for the s incorporation into transcripts.

Purification of transcripts. Conventional polyacrylamide gel electrophoresis is used for the purification of transcripts42. The s-containing transcripts can be detected through the glass plate during electrophoresis, using irradiation at ~350 nm wavelength from a handheld UV lamp.

Structural analysis. The s base emits fluorescence centered at 434 nm (fluorescence quantum yield = 0.41)30, characterized by two major excitation maxima (299 and 352 nm) at pH 7.0. The fluorescent intensity decreases with increased stacking interactions with neighboring bases, and thus the local conformational changes of RNA molecules can be traced by placing s at the target position. A typical example is the introduction of s into a thermally stable RNA hairpin with a GAAA loop RNA25; the fluorescent intensity of a GsAA loop hairpin is much greater than that of a GAsA loop hairpin, suggesting that the first A protrudes from the loop and, in contrast, the second A is stacked with the third A in the GAAA loop hairpin. This difference can be clarified by measuring the temperature dependence of the fluorescent intensity changes. In addition, the fluorescence melting temperature (T m f), obtained from the temperature-dependent fluorescent changes, also provides valuable information about the thermal stability of the local structure around the position of the probe. In this study, we show the s-base probing of yeast tRNAPhe, in which we chose two positions, U47 and G57, as s-modification sites in which to observe the tRNA L-shaped folding, depending on the magnesium concentration.

Controls. A transcript with the original natural base sequence is used as a control throughout the experiments. In gel purification of transcripts, the mobility of the s-containing transcript on the gel should be almost the same as that of the control transcript on the gel. To ensure that the introduction of s does not affect the global structure of the transcript, it is useful to carry out a comparison between the UV melting temperatures (T m) of the s-containing transcripts and the control transcript. For example, the T m values of the control, U47s and G57s tRNA transcripts are 65.5, 65.2 and 65.1 °C, respectively, in 50-mM sodium cacodylate (pH 7.2), 50-mM KCl and 2-mM MgCl2(ref. 25).


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Materials

REAGENTS


Caution For all reagents, follow the manufacturer's recommendations for the storage of materials. Wear proper safety equipment, including safety glasses and appropriate gloves for all experiments. It is important to take care to avoid nuclease contamination from conceivable sources.
  • Nuclease-free, deionized, sterile water (dH2O)
  • High-purity water (water, Milli-Q)
  • ElectroZap (Ambion, cat. no. AM9785). It effectively removes RNases, DNases and other proteins from laboratory instruments.
  • HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; Nacalai Tesque, cat. no. 17546-05)
  • Sodium hydroxide (Nacalai Tesque, cat. no. 31511-05)
  • Tris-HCl buffer solution (1 M, pH 7.6) (Nacalai Tesque, cat. no. 35436-01)
  • TE buffer solution (pH 8.0) (Nacalai Tesque, cat. no. 32739-31)
  • EDTA solution (0.5 M, pH 8.0) (Nacalai Tesque, cat. no. 14347-21)
  • Sodium acetate solution (3 M, pH 5.2) (Nacalai Tesque, cat. no. 31138-31)
  • Tris-borate-EDTA buffer (10×) (10× TBE Buffer; Nacalai Tesque, cat. no. 35440-44)
  • Ethanol (99.5%) (Nacalai Tesque, cat. no. 14713-95)
  • Urea (Amresco, cat. no. 0568-500G)
  • Glycogen solution (20 mg ml−1) from oyster, nuclease tested (Nacalai Tesque, cat. no. 17110-11)
  • SYBR Green II (Takara, cat. no. 50523)
  • Xylene cyanol FF (XC; Nacalai Tesque, cat. no. 36629-64)
  • Bromophenol blue (BPB; Nacalai Tesque, cat. no. 05808-61)
  • Potassium chloride (Nacalai Tesque, cat. no. 28538-75)
  • Magnesium chloride hexahydrate (Nacalai Tesque, cat. no. 20937-72)
  • Sodium chloride (Nacalai Tesque, cat. no. 31333-45)
  • sTP (10 mM) (Glen Research, cat. no. 81-3522-02)
  • NTP set (100-mM solutions) (ATP, CTP, GTP, UTP; GE Healthcare, cat. no. 27-2025-01)
  • Triton X-100 (Nacalai Tesque, cat. no. 35501-02)
  • Cloned T7 RNA polymerase (Takara, cat. no. 2540A; the 10× transcription buffer (400-mM Tris-HCl (pH 8.0), 80-mM MgCl2, 20-mM spermidine) and 50-mM DTT are included)
  • Acrylamide (monomer) (Nacalai Tesque, cat. no. 00861-01)
    Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
  • N, N′-methylenebisacrylamide (Nacalai Tesque, cat. no. 22407-52)
  • Ammonium persulfate (APS; Amresco, cat. no. M133-25G)
  • Tetramethylethylenediamine (TEMED; GE Healthcare, cat. no. 17-1312-01)
  • Template DNA strand containing Pa (prepared in-house (Step 1) or available commercially from custom oligonucleotide suppliers synthesizing DNAs using the Pa amidite (Glen Research, cat. no. 10-1523), such as Biosearch Technology, Eurogentec, IDT, Invitrogen, Metabion and Sigma-Custom products). See Experimental design, Figure 2 and Table 1.
  • Nontemplate DNA strand (prepared in-house (Step 2) or available commercially from oligonucleotide suppliers). See Experimental design, Figure 2 and Table 1.

EQUIPMENT

  • Ultrafree-MC centrifugal filter units with microporous membrane (Millipore, cat. no. UFC30GVNB)
  • Steriflip-GP filter units (0.22 μm, polyethersulfone, γ-irradiated; Millipore, cat. no. SCGP00525)
  • Microcon YM-10 (Millipore, cat. no. 42421)
  • Handy aspirator (ULVAC, cat. no. MDA-015)
  • Dessicator
  • pH meter
  • Parafilm
  • Autoclave
  • Vertical slab gel electrophoresis systems (Biocraft, cat. no. BE-620).
    Critical We typically use long glass plates (20 cm × 40 cm; thickness 1 or 2 mm) for higher resolution to separate oligonucleotides of different lengths.
  • PowerPac 3000 with temperature probe (100/120 V) (Bio-Rad, cat. no. 165-5059).
    Critical A temperature probe allows automatic and precise control of temperature during electrophoresis.
  • Luminescent image analyzer LAS-4000 (Fujifilm).
    Critical The use of UV LED with an L41 UV filter allows the direct detection of fluorescence from s-containing transcripts, without staining by SYBR Green II.
  • TLC plate silica gel 60 F.254 (Merck, cat. no. 5065-32137)
    Critical A UV fluorescent indicator enables the detection of a high concentration of oligonucleotides in a gel as shadows under 254 nm UV light, when the gel is placed on the TLC plate (UV shadowing).
  • Polaroid MP4 Land Camera (Polaroid) with a red filter and Polaroid film.
    Critical Standard-type cameras with the same equipment can also be used for UV shadowing.
  • Ultraviolet handheld lamp (Spectroline, cat. no. ENF-260C/12).
    Critical 365-nm UV allows the detection of s-containing transcripts, whereas 254-nm UV is used for UV shadowing to detect the gel bands of oligonucleotides.
  • High-speed microrefrigerated centrifuge (Tomy, cat. no. MRX-150)
  • Centrifugal concentrator (Tomy, cat. no. CC-105) with a low-temperature trap (Tomy, cat. no. TU-105)
  • UV/VIS spectrophotometer (Beckman Coulter, cat. no. DU-530)
  • UV cell (TOSOH Quartz, cat. no. T-9 M-UV 10-3)
  • FP-6500 Fluorescence Spectrometer (JASCO, cat. no. FP-6500)
  • Quartz cell with a Teflon cap (TOSOH Quartz, cat. no. T-607 ES3)
  • Dry thermo unit DTU-1B (TAITEC, cat. no. 14035)
  • Microincubator (TAITEC, cat. no. M-36)
  • Mild mixer (TAITEC, cat. no. PR-36)
  • Programmable thermal controller (MJ Research, cat. no. PTC-100)
  • Razor blade (Feather Safety Razor, cat. no. FAS-10)

REAGENT SETUP

  • 1× TBE buffer
    • Dilute 100 ml of 10× TBE buffer with 900 ml water. This can be stored at RT for several months.
  • 40% (wt/vol) acrylamide/bis stock solution
    • Dissolve 190 g acrylamide and 10 g N, N′-methylenebisacrylamide in water and prepare a final volume of 500 ml. Store at 4 °C with protection from light.
      Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
  • Denaturing 10% (wt/vol) acrylamide/bis stock
    • Mix 80 ml of 40% acrylamide stock, 32 ml of 10× TBE buffer and 134.4 g of urea and prepare a final volume of 320 ml with water. Store at 4 °C with protection from light. As for the acrylamide percentage, a 10% acrylamide gel is used for ~70-mer fragments, and the acrylamide gel percentage is changed depending on the nucleotide length43. The percentage of the polyacrylamide gel should be lower with longer oligonucleotides.
      Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
  • 10% (wt/vol) APS
    • Dissolve 1 g APS in 10 ml dH2O. This solution can be stored at 4 °C for several weeks.
  • Denaturing 10% (wt/vol) polyacrylamide gel
    • For a 20 cm × 40 cm × 1 mm gel, set up two glass plates with 1-mm-thick spacers. Mix 80 ml denaturing 10% polyacrylamide stock with 400 μl of 10% APS and 80 μl TEMED gently and quickly, and then pour the solution into the glass plate set. Insert a comb and cover with a Saran Wrap for protection against desiccation. For purification of oligonucleotides, preparing the gel several hours before use is recommended to ensure complete polymerization, although the gel usually polymerizes in about 30 min. We typically use gels with a thickness of 2 mm for the purification of chemically synthesized DNA fragments on a 200-nmol scale.
      Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
  • Gel-loading buffer (10 M urea in 1× TBE buffer)
    • Mix 10× TBE buffer (4 ml) and urea (24 g), and bring to a final volume of 40 ml using dH2O. As 10 M urea is difficult to dissolve, incubation at 37 °C is helpful. After filtration with a Steriflip-GP filter, store the solution in aliquots at −20 °C. A gel-loading buffer with a dye, such as 0.05% (wt/vol) XC or BPB, is also used as a marker.
      Critical Incubate the aliquot at 75 °C to completely dissolve the urea before use.
  • 1 mg ml−1 glycogen
    • Dilute 1 ml of glycogen solution with dH2O (19 ml). Store the solution in aliquots at −20 °C for up to a year.
  • 2.5 M sodium chloride (NaCl)
    • Dissolve sodium chloride (5.84 g) in water and bring to a final volume of 200 ml. Sterilize the solution by autoclaving or by filtration. This solution can be stored at RT for up to a year.
  • 10× annealing buffer (100-mM Tris-HCl (pH 7.6), 100-mM NaCl)
    • Mix 1 ml of 1 M Tris-HCl buffer solution (pH 7.6), 0.4 ml of 2.5 M NaCl and 8.6 ml of dH2O. Sterilize the solution by autoclaving or by filtration. This solution can be stored at RT for up to a year.
  • 70% ethanol
    • Mix ethanol (28 ml) with dH2O (12 ml). This solution can be stored at −20 °C for several months.
  • 1 M sodium hydroxide (NaOH)
    • Dissolve sodium hydroxide (2.0 g) in water (50 ml). Sterilize the solution by autoclaving or by filtration. This solution can be stored at RT for up to a year.
      Caution NaOH is corrosive. Handle with care.
  • 200-mM HEPES–Na (pH 7.0)
    • Dissolve HEPES (7.15 g) in dH2O (100 ml). Adjust the pH to 7.0 using 1 M NaOH (~10 ml) and bring to a final volume of 150 ml with dH2O. This solution can be stored at RT for several months.
  • 1 M potassium chloride (KCl)
    • Dissolve potassium chloride (7.46 g) in water and bring to a final volume of 100 ml. Sterilize the solution by autoclaving or by filtration. This solution can be stored at RT for up to a year.
  • Mensurative buffer (20-mM HEPES–Na (pH 7.0), 50-mM KCl)
    • For 50 ml, mix 200-mM HEPES–Na (5 ml, pH 7.0), 1 M KCl (2.5 ml) and dH2O (42.5 ml). This solution can be stored at RT for several months.
  • 160-mM magnesium chloride (MgCl2)
    • Dissolve magnesium chloride hexahydrate (1.63 g) in water to a final volume of 50 ml. Sterilize the solution by autoclaving or by filtration. This solution can be stored at RT for up to a year.
  • 0.2% Triton X-100
    • Mix Triton X-100 (10 μl) with dH2O (990 μl) (1%, vol/vol). Mix 200 μl of the 1% solution with dH2O (800 μl). Store the solution in aliquots at −20 °C for up to a year.
  • 25-mM NTP (each) mix
    • Mix equal volumes of 100 mM ATP, GTP, CTP and UTP solutions. This mixture can be stored at −20 °C for several months.
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Procedure

  1. Preparation of template DNA (for shorter than 80-mer transcripts)Timing: 2–3 dSynthesize34, 35 or purchase the nontemplate DNA strand (see Experimental design for further information, Fig. 2 and Table 1).
  2. Synthesize34, 35 or purchase the template DNA strand containing Pa (see Experimental design for further information, Fig. 2 and Table 1).
  3. Prepare a denaturing gel (10% (wt/vol) polyacrylamide–7 M urea gel) for the purification of each DNA fragment (see REAGENT SETUP).
    Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
    Critical step Ensure complete polymerization of the gel. After removing the comb, rinse the wells thoroughly.
  4. Place the gel in the electrophoresis apparatus and fill the upper and lower chambers with 1× TBE buffer.
  5. Before loading the sample solutions, run the gel for 30–60 min at 50 W (constant temperature mode: 50 °C), until the temperature of the gel reaches 45–50 °C.
  6. Dissolve each DNA fragment (nontemplate and template strands from Steps 1 and 2, respectively, at ~100 nmol) in 200 μl of dH2O and add 200 μl of gel-loading buffer.
  7. Mix the solutions and heat at 75 °C for 3 min to denature the DNA fragments.
  8. Turn off the power supply and flush out the urea leached from the wells.
    Critical step If the urea is not flushed out, then loading sample solutions by gravity is very difficult.
  9. Carefully load the sample solutions from Step 7 into the wells and load the marker dye solutions into unused wells to monitor mobility during electrophoresis.
  10. Run the gel until marker dyes reach predetermined positions. For example, on a 10% polyacrylamide–7 M urea gel, the positions of the XC and BPB markers correspond to those of ~70-mer and ~25-mer DNA fragments, respectively42.
  11. Turn off the power supply and remove the gel plate from the apparatus.
  12. Detach one glass plate and cover the gel with Saran Wrap.
  13. Turn the glass plate over and remove the other glass plate from the gel.
  14. Place the gel with Saran Wrap on a TLC plate.
  15. Identify the shadow bands corresponding to the desired DNA fragments on the gel with a handheld UV lamp (254 nm).
    Caution UV is dangerous to the skin and eyes. Wear protective gloves and glasses.
  16. Excise each band with a sterile razor blade, and transfer each gel slice to a 50-ml conical tube.
  17. Crush gel slices with a spatula, and add 5.5 ml of dH2O to each tube.
  18. Attach the tubes to the mild mixer and gently agitate the tubes at 37 °C for more than 10 h.
  19. Pass the eluted solutions through Steriflip-GP filters.
    Critical step Remove small gel slices before ethanol precipitation of the purified fragments eluted from the gel slices. These impurities are also precipitated and cause inaccuracies in concentration determination by UV absorbance.
  20. Divide the filtrate for each fragment (about 4.8 ml) into 1.2-ml aliquots in four 5-ml round-bottom tubes.
  21. Add 2 μl of glycogen (1 mg ml−1), 120 μl of 3 M sodium acetate and 2.5 ml of prechilled ethanol per tube.
  22. After mixing well, store the tubes at −20 °C for at least 1 h.Pause Point The samples can be stored at −20 °C for several days.
  23. Centrifuge at 4 °C for 40 min at 13,000g, and discard the supernatants.
  24. Add 500 μl of prechilled 70% ethanol per tube for washing the precipitate.
  25. Centrifuge at 4 °C for 5 min at 13,000g, and discard the supernatants.
    Critical step Do not disturb the DNA pellets.
  26. Evaporate the residual ethanol with a centrifugal concentrator.Pause Point Samples can be stored at −20 °C for several weeks.
  27. Add 100 μl of dH2O to each tube, and vortex.
  28. Combine the dissolved solutions for each fragment in a new 1.5-ml tube (total volume: ~400 μl).
  29. Determine DNA concentration from its UV absorbance. Add 5 μl of DNA solution to 600 μl of TE buffer for dilution. The total volume is dependent on your spectrometer. The molar extinction coefficient of the Pa nucleoside at pH 7 is about 7,000 at 260 nm.Pause Point Samples can be stored at −20 °C for several months.
  30. Dilute each DNA fragment to a final concentration of 25 μM in dH2O.
  31. Set up the mixture tabulated below in a 0.65-ml tube to anneal the corresponding template and nontemplate DNA strands. Heat the mixture to 95 °C for 3 min, and then cool it to 4 °C slowly (−0.1 °C per s), using a programmable thermal controller.
    Component Volume (μl)
    25-μM template 40
    25-μM nontemplate 40
    10× annealing buffer 10
    dH2O 10
    Pause Point Samples can be stored at −20 °C for several weeks.
  32. Trial transcription and condition optimizationTiming: 9 hIn 0.65-ml tubes, prepare the mixtures tabulated below for 20-μl transcription reactions, containing different concentrations of MgCl2, natural NTPs and sTP.
    Component Volume (μl ) Final concentration
         
    10× transcription buffer 2.0
    50-mM DTT 2.0 5 mM
    0.2% Triton X-100 1.0 0.01%
    10-mM sTP 2.0–4.0 1–2 mM
    25-mM (each) NTP mix 1.6 2 mM
    160-mM MgCl2 2.0–4.0 (additional) 16–32 mM
    100-mM GMP 0–2.0 0–10 mM
    10-μM template/nontemplate DNA 1.0 0.5 μM
    50 unit per μl T7 RNA polymerase 1.0 2.5 unit per μl
    dH2O up to 20  

    Critical step 1× transcription buffer originally contains 8-mM MgCl2. The addition of excess amounts of GMP allows the monophosphate to be incorporated at the 5′ termini of transcripts. Furthermore, in some cases, the addition of GMP may reduce the production of the full-length +1 products yielded by the random incorporation of an uncoded extra-natural base16.
    Critical step At first, the conditions should be based on those of the original transcription using templates without Pa and composed of only natural bases. Thereafter, optimize the MgCl2 and sTP concentrations, as the addition of sTP may change the balance of magnesium ions required for efficient transcription. To monitor the misincorporation rate of sTP opposite a noncognate natural base in the template, it is recommended to use the template without Pa for the reactions.
    Critical step Do not mix the transcription buffer (containing spermidine) directly with the DNA. Spermidine forms a complex with nucleic acids and may form an insoluble precipitate. In addition, avoid mixing high concentrations of MgCl2 and nucleotides directly, as this may result in a precipitate.
  33. Incubate the transcription reactions at 37 °C for 6 h.Troubleshooting
  34. Prepare a denaturing 10% (wt/vol) polyacrylamide gel for analyzing transcripts.
    Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
  35. Add 20 μl of gel-loading buffer to the reactions.Pause Point The solutions can be stored at −20 °C for several days; however, less time is preferable to get the best results.
  36. Set up gel electrophoresis according to Steps 4 and 5.
  37. Incubate sample solutions at 75 °C for 3 min.
    Critical step This procedure denatures the transcribed products.
  38. Load the sample solution and run the gel according to Steps 8–14. You can darken the room and illuminate the running gel with a handheld UV lamp (365 nm). During electrophoresis, s fluorescence can be detected through the glass plate at the positions of unreacted triphosphates and the products containing s, if the transcription yield is sufficiently high.
    Caution UV is dangerous to the skin and eyes. Wear protective gloves and glasses.
  39. Estimate the amounts of the full-length products from their band densities, by 254-nm UV shadowing on a fluorescent TLC plate. Check the fluorescence of s-containing transcripts by 365-nm illumination with a handheld UV lamp or with a luminescent image analyzer, LAS-4000 (method: fluorescence: DAPI mode). The bands of s-containing products sometimes show a bluish color under 254-nm UV shadowing, and light-blue fluorescence under UV light without the fluorescent TLC plate.
    Caution UV is dangerous to the skin and eyes. Wear protective gloves and glasses.Troubleshooting
  40. Large-scale transcription and purificationTiming: 1 dPrepare the 300-μl transcription reaction in a 1.5-ml tube, using the optimized amounts of MgCl2 and sTP.
    Component Volume (μl) Final concentration
    10× transcription buffer 30
    50-mM DTT 30 5 mM
    0.2% Triton X-100 15 0.01%
    10-mM sTP Variable Optimized
    25-mM (each) NTP mix Variable Optimized
    160-mM MgCl2 Variable Optimized
    100-mM GMP Variable Optimized
    10-μM template/nontemplate DNA 15 0.5 μM
    50 unit per μl T7 RNA polymerase 15 2.5 Unit per μl
    dH2O up to 300  
  41. Incubate the transcription reaction at 37 °C for 6 h.Troubleshooting
  42. Add 300 μl of gel-loading buffer to the transcription reaction.Pause Point The solutions can be stored at −20 °C for several days; however, less time is preferable to get the best results.
  43. Prepare a denaturing 10% (wt/vol) polyacrylamide gel (40 cm × 20 cm × 1 mm) for purification of the full-length products and set up gel electrophoresis according to Steps 4 and 5.
    Caution Acrylamide is a neurotoxin, and care should be taken to avoid exposure.
    Critical step This gel should be prepared several hours before use to ensure complete polymerization. Preparation the previous day is recommended.
  44. Electrophorese according to Steps 36–39 and identify the desired band on the gel by UV shadowing or s-base fluorescence.Troubleshooting
  45. Excise the band with a sterile razor blade, and transfer the gel slices to a 50-ml conical tube.
  46. Crush the gel slices with a spatula, and add 5.5 ml dH2O.
    Critical step With smaller amounts of gel slices, less dH2O is sufficient for the elution. Estimate the approximate volume of the slices and add 1–2 volumes of dH2O to the tube.
  47. Attach the tube to the mild mixer and gently agitate the tube at 37 °C for more than 10 h.
  48. Collection of purified transcripts after elutionTiming: 4 hCollect purified transcripts according to Steps 19–26. The pellet of RNA containing s shows light-blue fluorescence under 365-nm UV light.
    Critical step The presence of residual ethanol may cause adverse effects on the following experiment.Pause Point The pellet can be stored at −20 °C for several weeks; however, less time is preferable to get the best results.
  49. Add 50 μl dH2O to each tube, and vortex.
  50. Combine the dissolved solutions in a new 1.5-ml tube.
  51. Determine RNA concentration from its UV absorbance. The molar extinction coefficient of the s nucleoside at pH 7 is 8,200 at 260 nm.Pause Point The samples can be stored at −20 °C for several weeks; however, less time is preferable to get the best results.
  52. Buffer exchangeTiming: 4 hAdd 4 μl of 0.5 M EDTA (pH 8.0) to 196 μl of RNA solution (final concentration of EDTA: 10 mM).
  53. Incubate the mixture at 75 °C for 5 min, and then transfer the solution to a Microcon YM-10 centrifugal filter.
  54. Centrifuge the filter unit at 4 °C for about 1 h at 13,000g, until the residual volume is less than 20 μl.
  55. Add 200 μl of mensurative buffer (20-mM HEPES–Na (pH 7.0), 50 mM KCl).
  56. Repeat Steps 54 and 55 four times.
    Critical step Repetition is required for the removal of magnesium ions interacting with RNA.
  57. Concentrate the RNA by centrifugation, until the estimated sample concentration is higher than 2 μM.
  58. Transfer the recovered solution to a new 1.5-ml tube and determine RNA concentration from its UV absorbance.
  59. Prepare a 1-μM RNA solution by dilution with mensurative buffer.Pause Point Samples can be stored at −20 °C for several days; however, less time is preferable to get the best results.
  60. Divide the solution into 35-μl aliquots per tube for further fluorescence analyses.
    Critical step The volume is dependent on your spectrometer.
  61. Prepare 35 μl of 1-μM sTP in mensurative buffer as a reference sample.
    Critical step The volume is dependent on your spectrometer.
  62. Measurement of fluorescence changes with differing variablesTiming: 4–12 hSamples prepared in Steps 60 and 61 can now be used in different ways to assess the impact of varying conditions on fluorescence intensity and thus infer the local conformational structure. As an example, we provide here two options—follow option A to measure fluorescent melting profiles on temperature change and option B to measure fluorescent changes with different MgCl2 concentrations.
    1. Measurements of fluorescent melting profiles based on temperature change Timing: 12 h
      1. Add 35 μl of mensurative buffer containing 0.2-mM EDTA, 4-mM MgCl2 or 10-mM MgCl2 to the RNA solutions prepared in Step 60.
      2. Add 35 μl of mensurative buffer into the sTP solution prepared in Step 61.
      3. Incubate the solutions at 90 °C for 2 min, and then degas the solutions briefly under vacuum.
      4. Transfer the solution to a quartz cell with a Teflon cap.
      5. Measure fluorescence changes according to the increase in temperature (20–80 °C). The measuring conditions used with the FP6500 Fluorescence Spectrometer are as follows: band wavelength, 3 nm each; response, 0.5 s; sensitivity, high; wavelength range, 360–600 nm; data collection interval, 1 nm; excitation wavelength, 352 nm; scan speed, 1,000 nm min−1.
      6. Correct the fluorescent profiles of RNA molecules containing s by the temperature dependence of the reference (sTP) fluorescent intensity at 434 nm, using the following equation (Eq. 1)25:

        Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

        where Yct is the corrected intensity (at 434 nm) of each RNA molecule at t °C, Yt is the measured intensity (at 434 nm) of the RNA at t °C, Rt is the observed intensity (at 434 nm) of sTP at t °C and R20 is the observed intensity (at 434 nm) of sTP at 20 °C.


        Critical step The fluorescence-monitored melting curve of each RNA transcript is generally normalized by that of the nucleoside monomer of s (in this case, by sTP), because the quenching of s fluorescence by collision events with the solvent increases linearly at higher temperatures. When measuring fluorescence at the final 0.5-μM concentration, the background intensity at 434 nm is very low, and there is no need to subtract the background fluorescence spectrum for correction.
      7. From fluorescence intensity versus temperature curves, the fluorescence melting temperature (T m f) is calculated by the conventional method for determining the UV melting temperature44, using the first derivative of the thermal transition curve.
    2. Measurement of fluorescence changes with different MgCl2 concentrations Timing: 4 h
      1. Add 35 μl of mensurative buffer containing a series of MgCl2 concentrations (0, 10, 30, 50, 70, 100, 200, 500, 1,000, 1,500, 2,000 μM) to the RNA solutions prepared in Step 60.
      2. Incubate the solutions at 75 °C for 5 min, and then cool them to RT.
      3. Transfer each solution to a quartz cell.
      4. Set the cell in the fluorescence spectrometer (temperature setting: 20 °C), wait for 2 min and then measure the fluorescence of the sample.
        Critical step Use the same cell to avoid the error caused by differences between cells. Measure the fluorescence of the samples containing low MgCl2 concentrations first.
      5. Measure the fluorescence spectra of the buffer without the sample as the background. If the fluorescence intensity of the background affects that of the sample, subtract the background spectrum from the sample spectra.
      6. Plot fluorescent intensities at 434 nm against the concentrations of MgCl2. The fluorescence intensity changes reflect the effect of MgCl2 concentrations on the local conformation around the s-incorporated position.
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Timing

Steps 1–31, preparation of template DNA: 2–3 d
Steps 32–39, trial transcription and condition optimization: 9 h
Steps 40–47, large-scale transcription and purification: 1 d
Steps 48–51, collection of purified transcripts after elution: 4 h
Steps 52–61, buffer exchange: 4 h
Steps 62A and B, measurement of fluorescence changes with differing variables: 4–12 h

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Troubleshooting

Troubleshooting advice can be found in Table 2.


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Anticipated results

In this article, we show the site-specific s probing of the conformations of specific regions in the tRNA L-shaped structure, depending on the magnesium concentration and temperature.

T7 transcription of s-labeled tRNAs

Modified tRNAPhe molecules (100–300 pmol, 76-mer), containing s at either position 47 (47s) in the extra loop or 57 (57s) in the TΨC loop (Fig. 3), were prepared by transcription in the presence of 10-mM GMP, 1-mM each of NTP and sTP, and 24-mM MgCl2, with a 20-μl transcription scale using chemically synthesized DNA templates containing Pa at appropriate positions (Table 1) (refs. 25,38,45). As shown in Figure 4, we often observed higher transcription efficiency involving sPa pairing (47s and 57s in Fig. 4), relative to that of the natural-base transcription (WT in Fig. 4). Highly efficient transcription is achieved when Pa is located at any position except those within the 10 bases before both the 5′ and 3′ termini in DNA templates.

Figure 3: Tertiary structure of tRNAPhe and close-up views around the 47th and 57th bases (PDB ID: 1EVV).
Figure 3 : Tertiary structure of tRNAPhe and close-up views around the 47th and 57th bases (PDB ID: 1EVV).

(ae) The backbone is indicated in sky blue, the 47th and 57th base moieties are shown in green and Mg ions are shown by orange balls. Panels b and e are stereo views of a and d (walleye), respectively. This figure was created using PyMOL (http://www.pymol.org).

Full size image (84 KB)

Figure 4: Gel electrophoresis of transcripts generated by the unnatural base-pair system.
Figure 4 : Gel electrophoresis of transcripts generated by the unnatural base-pair system.

The main bands (shown by an arrowhead) are full-length tRNAs. Transcripts (76-mer) were observed by UV shadowing of the gel. 47s and 57s are the tRNA transcripts containing s at positions 47 and 57, respectively. WT and WT, −sTP are the products generated by the transcription of the control template with only the natural bases, in the presence and absence of sTP, respectively.

Full size image (17 KB)

Fluorescent intensity changes of s-labeled tRNAs at different temperatures and magnesium concentrations

Figure 5 shows the temperature- and Mg2+-concentration-dependent fluorescent intensity changes of 47s and 57s tRNAs, monitored at 434 nm with excitation at 352 nm. At low temperature (20 °C), the fluorescent intensity of 47s tRNA in the presence of 2-mM or 5 mM Mg2+ was significantly higher than that in the absence of Mg2+ (Fig. 5a). In contrast, the fluorescent intensities of 57s tRNA in both the presence and absence of Mg2+ were low (Fig. 5b). These results agree with the tertiary structure of the L-shaped tRNA, in which the base at position 47 protrudes toward the outside (Fig. 3d), but the base at position 57 is stacked with the neighboring bases (Fig. 3a). Thus, 47s yields the intrinsic fluorescence of s, and the fluorescence of 57s was strongly quenched. The thermal denaturation of the L-shaped structure also provided the characteristic fluorescent profiles of each position, depending on the degree of stacking between s and the neighboring bases in the denaturation process. The fluorescence melting temperatures (T m f) determined from the fluorescent profiles in Figures 5a and b were 67.5 (2 mM Mg2+) and 69.5 °C (5-mM Mg2+) for 47s and 69.5 (2-mM Mg2+) and 71.5 °C (5-mM Mg2+) for 57s. The T m f difference between 47s and 57s was 2 °C at each Mg2+ concentration, which is similar to the results obtained under different buffer conditions25. These results suggest that the stability of the local structure of the TΨC loop around position 57 in the L-shaped tRNA is slightly higher than that of the extra loop around position 47. A comparison with the UV melting temperature also provides useful information about the stability difference between the total structure and each local conformation25.

Figure 5: The fluorescent intensity of tRNA molecules containing s at the 47th or 57th position.
Figure 5 : The fluorescent intensity of tRNA molecules containing s at the 47th or 57th position.

(a) The fluorescent intensity profiles of the 47s tRNA in the presence of 0.1-mM EDTA, and either 2-mM or 5-mM MgCl2. Emission was monitored at 434 nm with excitation at 352 nm. (b) The fluorescent intensity profiles of the 57s tRNA in the presence of 0.1-mM EDTA, and either 2-mM or 5-mM MgCl2. (c) The fluorescent intensity of 47s tRNA at different MgCl2 concentrations. The fluorescent spectra, with the fluorescence spectrum of the buffer without MgCl2 subtracted as the background, are shown. Each intensity is normalized by the intensity at 434 nm without MgCl2 (relative intensity) = 100 × (intensity observed)/(intensity observed at 434 nm in the absence of MgCl2). (d) The fluorescent intensity change of the 47s tRNA depending on the MgCl2 concentration. The intensities at 434 nm in c are plotted. The regression curve is expressed as Y = 100 + X × 242.94/(X + 41.552), where Y is the relative intensity and X the MgCl2 concentration. The correlation factor is 0.9944.

Full size image (80 KB)

The Mg2+-induced fluorescence change of 47s (Fig. 5c) is represented by the equation of one Mg2+ binding to the complex between tRNA and several Mg ions (Eq. 2 and Fig. 5d). The K d value (42 μM) obtained here for the 47s tRNA transcript is similar to those reflecting the strong Mg2+ binding (4 and 16 μM) obtained by using the native yeast tRNAPhe, which was fluorescently modified at specific positions46.

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com



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Acknowledgments

This work was supported by the Targeted Proteins Research Program and the RIKEN Structural Genomics/Proteomics Initiative, the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant-in-Aid for Scientific Research (KAKENHI 19201046 to I.H., 20710176 to M.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author Contributions

Y.H. conducted most of the experiments and the data analysis; M.K. and I.H. conceived and designed the study, supervised the work and prepared samples; S.Y. supervised the work.

Competing financial interests: 

The authors declare  competing financial interests.

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