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RNA Scaffolds pp 169-179 | Cite as

Folding RNA–Protein Complex into Designed Nanostructures

  • Tomonori Shibata
  • Yuki Suzuki
  • Hiroshi Sugiyama
  • Masayuki Endo
  • Hirohide SaitoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1316)

Abstract

RNA–protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nano-objects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.

Key words

Ribonucleoprotein Kink-turn RNA L7Ae RNA–protein complex RNA nanostructures RNA nanotechnology RNP High-speed atomic force microscopy 

1 Introduction

Naturally occurring RNP complexes play an important role in modulating the structure and function of RNA molecules, which regulate a variety of cellular events [1, 2, 3]. For example, the bacterial ribosome consists of three ribosomal RNAs and approximately 50 ribosomal proteins that form complex RNP structures through a set of RNP interactions [4, 5]. Their sophisticated structure and function render RNP motifs as promising building blocks for a wide range of applications including synthetic biology and nanotechnology. Integration of RNP motifs into functional RNAs allows us to construct artificial RNAs with protein-controllable folding and function. We have previously reported that RNP complex-mediated nanostructures can be designed and constructed using RNA kink-turn (K-turn) motifs and ribosomal L7Ae protein [6]. The K-turn–L7Ae interaction bends the RNA motif by approximately 60° [7, 8, 9, 10], leading to the formation of equilateral triangular RNP nanostructures. In principle, desired functional proteins conjugated with L7Ae can be precisely arranged on a designed RNA scaffold of optimal size. Such RNP nanostructures, when attached to a functional protein module, can be used to detect mammalian cells [11]. Direct observation of native RNP interactions at high resolution is important for understanding the RNP folding and development of functional RNP nanostructures. HS-AFM is a powerful tool for capturing the molecular events of biological macromolecules such as DNA–protein interactions at high resolution [12, 13, 14, 15]. Thus, we used HS-AFM to observe the folding of the RNP complex into nanostructures at single RNP resolution and precise arrangement of proteins on RNA nanostructures of defined sizes.

In this chapter, we describe a strategy to construct and visualize equilateral triangular RNP nanostructures of different sizes. The size of the RNP nanostructures can be easily tuned by changing the length of the double helix between three K-turn motifs that are incorporated into the three vertices of the triangle. These RNP nanostructures can be analyzed biochemically and visualized directly by electrophoretic mobility shift assay (EMSA) and HS-AFM, respectively (Fig. 1).
Fig. 1

Schematic illustration of the preparation and evaluation of RNP nanostructures. The RNP interaction can be evaluated by EMSA. The folding of RNP nanostructures in solution can be directly visualized by HS-AFM

2 Materials

2.1 RNA synthesis

  1. 1.

    DNA templates and primers.

     
  2. 2.

    DNA polymerase: KOD-Plus-ver.2 (Toyobo, Japan).

     
  3. 3.

    Thermal cycler.

     
  4. 4.

    DNA purification kit: QIAquick PCR purification kit (Qiagen, Germany).

     
  5. 5.

    UV spectrometer: NanoDrop (Thermo Scientific, USA).

     
  6. 6.

    RNA polymerase: MEGAshortscript T7 kit (Amibion, USA).

     
  7. 7.

    Electrophoresis apparatus.

     
  8. 8.

    Electrophoresis buffer: 0.5× TBE (Tris–Borate–EDTA buffer).

     
  9. 9.

    Denaturing polyacrylamide gel: 10 % acrylamide–bis mixed solution (19:1), 8.3 M urea, 0.5× TBE.

     
  10. 10.

    Gel Indicator RNA Staining Solution, 100× (BioDynamics Laboratory Inc., Japan).

     
  11. 11.

    Elution buffer: 0.3 M NaOAc (pH 5.2), 0.1 % SDS.

     
  12. 12.

    Phenol, saturated with TE buffer (Nacalai Tesque, Japan).

     
  13. 13.

    Diethyl ether.

     
  14. 14.

    Ethanol.

     
  15. 15.

    80 % Ethanol.

     
  16. 16.

    3 M sodium acetate.

     
  17. 17.

    Milli-Q water (Millipore, USA).

     

2.2 Protein Synthesis

  1. 1.

    pET-28b(+)-L7Ae plasmid.

     
  2. 2.

    Competent cell: Rosetta(DE3) pLysS pRARE2.

     
  3. 3.

    Luria–Bertani (LB) medium.

     
  4. 4.

    Kanamycin.

     
  5. 5.

    Chloramphenicol.

     
  6. 6.

    1 M isopropyl β-d-thiogalactoside (IPTG).

     
  7. 7.

    Shaking incubator.

     
  8. 8.

    Sonicator.

     
  9. 9.

    Centrifuge.

     
  10. 10.

    Ni-NTA Superflow resin (Qiagen).

     
  11. 11.

    Lysis buffer: 20 mM Tris–HCl buffer (pH 7.6), 300 mM NaCl.

     
  12. 12.

    Wash buffer: 20 mM Tris–HCl buffer (pH 7.6), 300 mM NaCl, 10 mM imidazole.

     
  13. 13.

    Elution buffer: 20 mM Tris–HCl buffer (pH 7.6), 20 mM NaCl, 500 mM imidazole.

     
  14. 14.

    SDS-PAGE apparatus.

     
  15. 15.

    SDS running buffer: 25 mM Tris–Base, 191 mM glycine, 0.1 % SDS.

     
  16. 16.

    Dialysis system.

     
  17. 17.

    Dialysis buffer: 20 mM HEPES–KOH (pH 7.8), 150 mM KCl, 1.5 mM MgCl2, 5 % glycerol.

     
  18. 18.

    Stock buffer: 20 mM HEPES–KOH (pH 7.8), 150 mM KCl, 1.5 mM MgCl2, 40 % glycerol.

     
  19. 19.

    Bradford reagent: Bio-Rad Protein Assay (Bio-Rad, USA).

     
  20. 20.

    Microplate reader (Tecan, Switzerland).

     

2.3 EMSA

  1. 1.

    RNA solution (adjusted to 1 μM with Milli-Q water).

     
  2. 2.

    Protein stock buffer: 20 mM HEPES–KOH (pH 7.5), 150 mM KCl, 1.5 mM MgCl2, and 40 % glycerol.

     
  3. 3.

    L7Ae protein solution (adjusted to 10× concentration with protein stock buffer).

     
  4. 4.

    5× Binding buffer: 100 mM HEPES–KOH (pH 7.5), 750 mM KCl, 7.5 mM MgCl2.

     
  5. 5.

    Thermal cycler.

     
  6. 6.

    Gel loading buffer: 0.25 % bromophenol blue (BPB), 30 % glycerol.

     
  7. 7.

    Electrophoresis apparatus.

     
  8. 8.

    Electrophoresis buffer: 0.5× TBE.

     
  9. 9.

    Native polyacrylamide gel: X% Acrylamide–Bis Mixed Solution (19:1), 0.5× TBE.

     
  10. 10.

    SYBR Green I and II (Lonza, Switzerland).

     
  11. 11.

    Gel image scanner: Typhoon FLA-7000.

     

2.4 High-Speed Atomic Force Microscopy (HS-AFM)

  1. 1.

    Mica disks with a diameter of 1.5 mm (Furuuchi Chemical Corporation, Tokyo, Japan).

     
  2. 2.

    3-Aminopropyltriethoxy silane (APTES) for mica functionalization.

     
  3. 3.

    Ultrapure water for mica functionalization.

     
  4. 4.

    Solution of 50 nM RNP nanostructures prepared for the EMSA procedure (Subheading 3.3).

     
  5. 5.

    Imaging buffer: 20 mM Tris–HCl (pH 7.6), 10 mM MgCl2.

     
  6. 6.

    High-speed atomic force microscopy (Nano Live Vision, RIBM, Japan).

     
  7. 7.

    Cantilever (BL-AC10EGS, Olympus, Japan).

     

3 Methods

3.1 RNA Synthesis

All RNAs are synthesized by in vitro transcription using template DNAs and purified by denaturing PAGE (Fig. 1). The molecular design of a RNP nanostructure with an equilateral triangular shape has been reported [6, 11]. We designed equilateral-triangular RNP nanostructures of five different sizes (Fig. 2). The triangles have lengths of 15, 26, 48, 70, or 92 base pairs of double stranded RNAs that consist of long-RNA (L-RNA) and short-RNA (S-RNA). Hybridization between L-RNA and S-RNA leads to the formation of three K-turn motifs-containing RNA nanostructures (LS-15, LS-26, LS-48, LS-70, and LS-92).
Fig. 2

Design of RNP nanostructures of different sizes. The equilateral-triangular RNP nanostructures consist of three K-turn motifs and three RNA duplexes. The number of base pairs in the RNA duplexes can be easily changed to construct RNP nanostructures of different sizes. The length of the three sides, which contain the stem region of the K-turn motifs, is designed to be a multiple of 11

  1. 1.

    Synthesize template DNA using DNA polymerase (Toyobo, Japan) by PCR (see Note 1 ).

     
  2. 2.

    Electrophorese the reaction mixture with native polyacrylamide gel to confirm template DNA amplification.

     
  3. 3.

    Purify the PCR product using QIAquick PCR purification kit.

     
  4. 4.

    Measure the DNA concentration using NanoDrop.

     
  5. 5.

    Transcribe DNA template using MEGAshortscript T7 kit. In a final volume of 20 μL, mix Nuclease-free water, 150 nM DNA template, 2 μL of 10× Reaction buffer, 2 μL of 75 mM ATP solution, 2 μL of 75 mM UTP solution, 2 μL of 75 mM GTP solution, 2 μL of 75 mM CTP solution, and finally add 2 μL of T7 Enzyme mix. The reagents used here are accessories in MEGAshortscript T7 kit.

     
  6. 6.

    After incubation at 37 °C for 4 h, add 1 μL of TURBO DNase (contained in MEGAshortscript T7 kit) and incubate at 37 °C for 30 min.

     
  7. 7.

    Add 21 μL of Gel Loading Buffer II (contained in the MEGAshortscript T7 kit) and incubate at 95 °C for 3 min.

     
  8. 8.

    Electrophorese the sample with denaturing PAGE containing 8.3 M urea and 0.5× TBE.

     
  9. 9.

    Stain the gel with 1× Gel Indicator RNA Staining Solution until RNA bands appear (see Note 2 ).

     
  10. 10.

    Cut the band of the desired RNA and then crush the excised gel.

     
  11. 11.

    Add 600 μL of Elution buffer to the gel and then incubate at 37 °C for more than 2 h (see Note 3 ).

     
  12. 12.

    Recover the supernatant. Wash the recovered supernatant with 600 μL of TE-saturated phenol at three times and with 900 μL of diethyl ether.

     
  13. 13.

    Add 1,200 μL of 100 % ethanol and 50 μL of 3 M sodium acetate and then precipitate the RNA at −30 °C for more than 1 h.

     
  14. 14.

    Centrifuge at 20,400 × g for 30 min, remove the supernatant, and wash the pellet with 600 μL of 80 % ethanol.

     
  15. 15.

    After drying in air, dissolve in Milli-Q water. Measure the RNA concentration using NanoDrop.

     
  16. 16.

    Check the size and purity of the recovered RNA by denaturing PAGE and confirm the hybridization between L-RNA and S-RNA by native PAGE.

     
  17. 17.

    Store the RNA at −30 °C.

     

3.2 Protein Synthesis

L7Ae was prepared by conventional methods for protein preparation. The expression of L7Ae protein was performed by the transformation of E. coli. The L7Ae protein can be purified by affinity chromatography using polyhistidine-tag (His-tag).
  1. 1.

    Transform Rosetta(DE3) pLysS pRARE2 with pET-28b(+)-L7Ae plasmid.

     
  2. 2.

    Pick up a colony and then culture transformed E. Coli in 50 mL of LB medium containing 50 µL of 50 mg/mL kanamycin and 15 µL of 15 mg/mL chloramphenicol at 37 °C for >12 h.

     
  3. 3.

    Add 25 mL of the culture to 250 mL of LB medium containing 50 µg/mL kanamycin and incubate at 37 °C until OD660 reaches 0.5.

     
  4. 4.

    Add 250 μL of 1 M IPTG to the culture and incubate at 37 °C for 4 h.

     
  5. 5.

    Centrifuge the cells at 4,100 × g for 10 min and remove the supernatant.

     
  6. 6.

    Add 5 mL of Lysis buffer and then suspend the collected cells.

     
  7. 7.

    Sonicate the suspended cells on ice.

     
  8. 8.

    Heat the lysate at 80 °C for 15 min to denature endogenous proteins (see Note 4 ).

     
  9. 9.

    Centrifuge at 15,000 × g for 30 min and then filter the supernatant with a 0.45 μm filter.

     
  10. 10.

    Wash Ni-NTA resin with Lysis buffer and then load the supernatant onto the Ni-NTA resin-filled column.

     
  11. 11.

    Wash the resin with Wash buffer to elute proteins without His-tag.

     
  12. 12.

    Add 100 μL of 0.2 N NaOH to the column and then immediately wash the resin with Wash buffer (see Note 5 ).

     
  13. 13.

    Elute with Elution buffer.

     
  14. 14.

    Collect fractions and check the fraction containing L7Ae proteins by SDS-PAGE.

     
  15. 15.

    Dialyze the purified L7Ae protein solution in Dialysis buffer at three times.

     
  16. 16.

    Measure the concentration of L7Ae by the Bradford method.

     
  17. 17.

    Check the binding activity of L7Ae to box C/D K-turn RNA by EMSA.

     
  18. 18.

    Store the L7Ae protein in Stock buffer at −30 °C for short-term storage or at −80 °C for longer storage.

     

3.3 Electrophoretic Mobility Shift Assay (EMSA)

EMSA is a general tool for the investigation of the interaction between biological macromolecules. The formation of RNA–protein nanostructures can be analyzed by EMSA. Hybridization between L-RNA and S-RNA should form three box C/D K-turn motifs capable of binding to L7Ae. LS-RNAs of various sizes were assayed by EMSA in the absence and presence of L7Ae (Fig. 3).
Fig. 3

EMSA of RNP nanostructures. L- and S-RNAs interact with each other to form LS-RNAs of circular structures. In the case of LS-15 and 26, three up-shifted bands are observed in an L7Ae concentration-dependent manner (left ). The multiple RNP interactions of larger nanostructures (LS-48, 70, and 92) are indistinguishable by EMSA (right )

  1. 1.

    Prepare native polyacrylamide gel (see Note 6 ) and set up the electrophoresis apparatus. Perform a pre-run at constant voltage.

     
  2. 2.

    Mix 6 μL of Milli-Q water, 2 μL of 5× Binding buffer, 0.5 μL of each RNA solution (final concentration 50 nM).

     
  3. 3.

    After heating at 80 °C for 3 min, leave at room temperature for 10 min.

     
  4. 4.

    Add 1 μL of 10× L7Ae protein solution and incubate at room temperature for 30 min.

     
  5. 5.

    Add 2 μL of 6× loading dye and load the sample in the polyacrylamide gel. Electrophorese at constant voltage for an appropriate time.

     
  6. 6.

    Detach the gel from the electrophoresis apparatus and stain the gel with SYBR Green I and II (Lonza, Switzerland) for 10 min (see Note 7 ).

     
  7. 7.

    Observe the gel with Typhoon FLA-7000 (see Note 8 ).

     

3.4 High-Speed Atomic Force Microscopy (HS-AFM)

The obtained RNP nanostructures can be visualized by AFM, which is now a standard technique for visualizing nucleic acids nanostructures. We employed HS-AFM (Nano Live Vision, RIBM, Japan), which enables quick data acquisition of high-resolution images in liquid (Fig. 4). The following sections describe the preparation of samples for AFM imaging.
Fig. 4

Schematic illustration of AFM image (a) before and (b) after the addition of L7Ae. RNA nanostructures are heterogeneous structures in the absence of L7Ae due to flexibility of the K-turn motifs. However, the three K-turn motifs interact with L7Ae and bend by approximately 60°, leading to the formation of triangular RNP nanostructures. (c) Direct observation of RNP nanostructures in solution using HS-AFM. Single RNP interaction in the nanostructures is clearly observed (white arrows)

3.4.1 Preparation of Mica Surface

  1. 1.

    Prepare a 0.1 % APTES solution in ultrapure water.

     
  2. 2.

    Cleave the mica disc to obtain a fresh surface.

     
  3. 3.

    Deposit a drop (~2 μL) of 0.1 % APTES solution onto the freshly cleaved mica surface (see Note 9 ).

     
  4. 4.

    Rinse the surface with ultrapure water (~10 μL) after incubation for 5 min at room temperature (25 °C).

     

3.4.2 Sample Preparation for AFM Imaging

  1. 1.

    Dilute the pre-formed RNP nanostructures to ~5 nM by adding Imaging buffer (see Note 10 ).

     
  2. 2.

    Place ~2 μL of the sample solution onto the APTES-treated mica surface for 5 min.

     
  3. 3.

    Rinse the surface with Imaging buffer (~10 μL) to remove unbound molecules.

     

3.4.3 AFM Imaging of the Samples

  1. 1.

    Place the cantilever on the cantilever holder (see Note 11 ).

     
  2. 2.

    Fill the liquid cell with ~120 μL of Imaging buffer.

     
  3. 3.

    Align the laser focusing position so that the intensity of the laser light reflected back from the cantilever is maximized.

     
  4. 4.

    Align the photodetector position so that the reflected laser makes a spot at the center of the photodetector.

     
  5. 5.

    Mount the sample prepared in Subheading 3.4.2 on the scanner stage.

     
  6. 6.

    Mount the scanner over the liquid cell in which the cantilever is immersed in Imaging buffer.

     
  7. 7.

    Find the resonant frequency of the cantilever using a fast Fourier transform (FFT) analyzer.

     
  8. 8.

    Excite the cantilever at the resonant frequency by applying sinusoidal AC voltage.

     
  9. 9.

    Execute the approach until the software automatically stops the motor.

     
  10. 10.

    Adjust the set point voltage to ~75–95 % of the free oscillation amplitude.

     
  11. 11.

    Gradually decrease the set point voltage until the sample is clearly imaged (see Note 12 ).

     

4 Notes

  1. 1.

    We prepared double-stranded DNA templates for transcription by PCR. DNA templates of L-15, L-26, L-48, S-15, S-26, and S-48 are synthesized from two single-stranded DNAs with the corresponding sequences. In the cases of L-70, L-92, S-70, and S-92, these DNA templates are synthesized from four single-stranded DNAs through two-step PCR. The primer sequences have been reported [11].

     
  2. 2.

    Gel Indicator RNA Staining Solution can detect transcribed RNAs without UV irradiation. The staining time is 10–30 min, but depends upon the quantity of RNA.

     
  3. 3.

    Elution time depends on the length of RNA. When longer RNAs are eluted, we recommend incubation at 37 °C for more than 12 h (overnight) to maximize the recovery of RNAs.

     
  4. 4.

    Since L7Ae has thermostable, only endogenous proteins in E. coli are denatured and aggregated by heating.

     
  5. 5.

    The treatment of NaOH allows for the removal of contaminated nucleic acids in L7Ae. Contamination affects specific RNA–protein interactions and nucleic acid staining in EMSA. Nucleases can also be used to remove the contaminated nucleic acids.

     
  6. 6.

    The concentrations of the native polyacrylamide gels are changed in response to the size of the RNA and RNP nanostructures (LS-15, 10 %; LS-26, 7.5 %; LS-48, -70, and -92, 5 %).

     
  7. 7.

    The staining efficiency of RNA with SYBR Green I and II will be influenced by the RNA size, structures, and RNP interaction. Expanding the size of the RNA nanostructures will increase the staining efficiency.

     
  8. 8.

    Three up-shifted bands can be observed in small RNA nanostructures (Fig. 3 left; LS-15 and LS-26) depending on the L7Ae concentration. One up-shifted band is clearly observed in large RNA nanostructures (Fig. 3 right; LS-48, LS-70, and LS-92). This may be due to insufficient separation under our electrophoretic conditions.

     
  9. 9.

    APTES is used to coat the negatively charged mica surface. The treatment of APTES provides a positively charged mica surface, which immobilizes RNP nanostructures on the mica surface by electrostatic interactions.

     
  10. 10.

    The pre-formed RNP nanostructures are prepared using a procedure similar to that described in EMSA.

     
  11. 11.

    For HS-AFM imaging, small cantilevers are used. Small cantilevers (9 μm long, 2 μm wide and 130 nm thick; BL-AC10DS, Olympus, Tokyo, Japan) made of silicon nitride with a spring constant of ~0.1 N/m, and a resonant frequency of ~300–600 kHz in water and ~1,500 kHz in air are commercially available from Olympus. These cantilevers have bird beak-like tips, however, the apex of the tips are not sharp enough to obtain high-resolution images of RNP nanostructures in solution. Therefore, we use custom-made cantilevers having electron-beam deposited (EBD) tips at the top of the bird beak-like tips (BL-AC10EGS, Olympus, Tokyo, Japan).

     
  12. 12.

    The samples are imaged in Imaging buffer with a scanning rate of 0.2–1.0 frame/s (fps).

     

Notes

Acknowledgement

We thank Hirohisa Ohno and Eriko Osada (Kyoto University) to initiate the projects. We also thank Peter Karagiannis (Kyoto University) for critically reading the manuscript. This work was supported by the New Energy and Industrial Technology Development Organization (09A02021a) and a Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Robotics” (No. 24104002) from The Ministry of Education, Culture, Sport, Science, and Technology, Japan.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tomonori Shibata
    • 2
  • Yuki Suzuki
    • 1
  • Hiroshi Sugiyama
    • 1
  • Masayuki Endo
    • 3
  • Hirohide Saito
    • 2
    Email author
  1. 1.Department of Chemistry, Graduate School of ScienceKyoto UniversityKyotoJapan
  2. 2.Center for iPS Cell Research and Application (CiRA)Kyoto UniversityKyotoJapan
  3. 3.Institute for Integrated Cell-Material Sciences (WPI-iCeMS)Kyoto UniversityKyotoJapan

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