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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Abstract

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

Introduction

Four-stranded DNA or RNA G4 structures play critical roles in many important biological processes1. Many proteins are involved in G4 binding and regulation, including telomere binding proteins (telomerase, POT1, RPA, TEBPs, TRF2)1,2, transcription factors (nucleolin, PARP1)3, RNA processing proteins (hnRNP A1, hnRNP A2)4, helicases (BLM, FANCJ, RHAU, WRN, Dna2, Pif1)5, and DNA replication related proteins (Rif1, REV1, PrimPolymerase)6. Protein binding can stabilize or destabilize G4 structures; thus regulating the subsequent biological functions. The stability of G4 was measured by thermal melting using ultraviolet (UV) or circular dichroism (CD) methods7. However, such conditions are not physiological relevant and are difficult to apply to studying the effects of binding proteins7.

The rapid development in single-molecule manipulation technologies has enabled studies of folding and unfolding of a biomolecule, such as a DNA or a protein, at a single-molecule level with nanometer resolution in real time8. Atomic force microscopy (AFM), optical tweezers, and magnetic tweezers are the most commonly used single-molecule manipulation methods. Compared to AFM and optical tweezers9, magnetic tweezers allow stable measurements of folding-unfolding dynamics of a single molecule over days by using an anti-drift technique10,11.

Here, a single-molecule manipulation platform using magnetic tweezers to study the regulation of G4 stability by binding proteins is reported12,13. This work outlines the basic approaches, including sample and flow channel preparation, the setup of magnetic tweezers, and the force calibration. The force control and the anti-drift protocols as described in step 3 allow for long time measurements under various force controls, such as constant force (force clamp) and constant loading rate (force-ramp), and force-jump measurement. The force calibration protocol described in step 4 enables force calibration of < 1 µm short tethers over a wide force range up to 100 pN, with a relative error within 10%. An example of regulation of the stability of the RNA Helicase associated with AU-rich element (RHAU) helicase (alias DHX36, G4R1) that plays essential roles in resolving RNA G4 is used to demonstrate the applications of this platform13.

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Protocol

1. Preparation of G4 DNA for Single-molecule Stretching

  1. Prepare 5'-thiol labeled and 5'-biotin labeled dsDNA handles by PCR using DDNA polymerase on a lambda phage DNA template using 5'-thiol and 5'-biotin primers14 (Figure 1). Both dsDNA handles have high GC content (> 60%) to prevent DNA melting when DNA is held at high forces or during DNA overstretching transition15.
  2. Purify PCR products using a commercial purification kit and digest with BstXI restriction enzyme according to the manufacturer's protocol.
  3. Ligate G4 forming ssDNA, and flank ssDNA and dsDNA handles using T4 DNA ligase according to the manufacturer's protocol. Purify the ligated product by gel extraction using a commercial purification kit according to the manufacturer's protocol.

2. Preparation of Flow Channel

  1. Coverslip cleaning and surface functionalization
    1. Place bottom coverslips (#1.5, 22 mm × 32 mm) and top coverslips (#1.5, 20 mm x 20 mm) into cover glass staining jars (each jar can hold 7 pieces of coverslips, the volume is ~ 20 mL). Rinse the coverslips in the jars with distilled water 2-5 times.
    2. Add ~ 20 mL of 5-40% detergent solution into each jar, and then place in an ultrasonic cleaning bath for 30 min. Rinse with distilled water for > 10 times to remove the detergent.
    3. Dry the coverslips in the jars in oven (~ 150 °C; CAUTION, hot), or by N2 gas. Store the dried top coverslips in a dry cabinet.
    4. Use plasma (O2 gas) to clean the coverslips in the jar for 10 min. During the 10 min, prepare 20 mL of 1% (3-Aminopropyl)triethoxysilane (APTES) solution in methanol (CAUTION, toxic/flammable). Use a chemical fume hood to dissolve APTES in methanol.
      NOTE: Avoid humidity when storing APTES solution. Old APTES often causes problem in surface functionalizing of coverslips.
    5. Immediately after the plasma cleaning, add all of the 1% APTES solution into the jars and incubate for 1 h. Pour the waste into the waste bottle specific for 1% APTES methanol. Perform the methanol-related steps in the fume hood for flammable chemicals.
    6. Rinse the jars once with methanol and > 10 times with distilled water, and then dry by oven (~150 °C; CAUTION, hot). Store the APTES-coated bottom coverslips in a dry cabinet if not in use for up to two weeks.
      NOTE: After each sub-step from 2.1.1 to 2.1.4, the cleaning process can be paused before the next step.
  2. Assemble the flow channel
    1. Prepare two "spacers", i.e., parafilm or double-side tape (~ 4 mm × 20 mm) for each channel. Place the two pieces of spacers on a bottom coverslip along the long-edge. Place a top coverslip on the spacer, forming a flow cell in between (~ 10 mm × 20 mm area, Figure 2A, B).
    2. If parafilm is used as a spacer, place the flow channel on a heater (60-120 °C; CAUTION, hot) for 5-10 s while gently pressing the sides of the top coverslip to stick the two coverslips together by parafilm. The resulting flow channel has a height of ~ 100 µm, and thereby the volume of the channel is ~ 20 µL.
    3. Seal the long edge of the channel with silicone glue to avoid leakage. Use silicone glue to make a small sink-like structure at each open edge of the flow channel, which serves as an entry and an exit of solution.
      NOTE: The entry and exit can also be made by other ways, e.g., by adhering small plastic rings using wax.
    4. Store channels in a dry cabinet for up to 4 weeks.
  3. Tether DNA onto the bottom surface of the flow channel
    1. Dilute the amino-coated polystyrene beads (diameter: 3 µm) by 200X in distilled 1X phosphate buffered saline (PBS) buffer. Vortex the bead solution and then flow into channels. Incubate the bead solution in channels for ~ 30 min. Remove any unstuck beads by washing with 200 µL of 1X PBS buffer.
    2. Adjust (increase/decrease) the incubation time to achieve a surface density of 1-5 beads per 50 mm x 50 mm area. Store the channels deposited with the reference beads for up to 3 days.
    3. Dilute sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) powder into 1X PBS solution (~ 0.5 mg/mL). Vortex the solution and then flow into the channel.
      NOTE: Prepare fresh Sulfo-SMCC solution before use and add it immediately to the channel to avoid hydrolyzation of the SMCC.
    4. Incubate the SMCC solution in the channel for 30 min. Remove the SMCC solution by washing with a large amount (1 mL, ~ 50X the channel volume) of 1X PBS solution.
      NOTE: Wash the excess SMCC carefully. This step is very essential for the binding of DNA on the coverslip.
    5. DNA tethering
      1. Dilute the thiol-biotin labeled DNA into 1X PBS, with a resulting DNA concentration of ~ 0.3 nM. Gently pipette to mix the solution, flow the DNA solution into the SMCC-coated channel, and incubate for 30 min at room temperature (~ 23 °C).
    6. Gently wash away free DNA with 200 µL of blocking solution that contains 1X PBS with 10 mg/mL bovine serum albumin (BSA) and 0.01% 2-Mercaptoethanol.
    7. Block the channel surface by incubating the channel in blocking solution (10 mg/mL BSA, 0.01% 2-Mercaptoethanol) for > 2 h; after this step, the channel is ready for experiments. The prepared channel can be kept at 4 °C for ~ 1 day.
      NOTE: The blocking step is important for reducing the non-specific binding of DNA and magnetic beads to the coverslips.

3. Magnetic Tweezers Setup and Identification of Single dsDNA Tether

  1. Magnetic tweezers setup
    1. Start the magnetic tweezers control program. Here, the magnetic tweezers were controlled by an in-house-written LabVIEW program.
    2. Align the magnet centers before mounting the channel. Use a 4X objective lens to adjust the x- and y-axis of the magnets in the optical axis of the microscope.
    3. Use a computer-controlled motorized manipulator to move the magnets along the z-direction (Figure 2A) and set the distance as d = 0 (d: distance between the magnets and coverslip) when the magnets attach to a coverslip on the microscope.
    4. Program the movement of the magnets through the manipulator to achieve control of the force, including constant force (i.e., constant d) and time varying force F(t) (i.e., time varying d(t)).
    5. Use bright a light-emitting diode (LED) light source for back-scattered illumination of the bead through the objective. Collect the bead images at a sampling rate of 100 Hz by a charge-coupled device (CCD) camera.
  2. Sample setup and identify single dsDNA tether
    1. Tether formation
      1. Gently flow in 200X diluted M-280, paramagnetic beads in assay solution (100 mM KCl, 2 mM MgCl2, 10 mM Tris-pH 7.4) into a channel. Incubate for 10 min to allow the beads to bind to biotin-labeled DNA molecules immobilized on the SMCC-coated surface through the thiol-labeled end. Gently wash away un-tethered beads using 200 µL of standard reaction solution.
        NOTE: The M-280 beads show lower non-specific binding to the surface compared to other commercial magnetic beads such as M-270.
    2. Mount the channel onto the microscope stage. Search for the beads on the bottom surface using the 100X oil immersion objective.
    3. Select a reference bead on the surface and a moving tethered bead. Build the initial image libraries of both the reference bead and tethered bead at different defocus planes.
    4. Calibration of beads image
      1. Before experiments, use an objective piezo actuator to obtain images of both reference and tethered beads at different defocus planes spaced by 20-50 nm, which are stored as two separate bead image libraries and are respectively indexed with the defocus distance (Figure 2D).
    5. x, y, z position determination
      1. During experiments, determine the position of the bead in the x-y plane by the bead centroid. Determine the height change of the tethered bead by comparing the current bead image with those stored in the library. Use the auto-correlation function analysis of the power spectrum of the Fourier transform of the bead images16.
    6. Anti-drift feedback using reference bead
      1. During experiments, use the piezo to "lock" the distance between the objective and a specific reference bead image stored in the library through a low frequency feedback control, so that the stuck bead image has the best correlation with a specific image stored in the library (Figure 2D).
    7. Determine whether the tether is a single dsDNA molecule by applying ~ 65 pN force Determine a single dsDNA molecule if the tether undergoes the characteristic DNA overstretching transition17,18,19. Repeat the process until a single dsDNA tether is found. (Please refer to the next section for force calibration and DNA overstretching transition).

4. Magnetic Tweezers Force Calibration

  1. Directly determine the force (up to 100 pN) for long DNA (48,502 bp λ-DNA) molecules using bead fluctuation through:
    figure-protocol-10216
    , where kB is the Boltzmann constant, T is the temperature in Kelvin scale, δ2y is the variance of the bead fluctuation in the direction perpendicular to the magnetic field and the force direction. In this direction, the motion of the bead can be described as a pendulum fluctuation with a length of l + r0, where l is the end-to-end distance along the force direction (extension) of the DNA, and r0 is the radius of the bead10 (Figure 3A).
  2. Determine the calibration curve for force F as a function of magnets to bead distance d, and F(d) for different beads using the long λ-DNA. Usually, the distance between the magnet and the coverslip is used as the DNA tether length can be ignored. Fit the F-d curve by the double-exponential decay function:
    figure-protocol-11293
    , where the fitting parameters α1, α2, γ1, γ2 are determined by the magnetic tweezers and the parameter C is determined by the heterogeneous property of the magnetic beads. By shifting the C, the F-d curve obtained from different beads can be overlapped10 (Figure 3B).
  3. Calibration of parameter C for individual magnetic beads for short DNA experiments.
    1. Determining the force based on the fluctuation requires recording the bead position at a frequency higher than the corner frequency:
      figure-protocol-12020
      , where γ is the drag coefficient of the bead. For short DNA at high force, it requires recording frequency faster than 100 Hz, therefore, the force cannot be directly measured based on fluctuation with the camera. However, the parameter C can be determined by measuring the force at a low force range (< 15 pN) using fluctuation and fitting the data with a calibrated F-d curve to obtain the parameter C20.
    2. Alternatively, for experiments with dsDNA, determine the parameter C using DNA overstretching transition at a force of ~ 65 pN (Figure 3C)21,22,23 by recording the d position at which dsDNA showed extension jump (0.6X length increase of contour length). After determining the parameter C, calculate the force for the tethered beads.
  4. Control the loading rate by programming the movement of the magnets through the inverse function of F(d). The magnet should approach the sample double exponentially.
    NOTE: The relative error of force determined by such an extrapolation method is ~ 10%, and is mainly caused by the bead radius heterogeneity20.

5. Single-molecule Manipulation of G4 in the Presence and Absence of Binding Proteins

  1. Force-ramp experiments
    1. After identifying the dsDNA tether, perform a force-increase scan at a loading rate of 0.2 pN/s followed by a force-decease scan at -0.2 pN/s. After each stretching cycle, hold the DNA molecule at 1 pN for 30 s to allow the ssDNA to refold to G4.
  2. Force-jump experiments
    1. Cycle the force between 54 pN for 30 s under which a folded G4 could be unfolded, and 1 pN for 60s, under which a folded G4-15T could refold.
  3. Flowing protein solution
    1. Flow the protein solution when at ~ 20 pN force to avoid attachment of the beads to the coverslip. The DEAH-box RHAU helicase, which showed high specificity to the G4 structure, was used24. The recombinant Drosophila melanogaster RHAU (DmRHAU) helicase was expressed in Escherichia coli and purified as described previously25. The G4 unwinding activity of DmRHAU was assayed on a tetramolecular G4 DNA substrate13.
  4. Analyze the unfolding force using an in-house written Matlab program14.

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Results

The experiment setup for stretching a single G4 molecule is shown in Figure 4. A single-stranded G4 forming sequence spanned between two dsDNA handles was tethered between a coverslip and a paramagnetic bead. To find a single dsDNA tethered bead, an overstretching assay was performed by increasing the force at constant loading rates. Three types of measurements were often used for studying the folding and unfolding of biomolecules: (i) constant force measurem...

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Discussion

As described above, a platform for studying the mechanical stability of G4 DNA and the interactions of protein to G4 using single-molecule magnetic tweezers is reported. Accompanying the platform, highly efficient protocols of finding G4 DNA tether, and measurement of the folding-unfolding dynamics and stability of the G4 structure with nanometers special resolution are developed. The focal plane locking enables highly stable anti-drift control, which is important for detecting a small structure transition such as G4 (st...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Meng Pan for proofreading the manuscript. This work is supported by Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2012-T3-1-001) to J.Y.; the National Research Foundation through the Mechanobiology Institute Singapore to J.Y.; the National Research Foundation, Prime Minister's Office, Singapore, under its NRF Investigatorship Programme (NRF Investigatorship Award No. NRF-NRFI2016-03 to J.Y.; the Fundamental Research Fund for the Central Universities (2017KFYXJJ153) to H. Y.

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Materials

NameCompanyCatalog NumberComments
DNA PCR primersIDTDNA preparations
DNA PCR chemicalsNEBDNA preparations
restriction enzyme BstXINEBR0113SDNA preparations
coverslips (#1.5, 22*32 mm, and 20*20 mm)BMH.BIOMEDIA72204flow channel preparation
Decon90Decon Laboratories Limitedflow channel preparation
APTESSigma440140-500MLflow channel preparation
Sulfo-SMCCThermoFisher Scientific22322flow channel preparation
M-280, paramganetic beads,streptavidinThermoFisher Scientific11205Dflow channel preparation
Polybead Amino Microspheres 3.00 μmPolysciences, Inc17145-5flow channel preparation
2-MercaptoethanolSigmaM6250-250MLflow channel preparation
Olympus Microscopes IX71OlympusIX71Magnetic tweezers setup
Piezo-Z Stages P-721Physik InstrumenteP-721Magnetic tweezers setup
Olympus Objective lense MPLAPON-Oil 100XOlympusMPLAPON-Oil 100XMagnetic tweezers setup
CCD/CMOS cameraAVTPike F-032BMagnetic tweezers setup
Translation linear stagePhysik InstrumenteMoCo DCMagnetic tweezers setup
LEDThorlabsMCWHLMagnetic tweezers setup
Cubic MagnetsSupermagneteMagnetic tweezers setup
LabviewNational InstrumentsMagnetic tweezers setup
OriginPro/MatlabOriginLab/MathWorksData analysis

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G quadruplexMagnetic TweezersSingle molecule ManipulationFolding And UnfoldingBinding ProteinsDNA protein InteractionsHelicase ActivityDNA Tether FormationParamagnetic BeadsNanometer scale Dynamics

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