Single-molecule studies have extensively explored a variety of biological problems by following individual molecules going through their biochemical or biophysical reactions (1–3). Single-molecule assays have provided real-time snap shots of molecular events, resolving molecular sub-populations, transient intermediate states, rare molecular events, and non-uniform kinetics in the enzymatic reaction (4). In contrast to direct observation of probe-labeled enzymes on DNA using fluorescence microscopy (5), the most commonly used force spectroscopy, including optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM), can manipulate individual biomolecules by external forces in the range of 0.01–10,000 pN to unravel structural and mechanical properties (6). The force spectroscopy has been also used to precisely measure displacement as a function of force with high spatiotemporal resolution of sub-nanometers and sub-milliseconds to detect enzyme activities on substrates in DNA replication, DNA repair, transcription, and translation (7–11).
However, traditional force spectroscopy is difficult to parallelize measurement at one time, limiting monitoring of enzymatic activity by multi-proteins due to inefficient
In this mini review, we describe a simple, robust, low cost, and multiplexed single-molecule force spectroscopy to study processive enzyme activities on stretched-DNA substrates using a hydrodynamic force, called flow-stretching bead assay.
To monitor individual DNA in the smFS, DNA molecules are immobilized on the surface passivated with biotin-PEG (polyethylene glycol) via biotin-avidin interactions while the opposite end with digoxigenin is attached to a super-paramagnetic bead (2.8 μm in diameter) functionalized with anti-digoxigenin antibody. A steady buffer flow with a constant rate given by a syringe pump drags the bead linked to the tethered-DNA, resulting in extension of DNA (Fig. 2A). A damper made by a Falcon centrifuge tube that is filled with water and an air layer is installed between the syringe pump and flow chamber to filter high-frequency noises coming from mechanical fluctuation of the syringe pump (Fig. 2A). The position of beads is monitored to measure enzymatic activities on DNA substrates. Beads linked to immobilized-DNA molecules are visualized under a standard optical microscope and recorded by a charge coupled device (CCD) camera (Fig. 2A). Dark spots in the resulting image correspond to the beads (Fig. 2B). Optimal number of beads linked to DNA is ~300 per field of view under 10× objective. In the smFS, we can monitor enzyme activities on DNA substrates by measuring changes in the length of individual DNA molecules by imaging beads and tracking their position.
A flow chamber was constructed with a biotin-PEG functionalized cover slip, PEG-coated glass slide, double-sided tape, and tubing (Fig. 2C). A pair of inlet/outlet holes is drilled on a glass slide before it is passivated with PEG. A 100 μm thick double-sided tape is cut for a channel with 3 mm in width and 25 mm in length. Width and height of the channel must be maintained to perform experiments under an identical force once the external force is calibrated to flow rate by the syringe pump. A solution of streptavidin or neutravidin is evenly spread on the biotin-PEG cover slip and then washed with deionized water after 10 minutes of incubation. A chamber is made by putting the prepared cover slip and glass slide together with adhesive double-sided tape and then sealing with epoxy. Tubing is inserted into each inlet/outlet hole and then epoxy is applied to the junction between the hole and tubing. The flow chamber filled with a blocking buffer is ready to do a single-molecule flow-stretching experiment. For more detailed information on construction of the flow chamber, we refer to a demonstration movie published by the journal of visualized experiments (JoVE) (17).
The position of a bead is determined by two dimensional (2D) Gaussian fit to the intensity profile of the bead (Fig. 2D). The 2D Gaussian function is defined as
The smFS detects activities of DNA enzymes using the different elastic property of single-stranded (ssDNA) and double-stranded DNA (dsDNA). The ssDNA is more flexible (shorter persistence length) than dsDNA and easily forms secondary structure (hairpin-coil) by base pairing in the same strand. Thus, coiling of ssDNA results in shortening of dsDNA at low stretching forces (< 6 pN) (Fig. 3A). At forces higher than 6 pN that is sufficiently strong to break the secondary structure, the ssDNA is extended longer than dsDNA because distance between base pairs in ssDNA is longer than that in the double-helical dsDNA (Fig. 3A). Therefore, conversion from dsDNA to ssDNA or vice versa can be monitored through the motion of beads in the opposite direction (a decrease in total DNA length) or in the same direction as the flow (an increase in total DNA length) (Fig. 3B).
Bead position on the nanometer scale obtained from the pixel size of the CCD camera (effective pixel size = physical pixel size/magnification) is converted to the number of nucleotides (nt) transitioned from dsDNA (ssDNA) to ssDNA (dsDNA) by measuring the length of dsDNA and ssDNA. The ssDNA is prepared by denaturing the dsDNA. The dsDNA like 48.5 kilo-base pair (kb) long λ-phage DNA is incubated in 2 mM NaOH solution at 99°C for five minutes and then immediately quenched in a cold blocking buffer (~4°C) to prevent re-annealing. Distance (
Since the single-molecule kinetics study of λ-exonuclease (19), the smFS has been successfully used for studying enzyme activities by multi-protein complexes in DNA replication (20–22) and DNA repair (23, 24). During DNA replication, helicase transforms from dsDNA to ssDNA by unwinding duplex DNA at the replication fork, resulting in shortening of DNA (Fig. 4A). Single-strand binding protein (SSB) is associated with resulting ssDNA before DNA synthesis by DNA polymerase and is also involved in DNA repair and recombination. DNA polymerase in the replisome synthesizes DNA on resulting ssDNA as a template, resulting in lengthening of DNA (Fig. 4B) (20, 25). Basically, time trajectory of the bead position can provide unwinding or synthesis rate and processivity of enzymatic activity (Fig. 4A, B). The strand excision by exonuclease is a crucial step to repair DNA, resulting in transition of dsDNA to ssDNA (Fig. 4C). Using the smFS, Park
In addition to the detection of the transition of dsDNA (ssDNA) to ssDNA (dsDNA), since SSB binding to ssDNA extends ssDNA, but SSB-bound ssDNA is shorter than dsDNA, the smFS can visualize the SSB association and dissociation from ssDNA (21, 23). A transient intermediate state, appearing as a pause in the time trajectory, was also detected; the primer synthesis by primase (20) and the stoppage of the Tus-Ter complex in the termination of DNA replication (27). The smFS can visualize in real time the conformational change in dsDNA; the dynamic loop formation and release of the lagging strand during the Okazaki fragment synthesis (21).
The smFS is a powerful toolbox to study enzymatic activities in a multi-protein complex in various DNA-associated biological events. Especially, it is suitable for highly processive enzymes. However, for higher resolution (more information) of molecular activity in the protein complex, the smFS can be combined with fluorescence imaging. The correlative system such as OT-fluorescence microscopy (28–36) and MT-fluorescence microscopy (37–40) can visualize directly fluorescently-labeled enzymes on the substrate as well as the product as change in DNA length. In fact, Loparo
This study was supported by Global Research Lab Program through the NRF of Korea funded by the Ministry of Science and ICT (NRF-2017K1A1A2013241) (J.-B.L.).
The authors have no conflicting interests.
Multiplexed single-molecule force spectroscopy. (A) Holographic optical tweezers. (B) Multiplexed magnetic tweezers. (C) Acoustic force spectroscopy.
Schematic representation of single-molecule flow-stretching bead assay. (A) A setup based on a conventional optical microscope. A buffer solution including target proteins flows through the flow chamber with constant rate by a syringe pump. (B) ~300 beads (2.8 μm in diameter) attached to tethered-DNA molecules on the surface under 10× magnification objective. (C) Bead-DNA is immobilized on the streptavidin-coated surface that is passivated with biotin-PEG/PEG (1:100). A laminar flow stretched bead-DNA. (D) Intensity profile of a bead image shows a Gaussian distribution.
Flow-stretching bead assay using different elastic properties between dsDNA and ssDNA. (A) Force-extension curve for dsDNA and ssDNA. (B) Transition of dsDNA (ssDNA) to ssDNA (dsDNA) is shortening (lengthening). (C) The scheme of the length measurement of dsDNA and ssDNA at a given force.
Time trajectories of enzyme activities with flow-stretching bead assay. (A) Helicaese unwinds duplex DNA (dsDNA → ssDNA). Slope in time trajectory is the unwinding rate. Helicase dissociation drives reannealing of unwound strands (ssDNA → dsDNA). (B) The primer extension of DNA polymerases (ssDNA → dsDNA). (C) The strand excision by exonuclease (dsDNA → ssDNA).