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Relevance of the drag force during controlled translocation of a DNA-protein complex through a glass nanocapillary Roman Bulushev, Sanjin Marion, and Aleksandra Radenovic Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b03264 • Publication Date (Web): 22 Sep 2015 Downloaded from http://pubs.acs.org on September 23, 2015
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Relevance of the drag force during controlled translocation of a DNA-protein complex through a glass nanocapillary Roman D. Bulushev,† Sanjin Marion,‡ and Aleksandra Radenovic†,* †
Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland ‡ Institute of Physics, Bijenicka cesta 46, HR-10000 Zagreb, Croatia *Correspondence should be addressed to
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Abstract Combination of glass nanocapillaries with optical tweezers allowed us to detect DNAprotein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries. Keywords: DNA-protein complex, nanocapillary, nanopore, DNA translocation, optical tweezers, force measurements
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Introduction Large variety of DNA-binding proteins are constantly involved in numerous processes inside cells in order to maintain a regular cell cycle1. Identification and characterization of new proteins responsible for such processes as DNA transcription, replication, recombination, repair, and uptake require knowledge of their DNA-binding properties. Except for traditionally used bulk methods like electrophoretic mobility shift assay (EMSA)2, 3, DNA footprinting4 or enzyme-linked immunosorbent assay (ELISA)5, there are single-molecule techniques allowing for detection of DNA-protein complexes6. Among them one can mention fluorescence resonance energy transfer (FRET)7, optical and magnetic tweezers8-11, atomic force microscopy (AFM)12 and nanopore sensing13-23. Single-molecule methods provide additional information regarding unique properties of proteins that cannot be distinguished in bulk experiments. Several excellent examples include estimation of a single-step size of kinesin8, direct visualization of backtracking of RNA polymerase on DNA24, mechanism of DNA uncoiling by topoisomerase25, ATPinduced helicase slippage on DNA11, and influence of CpG methylation on stability of a DNA-nucleosome complex26. Combination of two single-molecule techniques solid-state nanopores and optical tweezers was performed to controllably translocate a DNA molecule through a nanopore and measure its charge in solution27-29. Later it was used to estimate the charge of RNA30, detect nucleoprotein filaments15 and DNA-bound ligands18, 19. Glass nanocapillaries are a cheap and easy-to-produce alternative to nanopores in membranes used for various application including detection of DNA31, 32, proteins33,
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and DNA-protein complexes23. They can be shrunken under SEM to diameters below 10
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nm35, providing better signal-to-noise ratio (SNR) in the current signal compared to nanopores in silicon nitride or 2D materials, like graphene32, 36. In addition, combination of nanocapillaries with optical tweezers does not require a sophisticated microfluidic sample cell and DNA translocation results in a lateral displacement of an optically trapped bead37, 38. The lateral stiffness of optical tweezers is known to be higher than the axial one and the measured signals are characterized by better SNR39. Recently a combination of nanocapillaries with optical tweezers was used to study mechanism of DNA relaxation40 and electroosmotic nanojets41. In contrast to nanopores, nanocapillaries have elongated conical shape, so that a potential difference between the cis and trans sides produces a significant electric field within first 100-1000 nm inside the opening42. Since the surface charge of nanocapillaries is negative at neutral pH, the electroosmotic flow (EOF) is directed outside of the opening when the positive electrode is in the trans chamber41, 43, 44. In addition, once a DNA molecule is inside a nanopore/nanocapillary the electrophoretic driving force (Fep) on the DNA is reduced due to the EOF-induced drag force (Fdrag) generated from the flow around its backbone42, 45. Thus, the total drag opposing translocation of the DNA is a superposition of flows of counterions originated from negatively charged glass walls and DNA. The electrophoretic force can be interpreted as the bare DNA linear charge density λ0 being reduced by some value λdrag, resulting in F = (λ - λ )ΔΦ , where ∆Φ is the potential difference between cis and trans electrodes. In this letter for the first time we used glass nanocapillaries combined with optical tweezers to detect DNA-bound proteins and demonstrate influence of the drag force acting on them inside nanocapillaries. In comparison to free translocation, addition
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of optical tweezers allows to stall a DNA molecule inside a nanocapillary, controllably pull the DNA outside, and measure the force acting on the DNA and DNA-protein complex. In addition to the force signals we present current signatures of DNA-bound proteins, providing additional information about the studied system. In glass nanocapillaries all detected DNA-protein complexes behaved as if they were less negatively charged than DNA. The electrophoretic force acting on RecA-coated DNA inside nanocapillaries was smaller than on the bare DNA and differed from the results previously obtained in nanopores15,
18, 19
. We proposed an explanation of these results
based on the EOF-induced drag force. We modified a stochastic model from Ref. 19 by adapting it to the specific geometry of nanocapillaries to obtain the effective charge of DNA-protein complexes. Experimental setup The setup used in this work combines nanocapillaries and optical tweezers (Fig. 1 a)42. Using the optical tweezers the surface of a polystyrene bead coated with DNAprotein complexes was positioned 2-4 µm away from the nanocapillary opening. The DNA molecules were driven inside the nanocapillary by the electric field. Insertion of a single DNA molecule was confirmed by a simultaneous change in the force and current signals (Fig. 1 b). The force measured in the system is proportional to the displacement of the optically trapped bead from its equilibrium position F = -k(z-r) , where k is the spring constant of the optical tweezers. After stalling a DNA molecule, the nanocapillary was moved away from the optically trapped bead using a nanopositioning stage until the DNA molecule was completely pulled out, e.g. force and current signals restored to the initial levels (Fig. 1 a). At the position corresponding to the protein attached to the DNA
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a sudden change in the force and current traces was observed followed up by their decay, while the nanocapillary was moved further away (Fig. 1 b). Theoretical model In
the
system
combining
nanocapillaries
and
optical
tweezers
the
electrophoretic force (Fep) pulling DNA inside the nanocapillary is balanced by the harmonic restoring force of the optical tweezers (Fot). These two forces act to extend the DNA, whereas the entropic force (polymer extension) of DNA opposes them. The force on the bead exerted by the optical tweezers is equal to the electrophoretic force (Fot = Fep), except when additional forces are introduced, i.e. the force on a DNA-protein complex during its translocation. The system was modeled with a pair of coupled Langevin equations for two state variables19, including the distance r of the bead center from the opening and the contour length of DNA s from the bead to the opening (Fig. 1 a). The evolution of the state variables is governed by the free energy of the system: (, ) = () + (, ) + !" () + # (, )
(1)
with contributions from the optical trap !" () = 1&2 '(( − )* , where Gwlc(r,s) – the entropic free energy of a worm like chain of DNA, GDNA(s) – the free energy of a charged DNA molecule in the nanocapillary, and Gp(s) – the free energy contribution from a protein bound to the DNA at the position sp (see section S2 of SI). The two Langevin equations were coupled by the DNA spring free energy Gwlc(r,s), which depends on both state variables r and s.
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We applied the stochastic model in the geometry of a nanocapillary extracted from images obtained with scanning electron microscope (SEM). Shrinking of a nanocapillary under SEM changes the shape of the tip to the geometry approximated by two truncated cones (Fig. S1), where T and t – the taper lengths of large and small cones, respectively, β and α – the opening angles of large and small cones, respectively, R0 – the radii of the opening. Applying the continuity condition for the electric field and potential inside two cones, the electrostatic potential inside the small cone can be expressed as: +, (-) = ./
0
+ ./
2 3
(,1 )4
(2)
and inside the large cone: +* (-) = ./
5 ,1
267 8
(3)
Here 9 = : ⁄(2;) and @ = A⁄(2;
