Piercen M.
Oliver
,
Jin Seon
Park
and
Dmitri
Vezenov
*
Lehigh University, Department of Chemistry, 6 E. Packer Ave, Bethlehem, PA 18015, USA. E-mail: dvezenov@lehigh.edu
First published on 19th November 2010
We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescence image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force–distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy.
Single molecule force spectroscopy (SMFS) has been used extensively for determining the nature of specific and non-specific interactions of biological molecules with surfaces or other biomolecules. The capability to measure the elastic properties of short polymers in an array format is important for the development of high throughput SMFS. Atomic force microscopy (AFM) and optical tweezers are the most widely employed force spectroscopy techniques and have enabled single molecule studies of stretching biological polymers and inter/intramolecular binding, including protein unfolding,11,12DNA/DNA and DNA/protein interactions,13,14 conformational changes in polysaccharides,15 mechanochemistry of single-bonds,16 and peeling of single DNA oligomers from solid surfaces.17 Despite their extensive use in force spectroscopy of biomolecules, optical tweezers and AFM are not readily amenable to implementation in a parallel format, since a different optical trap or AFM tip is needed for each polymer in the array. On the other hand, magnetic tweezers provide the means for parallel acquisition of molecular force-extension curves through measurement of the out-of-plane (z) position of multiple magnetic probes all undergoing simultaneous displacement under an applied external magnetic field.18–22 The technique allows for the capture of numerous force-extension curves of single polymer molecules within a field-of-view of an optical microscope. We are particularly interested in chip-based SMFS for DNA molecules, since our goal is to develop a platform capable of high-resolution measurements on short DNA oligomers. Advances in the introduction of chemical functional groups to synthetic and genomic DNA23 make it possible to use robust covalent attachment of DNA for prolonged, multi-cycle force spectroscopy on a massively parallel scale. With single nucleotide resolution, application of force spectroscopy to rapid genome sequencing can be envisioned.24
In this paper, we used high-resolution single molecule force spectroscopy based on magnetic tweezers with evanescent illumination (evanescent nanometry), also recently implemented by Liu et al.25 This technique has several practical advancements in terms of improved force and molecular extension sensitivity, low noise, and facile force calibration of each magnetic probe-biomolecule pair. Probe microspheres are synthesized in-house to optimize specific surface chemistry, reduce the surface roughness for uninhibited attachment of a single molecule, and maximize the dynamic range (i.e. fluorescent signal and magnetic force).
In any magnetic tweezers experiment, the inhomogeneity of the probes presents a problem for force calibration. Even for the most controlled probe synthesis, variations in magnetite content and diameter are expected. Hence, force calibration must be performed for each probe. Conventionally, one performs force calibration of magnetic tweezers using the equipartition theorem (or a fit to a full power spectrum of a probe's fluctuations). A probe's position z-variance 〈σ2z〉 can be used to derive the polymer rigidity (dF/dz), in order to get the probe force at a given electromagnet current:
![]() | (1) |
In this paper, we show that the freely jointed chain model for ssDNA stretching can accommodate several fitting parameters including parameters for probe force and information about the evanescent penetration depth. The simple model yielded consistent forces for any one probe and penetration depths consistent with independent calibration using a combined AFM/TIRF instrument. Using our experimental setup of electromagnetic tweezers with evanescent wave excitation, we are able to acquire stretching curves for individual molecules of ssDNA and detect hybridization at a resolution approaching a single nucleotide with the future goal of highly-parallel measurements.
A magnetic tweezers instrument that achieves observation of multiple probes, while simultaneously applying force using an electromagnet positioned above the sample, is most easily implemented using an objective-style TIRF microscope to create an illuminating evanescent field. Fig. 1 shows a schematic of the placement of the electromagnet, fluid cell with the sample, and TIRF objective in such a system. In spite of a typically greater force achieved by permanent magnets over electromagnets, we opted to use a stationary electromagnet, because re-positioning of the magnet during the experiment is not required, thus, eliminating noise due to mechanical movement of the positioning stage in the course of a force ramp. Relatively high fields (>0.1 Tesla) can still be achieved with the electromagnet (Fig. 2). The force ramp is easy to implement via a coil current ramp. Hysteresis of the magnetic field is negligible at the loading rates used in our experiments (see section S1 in the Electronic Supplementary Information, ESI†). The change in the incident angle θ of the laser beam enables selection of the penetration depth of the evanescent field.
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Fig. 1 Experimental setup for magnetic tweezers force spectroscopy with evanescent illumination. The sample is illuminated by a 532 nm laser beam through a high numerical aperture lens (NA = 1.45). The lateral position dx of the optical fiber from the laser source is adjusted with a motorized actuator and changes the position of the focused spot in the back-focal plane of the objective. The control of the laser spot position allows for adjustment of the laser angle θ incident to the glass/water interface. A thin layer (∼12.5 nm) of gold (yellow) is present at the interface for reaction with thiolated DNA. The evanescent field propagates from the glass/water interface to illuminate the tethered magnetic-fluorescent probes (inset). Force F is applied to the probes by an electromagnet placed above the fluid cell. The magnetic field B is modulated by a current i through the coil of the electromagnet. Fluorescent emission from the probesI is filtered from the incident laser radiation by a dichroic mirror and a high pass filter and detected by a digital camera connected to a computer. |
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Fig. 2 (a) Linear response of electromagnetic field and field gradient with applied current at a distance of 0.5 mm. (b) Magnetic field versus distance at 0.94 A applied current. The magnetic field gradient (dashed line) at a distance of 0.5 mm from the bead is ∼55 T m−1. (c) Probe fluorescent intensities as a function of distance at two actuator positions 0.150 cm (solid) and 0.200 cm (dashed). (d) Colorized probe image versus electromagnet current for stretching 200-mer ssDNA. Upon applying force to the probes, we observe a noticeable change in intensity, which depends non-linearly on the current applied to the electromagnet. |
Angle positioning can be readily achieved by altering the lateral displacement dx of the single-mode optical fiber connected to the laser source (λ = 532 nm). The setup provides essentially hands-free remote operation, being fully computer controlled except for the lateral stage positioning.
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Fig. 3 Reaction scheme of probe and DNA attachment to gold-coated glass coverslip. |
Fluid cells were constructed by placing a thin ring of 5-minute epoxy (Devcon 5 min Fast Drying Epoxy, Riviera Beach, FL) around the edges of the sample to adhere a 100 μm-thick mylar film with punched inlet and outlet holes. The chamber dimensions were approximately 45 mm × 10 mm × 0.2 mm. Blunt-ended needles (23 gauge, Henkel Loctite) were glued with 5-minute epoxy around the inlet and outlet holes for infusion of rinse buffer and activated magnetic force probes.
For attachment to ssDNA oligomers, the beads from 25 μL of a solution of synthesized force probes (1–2% solids) were washed three times with 1 mL of pH 5.4 100 mM MES (2-(N-morpholino)ethanesulfonic acid, MP Biomedicals) buffer with centrifugation and resuspended in the same buffer. 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were added and left standing for 1 h. The probes were washed 3 times with 1 mL pH 8.0 PBS buffer (10 mM with 150 mM NaCl) and resuspended in 0.5 mL pH 8.0 PBS buffer containing 0.1% Tween20 (Calbiochem). A solution of activated probes was infused in the fluid cell after an initial flush with pH 8.0 PBS 0.1% Tween20 and incubated for 1.5 h. Unbound beads were then removed from the surface by slowly infusing the same buffer. Further addition of 100 nM 50-mer reverse-complement centered on the 200-mer strand (Integrated DNA Technologies, Coralville, IA, USA) was conducted in a similar fashion.
The electromagnet was powered by a linear 24 V DC power supply modulated by a proportional power module (PWM950, Magnetic Sensor Systems, Van Nuys, CA, USA) through a 0 to 5 V input from a multifunctional USB I/O board (NI USB-6215, National Instruments). Control of the I/O board was enabled by a NIDAQ Tools XOP using custom code written in Igor Pro 6.1 (Wavemetrics, Inc.). Field measurements were made using a Hall effect sensor (Allegro Microsystems, Worcester, MA, USA) while moving the electromagnet on a motorized stage. The electromagnet displays a linear dependence on applied current and fields of just over 0.1 Tesla at close distances (Fig. 2a & b). On average, the electromagnet tip was about 0.5 mm above the probes during our experiments with a field gradient of ∼55 T m−1.
Evanescent field penetration depths d were later determined from a simultaneous AFM and TIRF experiment. A probe was glued to an AFM cantilever and immersed in pH 8.0 PBS 0.1% Tween20 above a gold-coated coverslip. The bead was moved from contact to 1 μm away from the surface while capturing a movie. Data at several angles, or actuator positions, were taken (Fig. 2c). The data were fit to exponentials and d was found as a function of incident angle (see section S3 in ESI for details†).
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Fig. 4 Applied electromagnet current (a) and raw probe intensity data (normalized to the intensity at i = 0 Amps) (b) versus time. Extension of DNA is indicated in blue and retraction in red. Intensity as a function of applied current is shown in (c). Three force loops are shown at different cycle times. Reducing the cycle time to 10 s introduces no hysteresis in the intensity-current retraction curves. There is, however, a hysteresis between the retraction and extension curves in all three cycle times. The hysteresis does not disappear until approximately 0.7 A of current is reached when the extension curve shifts to overlap the retraction data. |
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Fig. 5 Intensity-current data from retraction measurements fitted to eqn (6) in the text. (a) Plot of residuals for two intensity-current curves (raw data) indicating a noise level of less than 2% over all fits at high forces (i > 0.1 A). (b) The raw data (red) for two retraction curves; the black line is a fit from averaged data (not shown). Aside from normalization of each curve to its maximum intensity, no further modification was performed to align the data. Inset: expected variation of force with current based on eqn (4) and experimental data for magnetic field shown in Fig. 2a and 2b. (c) Averaged retraction data for four different dx laser positions (corresponding to different penetration depths). For clarity, the normalization in (c) is based on the fits to eqn (4), resulting in an Io value corresponding to the position of the surface (i.e. a value of I = 1 corresponds to z = 0 nm). Four retraction intensity-current curves were averaged for each laser position dx and then fitted to eqn (5). The standard deviation at each point is smaller than the size of the markers (<1% of the actual intensity value). Fitting results from this data set and others are listed in Table 1. |
An evanescent wave propagates into a medium with an exponential decay. Our numerical simulations of the fluorescent image of a microsphere excited by an evanescent wave show that the total intensity of the fluorescence also exhibits exponential decay with distance from the surface when captured with high NA objectives.28 Empirically observed z dependence (Fig. 2c) bears out this expected behavior of total image intensity in the range of interest (see section S3 in ESI†). Therefore, the probe distance from the surface z is a function of the measured intensity I, the maximum intensity I0 at contact, and the penetration depth d of the evanescent field:
![]() | (2) |
Typically, penetration depths can range between 70 nm and 200 nm or more, depending on the incident angle and refractive indices of materials at the interface. The exponential dependence of intensity on the probe position makes evanescent nanometry an ideal technique for measuring small extensions, especially since the penetration depths are on the order of the length of short (<500 bases) DNA oligomers and many proteins. Since the reference for z-displacement of the probe is the sample surface, and not other immobilized beads, a simple measure of the total intensity is all that is required to locate its out-of-plane z position. To measure the molecular extensions by evanescent nanometry, a calibration must be performed at the start of any experiment to determine the penetration depth of the field. We demonstrate that by fitting raw data to a known response of the macromolecule under tension (e.g. modeled as a freely-joined polymer) the ratio of the molecular contour length and penetration depth can be obtained as one such calibration. The method is only an absolute measure of the molecular extension if the contour length of the molecule (or part of the molecule) is known. Therefore, it will primarily be useful for applications where the change in the state of the molecule is of main interest.
Typical force calibration of optical or magnetic tweezers involves measurements of x, y or z-fluctuations for use in the equipartition theorem to determine the stiffness of the confining potential. The analysis of fluctuations, however, is problematic with pixel array (e.g.CCD) sensors, since one would have to correct for blurring effects (see section S6 in ESI†), if the exposure time is long compared to the system relaxation time (friction coefficient/stiffness ratio is 50–200 μs for our system). The exposure-time correction depends on the elasticity of the tether,29 complicating the analysis, since our exposure times are always kept at greater than 0.5 ms due to limits in signal-to-noise ratio in our system. We estimate that, using the fluctuation method, the forces in our system can be as much as a factor of 2–3 off even at very short exposure times (<100 μs) that still have reasonable signal to noise ratio (see section S6 in ESI†). In the case of evanescent nanometry, a photodiode detector would be the most straightforward high bandwidth solution for obtaining force information from z-fluctuations of a probe, but a more complex sensor, such as a CCD camera, is required for parallel analysis of multiple probes in a single field of view. Since our goal is to develop approaches to SMFS in an array format, we focus on the use of slow imaging sensors that have potential for future rapid acquisition of numerous force curves in parallel.
We observed that the relationship between applied electromagnet current i and magnetic force F is linear at forces above around 2–3 pN, whereas below 2–3 pN, we are in a non-saturating region. The full range of forces is described by the Langevin function, :
![]() | (3a) |
![]() | (3b) |
In eqn (3), Vbead is the volume of a single bead, ρbead and ρm are the densities of the bead and magnetite, respectively, MD is the domain magnetization of magnetite (4.46 × 105 A m−1), B is the magnetic field, and DNP is the diameter of the magnetite nanoparticles (∼10 nm). Since both B and ∇B are directly proportional to coil current i (see Fig. 2a), we can express changes in magnetic force with applied current by using a force sensitivity factor encompassing all of the constants from eqn (3):
F(i,SF,i0) = SF·![]() | (4) |
A widely accepted model to describe ssDNA stretching is the freely-jointed chain (FJC) model with finite segment elasticity:30–33
![]() | (5) |
![]() | (6) |
Due to an excluded volume effect (i.e. the spatial constraints of having two barriers—probe and surface—at opposite ends of the single-stranded DNA), a non-zero elongation at zero applied magnetic force is expected in our molecule-bead system. In addition, scaling predictions indicate deviation from simple polymer models in a low force regime.34 Therefore, at very low forces, the FJC model is not an adequate representation of the DNA tether and we fit our data only at a high stretching force regime (>1 pN). Our experimental results indicate that the bead intensity evens off at low applied force (or at an applied current of less than ∼0.04 A), meaning, along with the above considerations of subtleties of low force regime, that the crude normalization based on the maximum experimental intensity is not the quantity I0 from eqn (2). Instead, the value of I0 needs to be a fitting parameter, along with the force parameters, SF and i0, and the penetration depth d.
Fig. 5a shows an example of the fit applied to the data values in the range of 0.1 A to 0.94 A of current. The fit follows the functional form of eqn (5) well, as evidenced by the low residuals, which are (mostly) uniformly distributed around zero. Four intensity-current curves (from one movie sequence) were fitted for each of the four penetration depths captured on one probe (Fig. 5c). The results from fits to these magnetic tweezers intensity-current curves are summarized in Table 1.
dx/cm | L max ss /d | d/nm (Lmaxss = 116 nm) | S F/pN A−1 | I 0 |
---|---|---|---|---|
a The error indicated is the standard deviation of the fit coefficient. | ||||
0.125 | 0.992 ± 0.003 | 117.0 ± 0.3 | 25.4 ± 0.3 | 1.088 ± 0.003 |
0.150 | 0.907 ± 0.002 | 127.9 ± 0.3 | 25.8 ± 0.3 | 1.021 ± 0.003 |
0.175 | 0.836 ± 0.003 | 138.7 ± 0.5 | 24.2 ± 0.3 | 1.042 ± 0.003 |
0.200 | 0.834 ± 0.002 | 139.0 ± 0.3 | 24.0 ± 0.2 | 1.048 ± 0.002 |
The fitting function yields a ratio of contour length to penetration depth. Thus, one quantity must be known in order to get the value of the other. In this case, we used a known synthetic sequence (200 bases in length) and well-defined covalent attachment chemistry, so we were able to set the contour length to Lmaxss = 116 nm and obtain the values of penetration depth listed in Table 1. Any deviation of the contour length from its true value, due to pinning (i.e. changes in the point of attachment) or robust secondary structure, would alter the calculated penetration depth, although the results after addition of a complementary strand, discussed later, help corroborate this value of Lmaxss. In our case, the penetration depth values are within the expected range of those calculated from AFM/TIRF data (Fig. 6) and different by only a few nm for each dx setting. These deviations are expected, because a different sample, probe, and flow cell setup was used for AFM experiments, thus introducing possible slight changes in alignment (ESI, Fig. S5†).
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Fig. 6 Penetration depth versus incident angle calculated independently from AFM/TIRF data (black) and fits to eqn (6) (red). Dashed black line is a guide to the eye through TIRF/AFM results for d. |
An important result seen in Table 1 is the constancy of SF across different penetration depths. Although the penetration depths change, which corresponds to a change in dynamic range of our setup, SF is found precisely within less than 2 pN A−1 purely from fitting raw data to eqn (5). Thus, SF is truly independent of the penetration depth and precise knowledge of d for the analysis is not required (although knowledge of appropriate equation of state is required). Furthermore, a value of 10–30 pN A−1 is within the expected range of force values from probe magnetite loading and magnetic field measurements (ESI, Fig. S3†). The error in determination of d is around 0.3 nm, implying that we should be able to achieve the resolution of a single nucleotide (0.58 nm) in the length of the DNA molecule.
The low fit residuals give further justification for the use of a linear force sensitivity factor for our probes. We found that force sensitivity factors vary between beads due to the inhomogeneity in size of our probes and differences in the alignment of the magnet, but generally remain between 10 to 30 pN A−1. Interestingly, changes between the force-extension curves of ssDNA and double-stranded DNA (dsDNA) are most pronounced at very low force (<4 pN) and at higher forces (>10 pN) (see Fig. 7), which are well within range of the capabilities of our probes and the instrument.
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Fig. 7 (a) Experimental difference in intensity-current curves (four averaged retraction curves each) after hybridization of a 50-mer to the 200-mer, where the red curve is for fully ssDNA and black curve is for the partially hybridized strand. The scale is based on the initial intensity I0 set to 1 at z = 0. I0 values were obtained from fits to eqn (5) and (8) respectively. Lines indicate fits to data. Inset: same data plotted in terms of extension versus force using the parameters from fitting. The standard deviations of the averaged curves is less than their respective marker sizes. |
![]() | (7) |
In eqn (7), Kds is the unitless segment elasticity for dsDNA in 150 mM salt conditions (segment elasticity, 1000 pN, normalized by kBT/bds, where bds = 100 nm, or Kds = 24100).32 Although the correction for segment elasticity is small (∼2–3% at most), it is included for consistency with the model for ssDNA stretching. We combined eqn (5) and (7) and applied the logic behind the derivation of the fitting function for the single stranded DNA (see eqn (6)) to arrive at an analogous relationship for a mixed dsDNA-ssDNA model:34
![]() | (8) |
By fitting the ssDNA force curve before hybridization, the penetration depth (d = 139.0 ± 0.3 nm) was obtained along with the sensitivity factor (SF = 24.0 ± 0.2 pN A−1) (see Table 1 at dx = 0.200 cm). Since there is no realignment of either the electromagnet or TIRF angle during addition of the complement, we assume that these parameters do not change. The initial intensity (at i = 0 A), however, is expected to be different for single and partially double-stranded DNA due to appearance of the rigid fragment of dsDNA, so I0 was left as a fitting parameter. Using eqn (8), we found a double stranded fraction of χds = 0.252 ± 0.006 corresponding to 50.4 ± 1.2 bases in the 200-mer strand. This result is a difference of only 0.4 ± 1.2 bases from the theoretical fraction of double stranded character of χds = 0.25. Experimental knowledge of just two parameters, d and SF, from an initial fit to a ssDNA force curve is all that is required in order to determine the double-stranded fraction after hybridization. The initial intensity from the fits before and after hybridization changed from I0,ss = 1.048 ± 0.002 to I0,ds = 0.924 ± 0.001, which corresponds to an increase of ∼17 nm for the initial extension. Since the double stranded section behaves mostly as a rigid rod at low forces, the bead is effectively lifted away from the surface by the distance corresponding to the size of the dsDNA section ∼50 × 0.34 nm = 17 nm, in excellent agreement with the experiment. Therefore, the change in initial intensity is another possible method for detection of the hybridization event. Such a change in initial (zero applied force) intensity can be used to determine the extent of hybridization,10 but probably lacks the near single-base resolution that is offered by applying force and modeling a full mechanical response of the molecule.
The addition of a short primer to a longer ssDNA strand, which is to be sequenced, is a prerequisite for the initiation of the growth of the double-stranded segment using either polymerase or ligase. The 50-mer hybridized to the 200-mer is analogous to such a primer addition and allows for a convenient measure of the initial strand length in situations when the initial contour length is not known.
Since the length of the primer is known, we can factor it out in eqn (8) as follows:
![]() | (9) |
The hybridized intensity-current curve from Fig. 7 is fitted to eqn (9) with fit parameters of I0 and LssN, yielding values of 0.924 ± 0.001 and 86 ± 3 nm, respectively (the fit appears identical to what is shown in Fig. 7). The initial contour length Lssmax can be calculated by simply adding LssN + Lssprimer = 86 ± 3 nm + 29 nm = 115 ± 3 nm. Since a value of 116 nm is expected, this method of first adding a known primer appears to be a good way to estimate the contour length of the original ssDNA strand. Furthermore, the penetration depth can be calculated from R and Lssmax, and was found to be d = 138 ± 3 nm, within ∼1 nm of the expected value of 139 nm.
Once both the penetration depth and contour length of the ssDNA are determined, relative changes in the total hybridization of the ssDNA could be quantified from fitting a subsequent hybridization or base-addition event to eqn (8). If single nucleotide resolution can be achieved in this manner, application of this method to rapid genome sequencing using single nucleotide addition becomes a clear possibility.36 For example, in sequencing by synthesis, detection of extension of the double-stranded portion in a given single DNA molecule in the presence of polymerase and a single substrate (e.g. only dATP) is enough to detect that addition took place and, therefore, to make a base assignment in the original ssDNA molecule. The whole sequence can then be determined by cycling through the remaining three deoxynucleoside triphosphates and repeating the process until the full sequence is established. Force spectroscopy then effectively becomes a tool that detects elongation of the complementary DNA strand by polymerase and operates with single molecule sensitivity (like many fluorescence based systems currently in development).9 SMFS has the potential advantage over fluorescence-based methods in detections of runs of the same bases, because its response scales directly with the number of added bases (size of the double-stranded fragment).
While we designed the model sequence to eliminate the secondary structure as much as possible, future work on this system needs to incorporate the effect of transient secondary structure on force-extension behavior of short ssDNA oligomers. The presence of secondary structure can be mitigated with proper modification to the experiment or detailed analysis (e.g. accounting for shorter contour lengths due to hairpins). For example, one can either maintain a high temperature during the experiment (close to Tm) or focus on analyzing the data derived in the regime of high forces, i.e. above ∼15 pN—the threshold for DNA unzipping37,38—to completely eliminate contributions of secondary structure from experimental data. Since under normal conditions force-induced melting transition in dsDNA is initiated at around 65–70 pN,39 even under high force limitation there will be sufficient dynamic range to acquire force-extension data suitable for interpretation presented in this paper.
The drawback of the proposed technique is that, in the general case, only the ratio of the molecular counter length to evanescent wave penetration depth can be determined; however, applications of our approach to the detection of changes in molecular conformations (such as hybridization or ligation of the complementary DNA strand) can yield highly accurate values of the penetration depth and the polymer's contour length. As demonstrated here, inclusion of the known length marker (in the case of DNA, an oligomer of known sequence) can provide an alternative way of measuring the penetration depth via pre-determined changes to molecular size (e.g. due to hybridization with a primer or unfolding of a hairpin). While absolute extension measurements are possible with magnetic tweezers using reflection interference contrast microscopy,26 evanescent nanometry does not require a laterally resolved image, only the total intensity, making it possible to view many molecules in parallel with a low-resolution objective. Since magnetic tweezers are a straightforward and cost-effective platform for parallel analysis, potential single-base resolution opens the avenue for a possible rapid genome-sequencing device based on force spectroscopy detection of changes in DNA double stranded content, as described here.
The exponential distance dependence of the evanescent field, on the one hand, makes this technique extremely sensitive to small changes in conformations (with a resolution of 1–2 nucleotides), but on the other, it limits the method to relatively short molecules, <200 nm in a present setup, setting the upper limit for the length of a DNA molecule of interest at several hundred bases. While many next generation sequencing technologies operate on short (20–30 bases) reads, having the benefit of a reference genome, de novo sequencing requires long reads (hundreds of bases). The method described here already operates in this long oligonucleotide regime and it is possible to extend it further by moving to longer laser wavelengths and substrates with a lower index of refraction.
We acknowledge that more work needs to be done on optimization of the surface chemistry, specifically, to increase the number of active tethered microspheres, which is not routine and was not the purpose of this work. Extending the resolution to single-base addition will also require investigating the fluorophore composition of the probes in order to increase their intensity (i.e. signal to noise ratio) and extend their longevity, e.g. by using non-bleaching fluorophores such as quantum dots.
Footnote |
† Electronic supplementary information (ESI) available: SEM images, magnetic properties, and force versus field estimates for magnetic-fluorescent probes. Evanescent field penetration depth from simultaneous AFM/TIRF measurement. Noise estimate from immobilized probes. Sample raw intensity-current data. Conversion of intensity fluctuations to force. See DOI: 10.1039/c0nr00479k |
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