MspA nanopore as a single-molecule tool: From sequencing to SPRNT.

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a new tool for analyzing the motion of nucleic acids through molecular motors. With SPRNT, individual enzymatic motions along DNA as small as 40pm can be resolved on sub-millisecond time scales. Additionally, SPRNT reveals an enzyme's exact location with respect to a DNA strand's nucleotide sequence, enabling identification of sequence-specific behaviors. SPRNT is enabled by a mutant version of the biological nanopore formed by Mycobacterium smegmatis porin A (MspA). SPRNT is strongly rooted in nanopore sequencing and therefore requires a solid understanding of basic principles of nanopore sequencing. Furthermore, SPRNT shares tools developed for nanopore sequencing and extends them to analysis of single-molecule kinetics. As such, this review begins with a brief history of our work developing the nanopore MspA for nanopore sequencing. We then describe the underlying principles of SPRNT, how it works in detail, and propose some potential future uses. We close with a comparison of SPRNT to other techniques and we present the methods that will enable others to use SPRNT.

1. Introduction

Nanopores are emerging as a high-precision single-molecule tool. In its basic implementation, a nanopore device consists of a single pore within a membrane (Fig. 1). This membrane divides a salt solution into two wells called ‘cis’ and ‘trans.’ When a voltage is applied across this membrane, ion current flows through the pore. The magnitude of this ion current is the primary signal. Molecules of interest (i.e. DNA, RNA, peptides, nanoparticles, etc.) are drawn towards the pore and then through it by the electric field. As the molecules traverse the pore, they alter the ion current flowing through the pore. Depending on the degree to which the ion current is blocked, various properties of the molecule and its movement through the pore can be inferred. Generally, nanopores can be grouped into two categories: (1) ‘biological pores’ which are pore proteins such as alpha hemolysin (aHL) [1-4] or Mycobacterium smegmatis porin A (MspA) [5-7] embedded in a phospho-lipid bilayer and (2) solid-state pores, which are nanometer-scale holes drilled into thin membranes of silicon nitride [8-10], graphene [11-13], molybdenum disulfide [14], etc. Nanopores have been used to detect size and shape characteristics of proteins [15], separate polymers of different sizes and charges [16,17] or to separate two types of proteins [18].

Fig. 1

Schematic of the u-tube bilayer setup at various scales (originally described by Akeson et al. in 1999 [2], see Appendix A for detail on building and setting up such an experiment). The bilayer is established across the ~20 µm aperture. Once a bilayer is established, MspA is added to the cis solution until a single pore inserts. Once the operator detects an insertion, the remaining MspA is perfused from the cis well and the system is prepared for experiments. When a voltage is applied across the bilayer by two Ag/AgCl electrodes (as shown in c, not to scale), negatively charged DNA is drawn into and through the pore. While all experiments described below are performed using this single channel recording setup, it is possible to massively parallelize nanopore setups for higher throughput. Figure modified from [37].

In 1996, Kasianowicz et al. [1] proposed using a nanopore setup to sequence DNA. In this scheme, the applied voltage electrophoretically feeds single stranded DNA (ssDNA) into the pore. As the DNA passes through the pore, DNA bases within the pore block the ionic current. Because each base has differing size and charge distribution, it was proposed that each base has its own distinct ion current, allowing one to determine the DNA sequence by analyzing the current trace as DNA is drawn through the pore. Kasianowicz et al. found that they could translocate DNA strands through the pore and that the presence of DNA within the pore reduced the ion current. From these initial experiments, there emerged two clear challenges to the implementation of nanopore sequencing: researchers needed (1) a pore with the ability to distinguish between nucleotides with sufficient signal to noise and (2) a means of slowing the DNA translocation enough so that each base could be read for long enough [1].

In order to have single-nucleotide sensitivity, it is key that the size of the nanopore (diameter and length) is commensurate with the dimensions of the DNA bases [5,16]. Biological pores such as aHL [1,2,4,19-21] or MspA [5-7,22-27], with a geometry similar to the size of ssDNA nucleotides [5], have been used to study DNA.

2. MspA for sequencing DNA

MspA, in particular, proved nearly ideal for the analysis of single-stranded nucleic acid strands. This is because the pore constriction (the region of the pore with the highest ion current density and thereby highest sensitivity to nucleobase differences) is just 0.6 nm long and 1.2 nm wide [5] (Fig. 2). This compares well with the size and spacing of DNA nucleobases within ssDNA. In contrast, aHL has a long constriction resulting in several DNA sequence recognition sites spanning some ~12 nucleotides [20]. While it is possible to construct solid-state pores with dimensions similar to those of MspA, in graphene for instance [11-13], current fabrication techniques have not yet achieved the atomistic reproducibility inherent to biological systems and the efficacy of such solid-state pores for DNA sequencing has yet to be demonstrated.

Fig. 2

Mycobacterium smegmatis porin A (MspA). Wild type MspA contains negatively charged residues D90, D91, and D93. Butler et al. found that mutation of these residues to neutrally charged N allowed DNA to translocate through the MspA pore. Further mutation of the pore included positively charged residues within the vestibule (D118R, D134R, and E139K) and resulted in increased DNA interaction. Figure modified from [5].

2.1. Mycobacterium smegmatis porin A

MspA is an outer membrane protein that derives from Mycobacterium smegmatis. In 2008 Butler et al. proposed that its size and shape would make it a promising candidate for nanopore sequencing [5]. In initial experiments with wild-type MspA, DNA was unable to translocate through the pore. Butler et al. hypothesized that this was due to negatively charged aspartic acid residues that define the constriction of the pore (D90, D91, D93; Fig. 2). Mutating these residues to neutral asparagines resulted in clear ssDNA-induced current blockades. Further mutation of the pore to include positively charged residues within the vestibule (mutations D118R, D134R, and E139K; Fig. 2) significantly increased the rate of DNA capture by the pore allowing for a ~100-fold reduction in the minimum analyte concentration for the negatively charged DNA.

2.2. Base differentiation and single-nucleotide resolution

Early indications that DNA bases might actually create different blockage currents were shown with aHL [2-4,19,28]. In 1999 Akeson et al. translocated block homopolymer RNA strands consisting of 30 As followed by 70 C’s through an aHL pore and observed ssDNA translocation events that consisted of two distinct current levels, however they were unable to resolve individual bases. This confirmed what was apparent from the initial nanopore sequencing paper, that a method for slowing DNA translocation was needed [1]. In 2000 Henrickson et al. [19] tethered DNA to a large streptavidin molecule in order to hold DNA stationary within a nanopore. This technique was later used to demonstrate that each of the 4 bases created unique blockage currents within the aHL pore [3] however it was revealed that the region of sensitivity within the aHL pore was ~12 nt long [4,28], far too long to achieve the single-nucleotide resolution necessary for DNA sequencing.

As with aHL, ssDNA translocates through MspA far too quickly (>1 nt/µs) for individual bases to be resolved within the noise [5]. In 2010, Derrington et al. showed that MspA was capable of distinguishing between the four canonical bases when held within the pore by duplex DNA, which is too wide to fit through MspA’s constriction [6]. In 2011 Manrao et al. [7] showed that MspA was capable of distinguishing single bases using DNA nucleotides tethered to a streptavidin molecule (Fig. 3). By changing the location of a single nucleotide within a heterogeneous DNA strand held at different locations within the pore (Fig. 3a and c) Manrao et al. showed that MspA’s region of base sensitivity included ~4 nucleotides in and around the constriction and that the exchange of just a single DNA base could be detected. The ion current was even sensitive to the replacement of a C with methyl cytosine. These results showed that MspA’s ability to distinguish between bases was far better than observed in comparable experiments with aHL [4,20,21].

Fig. 3

Single nucleotide sensitivity. (a) Schematic of initial experiments with DNA held stationary in the pore by tethering it to a large streptavidin molecule. (b) These experiments showed that homopolymer strands containing each base had its own unique current. (c) Single nucleotide substitutions of nucleotides held within the pore at various positions shown in (a) such as a single C replacing an A at various positions along a homopolymer A DNA strand showed that the region of sensitivity for MspA was ~4 nucleotides wide, and that the substitution of a single base held within the constriction was detectable. (b) and (c) modified from [7].

2.3. Enzyme control

All that was needed then for nanopore sequencing was a means of slowing the DNA translocation through the pore; with such control, it was possible that the superior base recognition of MspA could enable nanopore sequencing. Just as nature provided almost ideal biological nanopores, nature again offers a solution in the form of molecular motors that walk along DNA (polymerases, helicases, translocases). Gyarfas et al. [29] pioneered such techniques, using at first T7 and Klenow fragment. However, the most success was later found with phi29 DNA polymerase (phi29 DNAP) [30,31] a highly processive DNAP from Bacillus subtilis phage phi29.

Rather than attaching the molecular motor to the pore, it is much easier to load the molecular motor onto the DNA and then draw the DNA into the pore until the enzyme rests on the pore. A key challenge is preventing enzyme extension of the DNA primer in bulk, before the DNA-enzyme complex is loaded onto the pore. To solve this problem, Cherf et al. [31] developed the ‘blocking oligomer technique’ (Fig. 4) wherein the phi29 DNAP is prevented from extending a DNA primer strand by the presence of a third DNA strand: the non-extendable blocking oligomer. The blocking oligomer hybridizes with the template DNA strand immediately 3′ of the primer strand. In bulk, phi29 DNAP binds to the template strand near the 3′ end of the blocking oligomer but is unable to extend the blocker due to non-complementarity and chemical modification of the blocking oligomer’s 3′ end [31] (it turns out that non-complementarity of the last ~7 bases of the blocking oligomer to the template strand is sufficient to prevent bulk activity [22]). When the template strand is threaded into the pore and the phi29 DNAP comes into contact with the pore rim, the force of the pore rim on the enzyme pushes the phi29 DNAP in the 5′-3′ direction through the duplex region created by the blocking oligo (this is the reverse of the direction that phi29 DNAP usually walks during polymerase activity). The phi29 DNAP, being forced backwards along the template strand, unzips the blocking oligo from the template strand one nucleotide at a time, thereby feeding the template strand into the pore from cis to trans one nucleotide at a time until the blocking oligomer is fully dissociated and the primer’s 3′ end falls into the phi29 DNAP’s active site. In the presence of MgCl2 and deoxynucleotide triphosphates (dNTPs), the phi29 DNAP then goes to work extending the primer DNA strand and pulls the template back out of the pore from trans to cis (Fig. 4b). Cherf et al. found that as the DNA moves through an aHL pore, the ion current shows distinct current steps corresponding to each of the phi29 DNAP steps along the DNA strand, however they could not discern the DNA sequence.

Fig. 4

Phi29 DNAP controls DNA translocation via the blocking oligomer technique. (a) DNA initially translocates from cis to trans as the electric force on the DNA pulls it through the phi29 DNAP unzipping the frayed end of the duplex DNA like a zipper until it arrives at a nick between the blocking oligomer and the primer strand, at which point the blocking oligomer dissociates. (b) This exposes the extendable end of the primer formed by the hairpinned 3′ end of the template strand. If proper buffer conditions are present, the phi29 DNAP begins extending the primer thereby pulling the template strand back out of the pore from trans to cis. (c) As phi29 DNAP steps DNA through the pore, a series of current levels are observed. Green and blue arrows indicate the current levels that correspond to unzipping and polymerase mode, respectively. (d) Because the durations of the observed current levels have stochastic durations, it can be more clear to depict the observed current levels without duration information so it can be displayed with the corresponding DNA sequence. As in Manrao et al. 2011 [7], the current is sensitive to approximately 4 nucleotides held within the pore constriction. In (d) the four nucleotides centered on a given current level are those that contribute most to the observed current, for instance, the nucleotides GGGT contribute most to the level indicated in (d). We call these four nucleotides a ‘quadromer.’ (c) and (d) modified from [22].

Manrao et al. applied this technique to control DNA motion through MspA (Fig. 4c), and observed better-resolved current steps with durations from milliseconds to seconds. Because of the superior base-resolution of MspA, the series of current amplitudes of each step could be associated with DNA sequence [22]. As in Manrao et al. 2011 [7], single nucleotide substitutions could be detected and approximately four nucleotides within the constriction influenced each observed current level (Fig. 4d). This work laid the groundwork for nanopore strand sequencing [32]. Subsequent studies using the MspA-phi29 DNAP system found that MspA could be used to directly detect epigenetic base modifications [23,25,26] and unnatural bases dNaM and d5SICS [27] within single DNA strands.

2.4. Making sense of long DNA reads

The ability to make long reads of single DNA molecules is one of the key attractions of nanopore sequencing. Long reads of several thousand bases were achieved using the same MspA-phi29 DNAP system described above [24]. This can be done by linking genomic DNA from bacteriophage phi X 174 with adapters that facilitate the loading of phi29 DNAP as in Manrao et al. 2012 [22,24]. In Laszlo et al. 2014, the adapter on one end of a linearized phi X 174 genome, contained a frayed end and the adapter on the other end contained a hairpin with a nick to enable the polymerase mode.

Because ~4 nucleotides within MspA’s constriction influence the observed current values, it was necessary to measure all 256 different sequence combinations of A, C, G, and T (we call each 4-nucleotide combination a quadromer, Fig. 5a). These measurements were conducted on a de Bruijn sequence [33,34] that contained 256 quadromers [24] (Fig. 5a).

Fig. 5

Quadromer map and alignment to sequence. (a) Graphical representation of quadromer map values aligned to the de Bruijn sequence used to measure all 256 quadromers. Each measured current value is centered on the four nucleotides that contribute significantly to its current value. CCGT corresponds to the first current level displayed, CGTC corresponds to the next current level, etc. (b) Subset of a raw current read of phi X 174 DNA. Average current levels are automatically detected and displayed in (c). (d) These current reads can be aligned to quadromer map based predictions of the reference sequence (using methods described in Appendix C). The ability to perform such alignments allows for association of enzyme behaviors with DNA sequence. Figure modified from [24].

The quadromer map was able to predict the current values that would be observed for other previously unmeasured DNA strands. Translation of the known phi X 174 reference DNA sequence into a series of current levels enabled alignment of the long DNA reads to the reference sequence (Fig. 5b). To make sense of these long reads, it was no longer practical to align the DNA sequences to the measured current levels by hand. Automated alignment techniques were used to associate nanopore current readings to DNA sequence. Such alignments can be used in high-confidence species identification and hybrid genome assembly [24] (using a single ~3800 base nanopore read as a scaffold for assembly of 100 base long Illumina reads, Fig. 5c). While species identification and hybrid genome assembly are not necessary for SPRNT, we list them here to demonstrate the ability of an MspA-based system to identify DNA sequences. As we will see, the ability to accurately identify where an enzyme is along a long DNA strand is a valuable aspect of SPRNT, enabling direct study of DNA-sequence-specific enzyme behavior. (For a full description on how nanopore current reads are preprocessed for alignment see Appendix B, for a full description on how these reads are then aligned to DNA sequence see Appendix C).

2.5. Nanopore sequencing outlook

Nanopore sequencing is now entering commercialization and several companies are pursuing nanopore technologies. Nanopore sequencing has intrinsic advantages of long reads and high speed in addition to its ability to directly detect non-canonical bases [7,21,23,25-27]. Such abilities will allow nanopore sequencing to answer questions accessible by few, if any, currently available sequencing technologies. Nanopores are already being applied to problems of rapid species identification [24,35] and the long reads make them useful for hybrid genome assembly [24,32]. Nanopore devices approaching the market are also remarkably portable [32] enabling them to be deployed rapidly in the field [36]. Challenges still remain with regard to read accuracy, sample preparation (for example delivering small, unamplified genomic samples to the pore), and throughput (currently available nanopore sequencers do not have enough throughput to tackle a human-size genome). Such challenges are the subject of research in academic and industrial laboratories.

2.6. From sequencing to SPRNT

A remarkable consequence of MspA’s sensitivity to small changes within its constriction is that the current is sensitive to not only the nucleotides’ identity but also their precise position along the axis of the pore’s constriction. For instance, DNA held slightly more towards cis within the pore results in a significant change in the observed current. Specific nucleotide sequences can be so sensitive to the exact nucleotide positioning that displacements of the DNA as small as 40 pm can be resolved on one ms time scales [37].[1] While such sensitivity to DNA registration within the pore may serve as a challenge to sequencing, it also enables use of MspA as a powerful new tool for analyzing the motion of nucleic acid processing enzymes at unprecedented resolution and therefore allows very detailed studies of how enzymes move along DNA. We cultivated this tool and called it Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) [37].

3. SPRNT

3.1. Historical use of nanopores for kinetics

Nanopores have been used to study single molecule kinetics for nearly two decades. In 2000, Henrickson et al. varied the applied voltage (thereby varying the applied force) to measure the varying dwell times of molecules held within an aHL pore at varying forces [19]. aHL was later used to measure the kinetics of DNA hairpin unzipping within aHL [39,40] and force dependent dissociation of DNA-exonuclease complexes [41]. Shortly thereafter, researchers used aHL pores to study the stepping kinetics of individual polymerases [30,42,43].

3.2. Introduction to SPRNT

While the use of nanopores to study single molecule kinetics is not new, the key advance that enabled SPRNT was the realization that the currents observed while DNA translocates through the pore can be directly related to the precise longitudinal position of DNA within the pore [37]. SPRNT takes advantage of this exquisite sensitivity to track the progression of DNA through enzymes.

The high spatial resolution of MspA is readily apparent when studying the effect of variations in the applied voltage [37]. Fig. 6 shows a comparison of current levels observed at 180 mV and 140 mV while phi29 DNAP passes DNA containing an abasic residue through the pore. The observed pattern of current levels at 140 mV appears shifted when compared to current levels at 180 mV. In addition to decreasing the current amplitude because of the decreased applied voltage, the voltage change shifts the envelope of the current levels; this shift corresponds to the DNA being held further to the cis side of the pore at 140 mV compared to 180 mV due to the reduced electric force on the DNA. Interestingly, the current levels observed at 140 mV lie along a spline interpolation of the observed 180 mV current levels (Fig. 6d). In other words, measured current level amplitudes can be predicted using a spline interpolant and knowledge of the DNA displacement. Conversely, measurement of the current levels and knowledge of the underlying smooth curve can be used to gain information about the position of the DNA within the pore. In regions where the current levels change significantly for small displacements of the DNA, SPRNT provides a high-precision measurement of DNA position within the pore. In some sequence contexts that exhibit large current differences between adjacent levels, SPRNT has resolution at or below 40 pm on ms time scales.

Fig. 6

Transduction of current to distance. (a) Regions of high current contrast can be used to measure DNA position precisely. Small uncertainties in measured current translate to small positional uncertainty. (b) Schematic depiction of DNA position within the pore at two different voltages; differences in the applied electric force result in different DNA extensions. (c) Current levels observed for phi29 DNAP controlled motion of DNA through MspA at 180 mV and 140 mV of applied potential. A cubic spline interpolant has been applied to each set of current steps. Note that, apart from a scaling factor, the shape of the spline is identical but the position of the spline has shifted a distance δ. (d) After a linear scale and offset is applied to the two splines, a horizontal displacement δ = 0.29 nt brings the two splines in line with one another. This experiment has two important results: (1) The current levels observed during single-nucleotide stepping of DNA through MspA lie along an underlying smooth curve that is well-approximated by a spline. (2) This spline provides a direct mapping from current to DNA position and we can use it to measure sub-nucleotide movement of DNA. Figure modified from [37].

Precision smaller than the diameter of a hydrogen atom may seem at first impossible or not meaningful given that the experiment takes place at room temperature in water. Naively, one might expect that Brownian motion of the DNA within the pore, Brownian hopping of the enzyme on top of the pore rim, and flopping of the bases along the axis of the pore may prohibit SPRNT’s exquisite spatial resolution. Even though the DNA’s position within the pore is fluctuating significantly more than the precision of the measurement, our measurements indicate that these fluctuations must occur on much faster time-scales than the measurement. This is evident in the experimental fact that the observed current follows a smooth curve and that, on the timescale of the measurement, the uncertainty in the observed current is relatively small compared to the change in current caused by a small translocation of the DNA. Thus, SPRNT is able to provide such a precise measurement of DNA position because it averages over these fluctuations.

3.3. Proof of principle application of SPRNT to another enzyme

In essence, SPRNT turns nanopore DNA sequencing on its head; instead of using enzymes to control the DNA’s motion through MspA in order to measure the DNA’s sequence, we are using our knowledge of the DNA sequence to measure the enzyme’s motions. To perform SPRNT, one needs precise knowledge of the underlying smooth curve that is associated with a given DNA sequence. Alignments of measured DNA levels using another enzyme can then be compared and aligned to this underlying smooth curve. Where along the curve aligned levels reside can then be translated to DNA position within the pore.

Derrington et al. [37] showed proof of principle demonstration of SPRNT to measure and compare the motions of phi29 DNAP and a Hel308 helicase from Thermococcus gammatolerans. Hel308 is a ski2-like superfamily II helicase/translocase that unwinds dsDNA in the 3° to 5° direction [44]. It is conserved in archaea and eukarya, including humans. Analysis of the crystal structure of Hel308 conjugated with a DNA substrate, in comparison with another superfamily II helicase VASA conjugated to an ATP analog, suggested that Hel308 walks along DNA via an ATP driven inchworm model [45].

SPRNT enabled the observation of this predicted [45], but previously unseen, two-step motion of Hel308. While the phi29 DNAP moves along the DNA strand in single-nucleotide steps via a Brownian ratchet model [31,43,46], Hel308 moves along the DNA strand in two steps per nucleotide, each about 0.5 nucleotides long (Fig. 7a and b; which seems to support a power stroke model proposed by Butner et al. [45]). By comparing these steps to phi29 DNAP, it became clear that the odd- and even-numbered steps correspond to DNA being held 0.14 ± 0.03 nt cis and 0.41 ± 0.03 nt trans of the closest observed phi29 DNAP level, respectively [37]. Because the spline interpolant is not a perfect approximation for the underlying smooth current curve, individual position measurements based off of this comparison to a spline through observed phi29 DNAP current levels carry systematic uncertainty. This systematic uncertainty can be corrected for by either averaging over several measurements at various positions along the DNA (as was done in Derrington et al.) or by improving the underlying curve through empirical measurements of the DNA at different positions within the pore. To achieve the highest spatial resolution with SPRNT, the shape of the underlying smooth current curve must be well understood. Even if this uncertainty makes it difficult to assign a precise position to the DNA, SPRNT’s ability to detect current changes associated with small conformational changes can still be used to gain insights into how enzymes work. For instance, while TIRF-FRET is capable of yielding distance measurements, it is commonly used only as an indicator of changes in enzymatic state [47].

Fig. 7

Comparison of two enzymes. (a) Levels observed when stepping the indicated DNA strand through MspA with phi29 DNAP controlling the DNA motion and (b) with helicase Hel308 tga controlling the DNA motion. The observed current patterns are similar, however the helicase takes twice as many steps. (c) Comparison of the half-lives of each level at two different concentrations of ATP. (d) Ratio of observed durations from (c). Interestingly, the duration of odd-numbered levels (orange diamonds) varies as [ATP] changes, while even-numbered levels (blue circles) remain unchanged. With the ability to resolve such small substates and observe their chemical dependencies, SPRNT has the potential to shed light on how such motors actually do their work. Figure modified from [37].

Variation of [ATP] can be used to better understand the origin of the two steps observed in Hel308 translocation [37]. Fig. 7c shows the median level duration at 10 µM and 1 mM ATP for the levels shown in Fig. 7b. Taking the ratio of these durations at these two [ATP]’s (Fig. 7d) makes the effect of changing ATP clear: the stepping rate for one of the steps is [ATP] dependent while the other is unaffected by [ATP].

3.4. Generalizing SPRNT to new enzymes

Many of the analysis tools developed for using MspA to sequence DNA are applicable to SPRNT. Setup of a single MspA channel in a bilayer is identical for a SPRNT experiment as it is for a sequencing experiment. The quadromer map developed is directly applicable to interpretation of SPRNT results and the code for analysis of SPRNT data is based on code developed for nanopore sequencing. There are two additional key considerations in adapting SPRNT for use with other enzymes:

It is generally necessary to stall enzyme activity in bulk to ensure progression only begins when the DNA threads into the pore. Without preventing this activity, experiments will run short as enzymes in bulk consume reagents. Strategies to prevent bulk activity may use blocking oligomers, or sections of DNA that serve as temporary road-blocks such as known enzyme pause sites, or stretches of abasic residues or carbon spacers, etc. that impede progression of the enzyme until it comes into contact with the pore. Other schemes will likely arise as the technology develops further.

How a given enzyme sits on the pore will effect where DNA resides within the pore constriction; it may rest higher or lower on the pore rim resulting in current shifts similar to those observed in our voltage stretching experiments. Such registration shifts can be easily adjusted for by using a spline interpolant to shift predicted levels appropriately. Related to this, is the question of the number of steps we expect to observe for a particular enzyme and how we expect them to be spaced. Such questions can be easily answered by use of a standardized DNA sequences for SPRNT. For instance, comparison of Fig. 7a and b clearly suggests the presence of two separate steps per nucleotide. Automated alignments of nanopore data to a prediction based on the quadromer map, as was done in Laszlo et al. [24], will be invaluable.

3.5. Comparison to other techniques

In Figs. 8 and 9 we compare the high spatiotemporal resolution of SPRNT to the spatiotemporal resolutions of other established single-molecule techniques used to study enzymes acting on DNA or RNA. With Gaussian-distributed noise, the spatiotemporal resolution of a given technique will follow a –½-power law relation between observable step-size and observable step duration [48] (Fig. 9). In other words, for a given technique there is a noise budget that is essentially a compromise between spatial and temporal resolution. By averaging for longer intervals, one can observe miniscule differences of state or by sacrificing spatial resolution, one can observe rapid single-molecule transitions. However, this also means some kinetic states are too small and fast to be observed with some techniques. Thus to observe some enzymes that take small steps, researchers have starved enzymes of reagents so that their steps were observable (Fig. 8d and “slowed E. coli RNAP” in Fig. 9) [49]. The spatiotemporal resolution of SPRNT should enable study of previously unobservable enzyme kinetics in conditions that more closely resemble those of the cell.

Fig. 8

Conversion of current trace to DNA displacement and comparison to optical tweezers, OT. (a) Raw current trace from SPRNT measurement of Hel308 helicase median filtered at 200 Hz (5 ms). (b) A piecewise cubic hermite interpolating polynomial (PCHIP) interpolation of current values observed for translocation of the same DNA through an MspA pore using phi29 DNAP. Spline uncertainty, as determined by bootstrapping of measured phi29 current values, is depicted via gray shading and is, in most places, too small to be visible in this graph. (c) The interpolation in (b) is used to translate the currents observed in (a) to DNA position. Pink shading denotes positional uncertainty introduced by statistical variation in the measured phi29 current values. Note that the relatively uniform noise seen in the current trace is either enhanced or suppressed due to the slope of the interpolant. Regions of high slope yield precise measurements of position on short time-scales while regions of low slope yield less precise position measurements. Nanopore data is displayed at 200 Hz. (d) Example OT data of single nt RNAP steps from [49] for comparison. Note, OT data in pink and black is median filtered at 20 Hz (50 ms window) and 1.33 Hz (750 ms window) respectively while nanopore data is median filtered at 200 Hz (5 ms window).

Fig. 9

Spatio-temporal resolution of SPRNT [37] as compared to optical tweezers, OT [48,49,54], magnetic tweezers, MT [51], and TIRF-FRET [55]. Many nucleic acid processing enzymes work on time-scales and step sizes that are too fast and too small to be resolved with current techniques. The spatiotemporal resolution provided by SPRNT may enable study of previously unobservable kinetic steps. SPRNT has already shown its ability to measure steps on time scales that exceed the spatiotemporal resolution of OT. To provide context, black diamonds indicate average step-sizes and durations for various enzyme steps observed with OT [48,49,56–59], black squares indicate enzymes measured with SPRNT [22,37], while circles represent estimated step-sizes and step durations for several other enzymes based on known stepping rates and suspected step size [60]. The square labeled “0.5 ms phi29 DNAP state” shows the spatiotemporal resolution required to resolve an observed single-step measurement from [37] that falls close to the noise limit. The enzyme and reference number from which it was taken are labeled.

It should be noted that spatiotemporal resolution is just one of several attributes that should be considered in single molecule experiments. For example, the ability to apply force [50] (or to apply no force) is often an important consideration. Table 1 compares other important aspects of various single molecule techniques individually [37,38,47,50,51]. Often single-molecule techniques are combined to observe different reaction coordinates in parallel [47].

Table 1

In addition to spatiotemporal resolution, the displacement range, force range, ability to be parallelized, and ability to apply torque may be important considerations when planning single-molecule experiments.

Technique	Displacement range (nm)	Force range (pN)	Massively parallelizable	Can apply torque?
SPRNT	0.04–105	15–60	Yes	No
Optical Tweezers	0.1–105	0.1–100	No	Yes
AFM single molecule probe	0.5–105	10–10,000	No	No
TIR-FRET	2–10	N/A	Yes	No
Magnetic Tweezers	0.5–105	0.001–10,000	Yes	Yes

Similar to other single molecule techniques, performing SPRNT may affect the enzyme’s behavior compared to its in vivo behavior. In particular, forces applied to the enzyme by pulling on the DNA strand and the balancing force where the pore rim comes into contact with the enzyme may alter the enzyme’s function. This is similar to optical tweezers where attachment of an enzyme to a bead may affect its function.

Depending on the voltage applied, SPRNT applies forces from 15 to 60 pN in a force-clamped mode. We estimate that MspA applies a force of ~35 ± 10 pN to the DNA when 180 mV is applied across the membrane. More accurate force measurements with SPRNT will require direct and independent force calibration.

3.6. Discussion

The ability to detect DNA displacements as small as 40 pm on millisecond time scales establishes SPRNT as a new tool for probing nucleic acid-protein interactions with unprecedented spatiotemporal resolution. Such resolution enabled detection of two distinct substates within a single ATP hydrolysis cycle of a Hel308 helicase [37] that would have been unobservable with other techniques (Fig. 9). SPRNT’s high spatial precision makes it necessary to consider what exactly the DNA motion means: the measured DNA motion is a result of motion of the DNA relative to the active site of the enzyme plus the motion of the enzyme caused by conformational changes that move the enzyme relative to MspA. Interpretation of SPRNT data must take the origin of the DNA motion into account.

In addition to the ability to measure small DNA displacements, SPRNT provides a simultaneous measurement of an enzyme’s absolute location relative to the sequence of the DNA strand. This enables real-time study of sequence-dependence of enzyme activity. Hints of sequence dependence are already apparent in the SPRNT level duration data in Fig. 7c. Steps that are ATP-dependent or ATP-independent appear to come from statistically significant distributions depending on where along the DNA the enzyme is.

It’s worth noting that the MspA protein and phospholipid bilayer is remarkably robust to changes in experimental conditions. MspA is unchanged in a range of salt conditions from 0 M to 2 M and pH from 0 to 14 [52]. MspA retains pore-forming ability after extraction for 30 min at 100 °C and subsequent 15 min incubation at 80 °C in 2% SDS. While the presence of at least some salt is necessary to achieve an ionic current signal, the use of asymmetric salt conditions [53] should enable enzymatic studies at any desired condition.

SPRNT compares well with other single molecule techniques however it is less generalizable than other single-molecule tools. At present, force can only be applied between a single ssDNA strand and an attached molecule. Experiments that require pulling on microtubules or dsDNA are not yet feasible and complicated experimental geometries that require pulling on multiple DNA strands may be undoable without combining SPRNT with other single-molecule techniques. While SPRNT is currently limited to use with nucleic-acid processing enzymes, it is possible that strategic tethering of a ssDNA probe strand to particular locations on an enzyme could be used to reveal small conformational changes of those enzymes as they function.

SPRNT is at an early stage in its development and there is still work to do to make SPRNT more adaptable to new enzymes and experimental configurations. For example, DNA orientation within the pore affects the observed current (5′ threaded first or 3′ threaded first, see Fig. 3b vs. Fig. 3c). As such, a new quadromer map for 3′ feeding will be needed for certain experiments. Measurements of RNA motion through the pore are likely possible [2] but RNA will require its own set of quadromer maps. Development of DNA sequences that result in large current swings for small displacements will be necessary to maximize SPRNT’s resolution. We have already used MspA’s sensitivity to abasic residues to provide high precision position measurements and it is possible that current patterns caused by unnatural bases will yield even higher resolution. Improvements are also necessary in our understanding of the underlying smooth curve. While a cubic spline is a good interpolation to find the DNA position, it seems unlikely that a spline is the true mathematical form of the underlying curve. As such, the spline introduces small systematic error in places where the underlying curve has curvature (regimes that appear linear likely have less error). Theoretical work that accurately predicts nanopore ion current values with DNA held at various positions or empirical measurement of a known optimal DNA sequence held at different positions will reduce this systematic. Finally, massive parallelization will make SPRNT a high-throughput single-molecule technique, capable of obtaining thousands of single-molecule data-traces within minutes. As such, SPRNT may become a useful drug-discovery platform that enables exploration of how small molecules affect enzymatic reactions.