initially , a capturing voltage 
 of 350550 mv is applied until threading of a  2050 
 bp - long nucleosomal dsdna tail into a 2.62.8 nm diameter pore 
 is detected ( i ) . the current drop that marks dna threading signals 
 a hardware trigger to reduce the voltage ( see materials 
 and methods section ) and then ramp it upward at a constant 
 loading rate ( 2.5 v / s40 v / s ) ( ii ) . because the nucleosome 
 dimensions ( 11 nm ) are larger than the pore dimensions , the 
 electrophoretically pulled dsdna tail gradually strains the complex 
 until the nucleosome is ruptured , after which dna is rapidly ( 1050 
 s ) released from the pore ( iii ) .
the release event , which signals 
 nucleosome rupture , is detected as a fast opening transition in the 
 ionic current . in all of our experiments
we have used a narrow range 
 of pore dimensions ( diameters of 2.62.8 nm , effective pore 
 lengths of 58 nm ) , as determined via the ionic current traces .
ii : 
 a potential ramp is triggered , applying increasing force on the nucleosome . 

iii : when the nucleosome unravels , the dna molecule escapes from the 
 pore and a stepwise increase in the current signal at a specific transition 
 voltage is observed .
light and bold red shaded areas on the nucleosomal 
 dna represent off - dyad and on - dyad sites of interaction , respectively . 

( b ) representative current trace of a capture event at 400 mv voltage ; 
 red arrow indicates trigger level .
( c ) transmission electron microscope 
 ( tem ) image of one of the 2.8 nm silicon nitride nanopores 
 used in our experiments .
( d ) finite - element simulations ( comsol ) of 
 the electric field of a pore with dimensions as in ( c ) at a capture 
 voltage of 550 mv ( log scale , v / cm ) .
( e ) same as in ( d ) , except the 
 voltage is reduced to 100 mv after nucleosome capture .
as observed in constant voltage 
 nanopore experiments , nucleosome - associated 
 current blockades feature a multilevel structure with one or more 
 shallow current blockade levels that precede a deep blockade level . 

we attribute the shallow levels to nucleosome interactions with the 
 pore before threading of the dna tail into the pore lumen has occurred 
 and the deep blockade level to capture of a dna tail . therefore , in 
 our acquisition protocol , we set the trigger level for nucleosome 
 capture to a value that suggests complete dna threading , as shown 
 by the red arrow in figure 1b ( for more details 
 see supporting information figure s1 ) .
a representative transmission - electron microscopy ( tem ) image of 
 one of the pores used in our experiments is shown in figure 1c .
our used pore diameters ( 2.8 nm ) are 
 only slightly larger than the cross - sectional diameter of a dna molecule 
 ( 2.2 nm ) , which efficiently excludes nucleosome entry into 
 the pore . however , for this measurement a dna tail must be properly 
 oriented in order for dna threading to occur , and consequently , we 
 have found that nucleosome capture requires much higher voltages as 
 compared with free dna capture . to illustrate 
 the impact of applied voltage on nucleosome capture , we performed 
 finite - element simulations . in figure 1d
, the 
 color map shows the electric field distribution when a capturing voltage 
 of 550 mv is applied ( log scale ) . at this voltage ,
the electric field 
 above the pore is large enough to electrophoretically trap the nucleosome 
 at the pore mouth until its dna tail is threaded . upon hardware - based 
 detection of tail threading the voltage is reduced to 100 mv ( figure 1e ) , which produces a finer , more localized electrophoretic 
 force to the dsdna tail at the onset of the force spectroscopy experiment . 

in addition to high - throughput , this active control approach allows 
 us to access a lower range of voltages , in which we expect an increased 
 sensitivity to alterations in biomolecular interactions .
we first performed force spectroscopy studies 
 on nucleosomes assembled from sea urchin 5s rdna 
 ( total dna length = 208 bp ; see supporting information figure s2 ) .
this positioning sequence is known to form nucleosomes 
 with a relative energy gain of 0.5 kcal / mol compared to a pool of 
 random dna sequences . for each detected 
 capture event , we recorded the ionic current before and during the 
 triggered voltage ramp .
a representative set of 50 overlaid current 
 traces obtained in a 5s nucleosome unraveling experiment is shown 
 in figure 2a .
for the sake of discussion , we 
 highlight a model trace in white . at the beginning of the voltage 
 ramp
the current was in a low state that corresponds to an occupied 
 pore . at some critical voltage ( 235 mv for the model trace ) 
 an opening transition
the 
 red background in the image reflects the relative frequency of unraveling 
 at each particular voltage range during the experiment ( n = 314 ) . in rare cases ,
the pore remained in its blocked state throughout 
 the voltage ramp ; such events were not considered in our analysis . 

apart from these long - lived events , the traces show two distinct regions 
 in which events are frequently seen . below the traces in figure 2a ,
we show the distribution of transition voltages 
 shown , which reveals a dominant population centered at 230 
 mv that we attribute to nucleosome unraveling .
in addition to this 
 well - defined population , we observe the tail of a second minor population 
 that vanishes as the voltage reaches 150 mv . by performing a control 
 measurement using a 250 bp dsdna fragment
, we were able to assign 
 the minor population to free dsdna , which is also observed in our 
 experiments , although infrequently and at unmeasurably low peak voltage 
 values .
because our sample contains both free dna and nucleosomes , 
 we tuned our experimental conditions so that the relative capture 
 frequency of nucleosomes to dna is maximized . by performing this experiment 
 using various loading rates ( i.e. , voltage ramp speeds )
, we found 
 that the peak voltage ( vpeak ) of nucleosome 
 unraveling scales with the logarithm of the loading rate in the range 
 of 2.540 v / s ( see figure 2b ) . in figure 2c , we calculated the nucleosome lifetime as a function 
 of bias using eq 1 ( see materials 
 and methods section ) , where only events assigned to the nucleosome 
 population in the transition voltage distribution were taken into 
 account .
all data obtained from three different pores at various loading 
 rates collapse onto a single master curve , confirming the applicability 
 of the transformation defined by eq 1 .
we also evaluated the possible influence of 
 capture voltage on the unraveling statistics by performing measurements 
 at two different capture voltages ( 400 mv and 550 mv ) for identical 
 ramp parameters ( 20 v / s , 50 mv starting voltage ) .
although we found 
 the rate of nucleosome capture to increase at the higher voltage , 
 in figure 2b , we show that the most probable 
 nucleosome unraveling voltage for the 20 v / s ramps are independent 
 of the capture voltage , implying that our capture conditions are not 
 nucleosome - destructive . finally , when comparing the obtained lifetimes 
 with our previous constant voltage data , we find a systematic trend in which constant voltage lifetimes are 
 always lower than our ramp data ( figure 2c , 
 black squares ) .
these consistently higher values obtained in constant 
 voltage experiments arise from the way in which we performed our analysis : 
 for the constant voltage data , we measured the full duration of the 
 event including the shallow current level at the beginning , whereas 
 in voltage ramp experiments , any prethreading time is not taken into 
 account .
( a ) top : current traces acquired 
 during triggered potential ramps in the presence of nucleosomes assembled 
 from the sea urchin 5s sequence ( subset of 50 representative traces 
 with an opening transition , 20 v / s loading rate , typical trace highlighted 
 in white ) .
red color shading : distribution of opening transitions 
 for the complete data set ( 314 detected transitions ) .
bottom : corresponding 
 transition voltage histograms for the data set ( red ) and for a 250 
 bp dna control ( gray ) .
( b ) peak position of the nucleosome - attributed 
 population as a function of loading rate . dashed
v / s , the peak position is 
 shown for two experiments with different capture voltages ( 400 mv 
 and 550 mv ) .
( c ) voltage - dependent lifetime of 5s nucleosome structures 
 determined from measurement data using eq 1 . 

data from three different pores ( triangles , diamonds , and circles ) 
 at loading rates of 2.5 v / s ( red triangles with gray border ) , 6.7 
 v / s ( diamonds ) , 20 v / s ( red triangles with black border and open triangles ) , 
 and 40 v / s ( gray spheres ) are shown . data from constant voltage nanopore 
 experiments are shown for comparison 
 ( black squares ) .
pores with diameters d = 2.62.8 
 nm and effective length heff = 58 
 nm were used .
our findings give rise 
 to a rather straightforward interpretation : logarithmic regimes of 
 the most probable transition voltages across a wide range of loading 
 rates are associated with an irreversible crossing of a single energy 
 barrier at a fixed location along the unbinding pathway .
likewise , the validity of the transformation used for the voltage - dependent 
 lifetime representation of our data ( eq 1 ) is 
 coupled to the assumption of an escape over a single barrier . as nanopore 

force spectroscopy experiments do not provide information on the unraveling 
 coordinates , escape over individual energy barriers within a multibarrier 
 landscape can not be resolved .
thus , even for a multibarrier energy 
 landscape , our findings imply that one distinct energy barrier limits 
 the transition across our experimental bias range . should multiple 
 barriers with comparable energy govern the transition , the lifetime 
 transformation shown in eq 1 would not reflect 
 lifetime values at constant voltage . to explain our observation of 
 a single barrier
, we turn to the prior literature : a recent study 
 by hall et al .
in which nucleosomal dna 
 was unzipped using optical tweezers has identified three major regions 
 of interaction that can be categorized as on - dyad and off - dyad interactions . 

whereas on - dyad interactions that are localized at the h3h4 
 tetramer were found to be the strongest , off - dyad interactions are 
 significantly weaker .
further , single - molecule fret studies have revealed a salt - induced decrease in off - dyad 
 stability .
this salt dependence is presumably due to a shift in the 
 balance of dna bending and electrostatic interaction with the histone 
 core , which is screened more efficiently at elevated salt concentrations . 
 given that our experiments were conducted at an ionic strength that 
 is similar to
that inside of a eukaryotic nucleus , we conclude that the nucleosome dyad is the most energy - costly 
 barrier of the unraveling process in our experiments .
next , we studied the sensitivity of electrophoretic force spectroscopy 
 to positioning sequence by comparing the 5s nucleosome to a similar 
 nucleosome construct assembled from the widom 601 clone ( total dna length = 189 bp ; see supporting information figure s2 ) .
compared to 
 5s rdna , a relative energy gain of 2.8 kcal / mol was determined for 
 this selex - generated positioning sequence , which is known to form 
 the most stable and most accurately positioned nucleosome assembly . 
 as its selection was mainly driven by the interaction of its central 
 70 bp with the h3h4 tetramer , dyad interactions are particularly strong for this sequence . in 
 figure 3 , we display raw ramp traces for 5s 
 ( red ) and widom 601 ( blue ) nucleosomes under identical conditions 
 ( loading rate = 40 v / s ) . in agreement with its higher stability ,
we 
 observe for 601 nucleosomes considerably higher transition voltages 
 than for 5s nucleosomes : vpeak is 260 
 mv for the 5s sample , whereas it is 330 mv for the 601 sample .
we 
 note that full dna tail threading was achieved in all experiments , 
 as indicated by the similar changes in conductance levels between 
 open and occupied pore states in all experiments ( g = 2.6  0.1 ns ) .
top : current traces acquired during triggered potential 
 ramps in the presence of nucleosomes assembled from the sea urchin 
 5s ( red ) and the widom 601 sequence ( blue ) ( subsets of 50 representative 
 traces with an opening transition , 40 v / s loading rate ) . color shading : 
 distribution of opening transitions for the complete data sets ( 703 
 and 1547 detected transitions for 5s and 601 , respectively ) .
bottom : 
 corresponding transition voltage histograms for 5s nucleosomes ( red ) , 
 601 nucleosomes ( blue ) , and a 250 bp dna only control ( open gray bars ) . in figure 4a ,
voltage - dependent lifetime data are analyzed for both positioning 
 sequences by compiling data from experiments using multiple pores 
 and different loading rates .
similarly to the 5s nucleosomes , unraveling 
 lifetimes for the widom nucleosomes at different loading rates ( 10 
 v / s and 40 v / s ) fall on the same master curve .
furthermore , triggered 
 constant voltage experiments ( see figure 4a 
 gray symbols and supporting information figure s3 ) reveal a good agreement of lifetimes with transformed 
 ramp data .
despite various potential pitfalls from pore - to - pore variability 
 and possible ionic strength fluctuations , we are able to resolve both 
 positioning sequences , and we find that widom 601 lifetimes are universally 
 5-fold longer than 5s lifetimes . nucleosome lifetime comparison . 

( a ) voltage - dependent lifetime of nucleosomes assembled from the widom 
 601 ( blue symbols ) and the 5s positioning sequence ( red symbols , shown 
 for comparison ) .
the 601 data was obtained from three different pores 
 at loading rates of 10 v / s ( triangles ) and 40 v / s ( diamonds and circles ) . 

( b ) voltage - dependent lifetime of nucleosomes 
 assembled from methylated dna ( mcpg ) using the widom 601 ( open diamonds ) 
 and the 5s positioning sequence ( open circles ) .
scatters in lifetime data for the same nucleosome 
 reflects pore - to - pore variability .
both dna sequences we study here 
 contain > 10 cpg sites in their positioning sequences ( see supporting information figure s2 ) .
first , we 
 used a methyltransferase to methylate cpg sites on the unassembled 
 dna of both positioning sequences ( 5s rdna and widom 601 , for details 
 on the methylation protocol , see materials and methods section ) . after confirming dna methylation via methylation - specific 
 digestion
, we reconstituted nucleosomes from the methylated dna and 
 histones and performed force spectroscopy on the assembled nucleosomes . 
 in figure 4b
, we present lifetime vs voltage 
 data for cpg methylated ( mcpg , open markers ) and unmethylated ( cpg , 
 solid markers ) nucleosomes . for the widom 601 data , we clearly see 
 that methylation does not impact the overall nucleosome stability , 
 as indicated by the similar lifetime vs voltage trajectory . for the 
 5s sequence ,
we find a slight decrease in stability that is marked 
 by a less than 2-fold reduction in mean lifetimes in the 100150 
 mv voltage range .
however , these experiments on both sequences show 
 that the difference in stability upon methylation in both nucleosomes 
 is far smaller than differences we observed for the 5s and widom 601 
 samples , for example .
though a more exhaustive study to ascertain 
 the generality of this result , our data for these sequences suggests 
 a minor impact of methylation on nucleosome packaging and stability .
in this work , we investigated the 
 influence of sequence and cpg methylation on the stability of unlabeled 
 mononucleosomes .
nanopore - based electrophoretic force spectroscopy 
 was used here to capture a nucleosomal dna tail and gradually unravel 
 it under increasing force .
in contrast with experiments that are conducted 
 at constant voltage , the use of dynamic force spectroscopy was helpful 
 for our nucleosome study because a large bias is typically required 
 for dna tail capture , whereas the smaller bias regime allows our technique 
 to probe behavior under a weaker electrophoretic force , in which longer 
 nucleosome lifetimes are obtained .
the automated approach in our experiment 
 offers high throughput without requiring any chemical labeling or 
 long tail dna handles ( 2030 bp tails are sufficient for our 
 study ) .
results for the sea urchin 5s rdna positioning 
 sequence align reasonably well with prior constant force nanopore 
 experiments from our group .
we have observed 
 a single logarithmic loading rate dependence on the peak unraveling 
 voltage , which supports a mechanism that involves crossing of a single 
 energy barrier , which we interpret to reflect the most dominant interaction , 
 that is , of nucleosomal dna with the h3h4 tetramer in the 
 dyad region . for the high - affinity widom 601 positioning sequence , 

we obtain lifetimes that are half an order of magnitude higher , and 
 despite minor pore - to - pore scatter , our method demonstrates the required 
 sensitivity to discern these two positioning sequences .
we demonstrated 
 its use by showing for the first time nucleosome unraveling experiments 
 on cpg methylated dna sequences .
surprisingly , for the sequences we 
 tested , cpg methylation did not affect the nucleosome assembly nor 
 unraveling trajectories , which suggests that dna methylation plays 
 alternative role in nucleosomal maintenance , for example , transcription 
 factor binding modulation . whereas methylation did show an effect 
 on off - dyad nucleosome equilibrium dynamics in recent fluorescence - based 
 studies and for some periodic cpg patterns in a mnase digestion assay
, our method implies that the central h3h4 
 tetramer interactions on the dyad region are hardly affected by dna 
 methylation .
because in the dyad region the dna is less bent , we expect that changes in dna mechanical properties will not greatly 
 influence the nucleosome stability , and 
 thus , our observations appear consistent with previous findings .
this 
 confirmation of methylation - independent nucleosome stability also 
 points to other possible mechanisms by which dna methylation alters 
 gene expression , for example , modulating the binding of transcription 
 activators / repressors . due to
its high throughput and simplicity , 
 our technique is well suited to screen different epigenetic markers 
 and assess their influence on nucleosome stability under external 
 force .
nanopore - based force spectroscopy can probe biomolecular 
 interactions with high throughput and a unique geometry , as compared 
 with optical / magnetic tweezers and atomic force microscopy .
much like 
 the action of processivity - enhancing motors in prokaryotes and eukaryotes , 
 our method applies either a constant or a time - varying force to a 
 nucleosomal dsdna tail in order to displace histones that interact 
 with the pulled dna .
we have found little to no impact of dna methylation 
 on nucleosome stability , which suggests that histone modifications 
 play a greater role on these systems .
fortuitously , the label - free 
 nature of our method is ideally suited for studying the influence 
 of histone core modifications such as methylation , acetylation or 
 phosphorylation on nucleosome structure , which is highly challenging 
 for other single - molecule experiments due to many chemical modifications 
 that are required .
the substrate membrane 
 devices were prepared from 500-m - thick 100 
 silicon wafers passivated with a 2.5 m sio2 thermal 
 layer and coated with a 40 nm - thick silicon nitride layer ( lpcvd - grown ) . 

freestanding silicon nitride membranes were released in each device 
 using optical lithography and wet chemical etching , and membranes 
 were thinned using e - beam lithography and reactive ion etching .
finally , 
 nanopores were drilled by exposing the membrane to the highly focused 
 beam of a transmission electron microscope ( jeol 2010feg ) .
nanopore 
 devices were cleaned by treatment with a hot piranha solution and 
 a copious water rinse prior to each experiment .
purified histones from the epimark nucleosome assembly 
 kit ( neb e5350 ) and either 5s rdna ( neb ) or biotinylated nucleosome 
 assembly 601 sequence dna ( widom 601 ) ( epicypher 18 - 0001 ) were combined 
 following the dilution assembly protocol for 25 pmol .
briefly , 5 m 
 nacl was mixed with either unmethylated or methylated dna ( methylation 
 of widom 601 dna described below ) and dimer and tetramer histone proteins 
 for each reaction .
additional amounts of 10 mm tris were added to 
 dilute nacl as per the protocol .
gel shift was completed by running 10 l 
 of the assembly reaction on a 6% polyacrylamide gel , staining the 
 dna with sybr - safe ( life technologies s33102 ) , and imaging with a 
 chemi - doc imager ( biorad ) ( supporting information figure s4 ) .
dna was methylated using cpg methyltransferase 
 ( m.sssi ) ( neb m0226 m ) .
specifically , 15 g of dna was incubated 
 with 640 m s - adenosylmethionine ( sam ) and m. sssi enzyme ( 5 
 l of 20 u/l ) in the provided buffer for 4 h at 37 c . 

another 2.5 l of 32 mm sam was added and the incubation extended 
 for an addition 2 h at 37 c .
dna was purified with the genejet 
 pcr extraction kit ( thermo scientific k0701 ) following the provided 
 protocol except the dna was eluted with water .
dna was quantified , 
 lyophilized , and resuspended at the appropriate concentration ( 10 
 m ) .
methylation of dna was determined through restriction digestion 
 with the mspi ( neb r0106 m ) and hpaii ( neb r0171 m ) 
 isoschizomer restriction enzymes as per the provided protocols .
both 
 of these enzymes target ccgg sequences for digestion , but only hpaii is blocked when the inner cytosine is methylated . 

the nucleosome assembly 601 dna sequence contains one ccgg site near 
 the center of the sequence .
digested samples were run on a 6% polyacrylamide 
 gel to detect the digested dna fragments ( supporting 
 information figure s4 ) .
a cleaned nanopore chip was mounted between two aqueous compartments 
 using a fast - curing silicone elastomer ( ecoflex 5 , smooth - on inc . ) . 

each compartment was filled with buffer ( 265 mm kcl , 83 mm nacl , 1 
 mm edta , 10 mm tris , ph 8) and an ag / agcl electrode .
ionic current 
 was driven through the nanopore and recorded using an axopatch 200b 
 ( molecular devices ) patch clamp amplifier , interfaced to a computer 
 using synchronized ni pci-6230 ( potential ramp output ) and ni pcie-6351 
 ( current acquisition ) daq cards ( national instruments ) . using custom 
 labview code
, the card s built - in fpga chip was programmed 
 to trigger voltage ramps on falling edges in the current signal with 
 selected threshold levels and with a response time in the microsecond 
 range ( without the measurement cell attached ) .
the true response of 
 our system ( including the measurement cell ) is given by its rc time 
 constant , typically 300 s .
at the end of each ramp , 
 a reverse bias pulse was applied ( 400 mv for 2040 
 ms ) to prevent long - lived pore blocks .
current recordings were filtered 
 at 100 khz using the amplifier s built - in bessel filter and 
 digitized at sampling rates of at least 250 khz .
the raw current 
 traces were median - filtered using a 4080 s time window , 
 and a derivative - based step detection algorithm was used to identify 
 the transition voltages .
traces corresponding to clogged states of 
 the pore , as indicated by a reduced open pore current prior to capture 
 were removed automatically beforehand . as demonstrated previously , 
 the lifetime as a function of bias voltage can be obtained from unfolding / unraveling 
 voltage distributions using the expression1where p(v|v ) is the unraveling probability as a function of 
 voltage v and loading rate v. this expression is not based on a specific functional form of (v ) and provides model - independent estimates of (v ) from the measurement data . however , it is based 
 on the assumption that the underlying transition is irreversible and 
 can be described as the escape over a single energy barrier under 
 quasi - adiabatic conditions , that is , intrinsic thermal relaxation 
 processes are assumed much faster than the time scale of the loading 
 rate .
if the transition is governed by multiple energy barriers , this 
 transformation will not reflect constant voltage behavior and data 
 obtained at different loading rates are not expected to collapse on 
 a master curve .
 nucleosomes 
 are the fundamental repeating units of chromatin , and dynamic regulation 
 of their positioning along dna governs gene accessibility in eukaryotes . 
 
 although epigenetic factors have been shown to influence nucleosome 
 structure and dynamics , the impact of dna methylation on nucleosome 
 packaging remains controversial . 
 
 further , all measurements to date have been carried out under zero - force 
 conditions . in this paper , we present the first automated force measurements 
 that probe the impact of cpg dna methylation on nucleosome stability . 
 in solid - state nanopore force spectroscopy 
 , a nucleosomal dna tail 
 is captured into a pore and pulled on with a time - varying electrophoretic 
 force until unraveling is detected . 
 this is automatically repeated 
 for hundreds of nucleosomes , yielding statistics of nucleosome lifetime 
 vs electrophoretic force . the force geometry , which is similar to 
 displacement forces exerted by dna polymerases and helicases , reveals 
 that nucleosome stability is sensitive to dna sequence yet insensitive 
 to cpg methylation . 
 our label - free method provides high - throughput 
 data that favorably compares with other force spectroscopy experiments 
 and is suitable for studying a variety of dna  protein complexes . 
Results
Discussion/Conclusion
Materials and Methods