protein and solid - state 
 nanopores have been utilized as sensors 
 to detect dna , rna , and proteins . in the past decade , the protein 
 nanopore -hemolysin ( -hl ) has been well characterized 
 and utilized as a sensor for biomolecules and a platform for label - free 
 dna sequencing .
furthermore , -hl has been employed to study the kinetics 
 of dna base pair unzipping for hairpin ( intramolecularly base - paired ) and duplex ( intermolecularly base - paired ) structures under an applied voltage .
various techniques , including 
 magnetic and optical tweezers and atomic force microscopy ( afm ) , have been utilized to determine the force required to unzip dna 
 or rna secondary structures .
in contrast , the -hl nanopore provides a label - free 
 method to probe dna molecules when electrophoretically driven through 
 the channel .
the capture of dna molecules leads to a perturbation 
 in the ion current through the -hl nanopore that is readily 
 detected .
the -hl nanopore is composed of a wide vestibule 
 and a narrow 
 -barrel .
the diameter of the -barrel 
 ( 1.4 nm ) allows translocation of single - stranded 
 dna or rna ( 1 nm ) ; however , larger structures , 
 such as hairpins and g - quadruplexes , have to unzip before they are 
 driven through the nanopore by a voltage bias .
the current blockage 
 level and the time it takes to unzip can provide information about 
 the identity and the stability of the dna or rna secondary structures . recently , duplex unzipping through 
 the -hl ion channel has attracted much interest , and the unzipping 
 kinetics and base pairing energy of duplex dna have been extensively 
 explored .
studies undertaken by deamer , akeson , and co - workers found 
 that 
 the interaction between terminal hairpins ( without tails ) and the 
 -hl nanopore led to a unique current modulation pattern when 
 the hairpin interacted with the constriction zone on the cis side .
later , fishhook hairpins 
 ( a terminal hairpin with one single - stranded tail ) were used to study 
 the kinetics and mechanism of hairpin unzipping in the -hl 
 nanopore .
more recently , the reverse ( trans to cis ) translocation dynamics of an internal hairpin 
 ( hairpin with two tails ) was investigated by nanopore force spectroscopy .
backward translocation ( trans to cis ) through the -hl pore combined with 
 theoretical modeling was considered as a promising approach for label - free 
 single - molecule analysis of dna and rna folds .
the kinetics of hairpin unzipping in the -hl nanopore 
 has been extensively studied , with an assumption that internal hairpins 
 enter the vestibule of -hl in the same fashion as a fishhook 
 hairpin , i.e. , with the base of the duplex region positioned on the 
 lower floor of the protein vestibule and with one overhanging trail 
 threaded into the narrow -barrel .
nevertheless , the location of hairpin unzipping remains unestablished 
 for internal hairpins , and whether or not internal and fishhook hairpins 
 unfold by the same mechanism in the nanopore experiment is not clear . 

because internal dna hairpins are a common secondary structure observed 
 in single - stranded dna , it is important to understand the behavior 
 of these secondary structures and to explore the limits of nanopores 
 for gaining information about nucleic acid structure and dynamics . in the present work , an -hl nanopore was constituted in 
 a lipid bilayer suspended across the orifice of a glass nanopore membrane 
 ( gnm ) .
dna oligomers were driven through 
 the -hl nanopore by an electrophoretic force from the cis to the trans side . the duration and 
 current level while the oligomer blocked the nanopore correspond to 
 the unzipping time and blockage current , respectively .
we examined 
 the effect of duplex stem length , sequence , and single - stranded tail 
 length on the unzipping behaviors of a series of internal and fishhook 
 hairpins .
the unzipping characteristics of internal hairpins turned 
 out to be very different from those of analogous fishhook hairpins , 
 indicating they have different mechanisms of unzipping in the -hl 
 nanopore .
not only was the time of unzipping markedly affected by 
 the unzipping mechanism , but the current levels observed during the 
 two processes of denaturation were also distinctly different .
a custom - built , high - impedance , 
 low - noise amplifier and data acquisition system , designed and constructed 
 by electronic biosciences ( ebs ) , san diego , ca , was used for the current  time 
 ( i  t ) recordings . for all translocation studies , 

the dna hairpin ( 2 nmol , 10 m ) was added and > 1000 events 
 were 
 collected for each voltage with a 100 khz low pass filter and a 500 
 khz data acquisition rate .
the composition of the buffered electrolyte 
 solution was 1.00 m kcl , 10 mm pbs , and 1 mm edta ( ph 7.4 ) . the oligodeoxynucleotides 
 were synthesized from commercially available phosphoramidites ( glen 
 research , sterling , va ) by the dna - peptide core facility at the university 
 of utah , followed by purification using a semipreparation ion - exchange 
 hplc column with a linear gradient of 30100% b over 30 min 
 while monitoring absorbance at 260 nm ( b = 20 mm tris , 1 m nacl , ph 
 8 in 10% ch3cn/90% ddh2o ; a = 10% ch3cn/90% ddh2o ; flow rate = 3 ml / min ) .
the purities of the 
 oligodeoxynucleotides were determined by reinjecting the purified 
 samples on an analytical ion - exchange hplc running the previously 
 mentioned buffers and method with the exception that the flow rate 
 was 1 ml / min .
the glass 
 nanopore membrane 
 ( gnm ; with radius 800 nm ) was fabricated as previously reported .
1,2-diphytanoyl - sn - glycero-3-phosphocholine 
 ( dphpc ) bilayers spanning across the orifice of the gnm were prepared 
 as previously described .
the protein 
 -hl was diluted to a 1 mg / ml solution in ultrapure water ( 18 
 mcm ) , and the dphpc was dissolved in decane to a concentration 
 of 10 mg / ml , both of which were stored at 80 c .
a pipet 
 holder with a pressure gauge and a 10 ml gastight syringe were used 
 to attach the gnm to the dc system .
two ag / agcl electrodes were positioned 
 inside and outside of the gnm to apply a voltage .
a plastic pipet 
 tip was used to paint the dphpc solution ( 1 l , 10 mg / ml ) on 
 the gnm surface .
after addition of monomer -hl ( 0.2 l , 
 1 mg / ml ) , a pressure was applied to form a suspended bilayer , followed 
 by reconstitution of a single -hl nanopore in the bilayer . 

populations of > 1000 deep blockage events were collected for most 
 of the experiments , and > 500 deep blockage events were collected 
 for 
 those hairpins that took > 0.5 s to unzip .
t traces were refiltered to 2 or 10 khz for presentation depending 
 on the duration of single events . due to the fact that different hairpins 
 may have very different unzipping time and distributions , different 
 numbers of bins ( 30100 )
as a preliminary 
 study , one fishhook hairpin ( f - hp12 - 1 ) and one 
 internal hairpin ( i - hp12 - 1 ) were designed to examine their behavior 
 in the -hl nanopore .
( note : f = fishhook ; i = internal ; 12 
 = base pairs ( bp s ) in the stem ; the last number represents 
 sequence variations studied ; see figure 1 . ) 

both hairpins have exactly the same loop , stem sequence , and tail 
 length ( figure 1 ) . according to kasianowicz s 
 work , a 30-mer poly-2-deoxycytidine ( dc30 )
therefore , the internal hairpin was designed 
 to have a dc30 tail at both the 3 and 5 
 ends , while the fishhook hairpin was designed to have only one dc30 tail at the 3 end .
next , these two hairpins were 
 subjected to identical conditions in the nanopore , while monitoring 
 the time ( t ) and blockage current ( i d ) during unzipping and translocation . according to a 
 previous work ,
the current levels of events that are more blocking 
 than 25% of the open channel current ( io ) are considered to be translocation events ( i d ) ; therefore , we only show and 
 analyze deep blockage events in this work .
the long events that are 
 less blocking than 30% of io were attributed 
 to loop entry events where the loop of a hairpin enters the nanopore 
 and can not unzip ; thus , they have to escape from the cis side of the channel .
this observation was verified by experimentation 
 on a dumbbell hairpin that can only enter via the loop , which leads 
 to only long blocks with currents >
30% io ( figures s1s4 , supporting information ) similar to the ion currents observed with f - hp12 - 1 and i - hp12 - 1 .
first , the durations of the unzipping / translocation events for 
 the fishhook and internal hairpins were dramatically different ( figure 1 ) .
the distributions of unzipping times for f - hp12 - 1 
 and i - hp12 - 1 were well fit to an exponential decay function ( figures 
 s1 and s4 , supporting information ) with 
 a mean time constant (  ) that is reported for each hairpin s 
 unzipping time , and the reported errors were determined from the fitting 
 function errors . the fishhook hairpin required 64  2 ms to unzip 
 and translocate the pore , while the internal hairpins took 2.6  
 0.1 ms under the conditions studied ( 1.00 m kcl and 100 mv bias ) . 
 in other words ,
the duration for the internal hairpin unzipping and 
 translocation was 20 times shorter than that for the fishhook 
 hairpin .
second , the blockage current i d was different between the internal and fishhook hairpins .
the deep 
 blockage current i d for the internal hairpin 
 was 18  1% of io , while the i d for the fishhook hairpin was 11  1% 
 of io . considering these two hairpins 
 have identical stem and loop sequences ,
sequence of the 
 internal hairpin = 5-c30 cgc ggc att aaa gtta ttt 
 aat gcc gcg c30 - 3. sequence of the fishhook hairpin 
 = 5-cgc ggc att aaa gtta ttt aat gcc gcg c30 - 3. 
 experimental conditions : 1.00 m kcl , 10 mm pbs , ph 7.4 , 22.0  
 0.5 c . to further support these 
 results , the internal and fishhook hairpins 
 were mixed together and unzipped using the same -hl nanopore . 

these experiments confirmed that the fishhook hairpin was 6% 
 more blocking than an internal hairpin in the same protein channel 
 ( figure 2 ) .
consistent with our previous results , 
 it took at least 20 times longer ( 64  2 ms vs 2.6  0.1 
 ms ) to unzip the fishhook hairpin than the internal hairpin ( figures 1 and 2 ) .
in addition , there 
 are two peaks in the current plot for unzipping the internal hairpin , 
 in which one was 17  1% of io and 
 the other was 19  1% of io ; this 
 observation is consistent with the ability of this hairpin to unzip 
 from either the 5 or 3 side depending upon the initial 
 terminus captured and threaded into the nanopore . under the same conditions ,
the fishhook hairpin gave a 
 single blocking current of 11  1% of io , consistent with only 3 entry .
the fact that the i d of the fishhook hairpin , which must enter 
 from the 3 terminus , does not match with either of the i d levels for the internal hairpin is additional 
 evidence that these two hairpins unravel by different mechanisms . 

the other key difference between these hairpins was observed in the 
 current distributions ; the fishhook hairpin displayed a narrow distribution 
 in current blockage levels ( figure 2a and c ) , 
 while the current distributions observed for the internal hairpin 
 were broader ( figure 2b and c ) .
specifically , 
 for the internal hairpins , the short duration events were more blocking 
 than the longer duration events , leading to a tail toward the more 
 blocking side ( figure 2b and c ) . given that 
 these two hairpins have exactly the same stem and loop , and the fact 
 that they produced different blockage currents and unzipping times , 
 suggests they unzip by different mechanisms .
current and time plots 
 for unzipping experiments with internal 
 and fishhook hairpins at 80 mv ( trans vs cis ) .
the left column is the current histogram showing the 
 ratio of id / io .
the right column shows scatter plots of id / io vs t. ( a ) 
 unzipping of fishhook hairpin alone ; ( b ) unzipping of internal hairpin 
 alone ; ( c ) unzipping of both fishhook and internal hairpins using 
 the same protein nanopore .
experimental conditions : 1.00 m kcl , 10 
 mm pbs , ph 7.4 , 22.0  0.5 c .
the data were binned differently 
 for presentation purposes . when the unzipping events for the internal hairpins were 
 examined 
 more closely , two deep ion current blockage levels , ia and ib , were identified 
 ( figure 3a ) . for the internal hairpins , the 
 deep blockage current levels measured for ia and ib were 18  1 and 13  
 1% of io , respectively ( figure 3a ) .
a single unzipping event began with a deep blockage ib1 , with a duration of 10  5 s , 
 followed by a shallower blockage , ia , 
 with a duration of 37  2 ms , and ended with another deep blockage , ib2 , that had a duration of 150  30 s 
 at 80 mv ( trans vs cis ) .
this current 
 pulse pattern io  ib1  ia  ib2  io was 
 observed in 50  10% of the total events recorded .
the only deviation 
 from this pattern was a loss of the ib1 pulse in the remaining events ( i.e. , a io  ia  ib2  io pulse pattern ) , 
 presumably because it was too short to be detected with the 100 khz 
 low - pass filter used in these studies ( figure 3a ) .
a voltage - dependent study for internal hairpin unzipping was 
 conducted on the internal hairpin ( figure 3b ) .
the unzipping time distributions observed for the internal hairpins 
 all gave a single exponential decay curve with a mean time constant , 
  , that decreased as the voltage was increased ( figure s5 , supporting information ) . the exponential time 
 distribution signifies that a first - order process is occurring during 
 unzipping ; in previous studies , the kinetic process was proposed to 
 be unzipping of the dna secondary structure ( i.e. , hairpin or duplex ) .
in contrast to the data obtained for the internal 
 hairpin , only one deep blockage current level ( i d ) was identified for the fishhook hairpins ( 11  1% , 
 figures 1 and 2a ) .
constant for the fishhook 
 hairpins decreased with increased voltage ( figure s6 , supporting information ) . in conclusion , these 
 data identify two key parameters that differ between the internal 
 and fishhook hairpins : the unzipping time and the current blockage 
 pattern .
t trace for unzipping the internal 
 hairpin i - hp12 - 1 at 100 mv ( trans vs cis ) .
( b ) voltage - dependent unzipping time constant  for i - hp12 - 1 . 

( c ) histogram of unzipping time for the entire event and exponential 
 decay fitting ( red line ) for i - hp12 - 1 at 80 mv ( trans vs cis ) .
( d ) voltage - dependent time histograms 
 and gaussian fits ( green curve ) for only the ib2 portion of i - hp12 - 1 events .
( e ) comparison of the voltage - dependent tmax ( peak values from gaussian distributions ) 
 for dc87 ( purple ) and ib2 of 
 i - hp12 - 1 ( green ) .
( f ) blocking current distribution for ib2/io and id / io for i - hp12 - 1 ( green ) 
 and dc87 ( purple ) , respectively .
experimental conditions : 
 1.00 m kcl , 10 mm pbs , ph 7.4 , 22.0  0.5 c .
next , the blocking current pattern was studied 
 more closely to 
 determine the significance of each current level .
it was established 
 that the internal hairpin unfolded and translocated in the studies 
 above ; therefore , the following model is proposed to describe the io  ib1  ia  ib2  io current pattern .
given that the initial current 
 changed from io  ib1 , this identifies ib1 as 
 the current associated with initial threading of the dc30 tail into the nanopore channel .
consistent with this hypothesis 
 is the similarity in the ib1 current ( ib1 = ib2 = 13  
 1% io ) with the blocking current observed 
 for free translocation of a long homopolymer of dc that has a blocking 
 current of 12  2% under the same experimental conditions . to support this claim , a control experiment was 
 conducted in which a dc87 strand was allowed to translocate 
 while monitoring the current and time ( figure 3e and f ) . in this control experiment ,
the same blocking current was 
 observed for dc87 as monitored for ib1 of the internal hairpins unzipping , thus supporting our 
 claim .
the next current identified during the unzipping process of 
 the internal hairpin was ib1  ia , with ia having 
 a higher residual current ( 18  1% io ) .
we hypothesize this current corresponds to the stalling of the 
 internal hairpin outside the vestibule of -hl with one dc30 tail extending through the protein channel .
support for 
 this claim comes from previous reports that monitored the current 
 of poly dc strands when they were immobilized in the nanopore by a 
 biotin  streptavidin complex . in these studies ,
the residual 
 current of poly dc was 16% of io , which is higher than observed for free translocation of poly dc 
 ( 13% io , figure 3f ) .
consistent with stalling of the 
 hairpin in the channel , ia gives a larger 
 residual current supporting the immobilization - like state of the internal 
 hairpin .
moreover , it seems unlikely that both the 3 and 5 
 termini of the internal hairpin would thread into the vestibule of 
 -hl during the same event . on the basis of our model
, we hypothesize 
 that the internal hairpins presented here remain outside of the vestibule 
 during the unzipping process .
therefore , the last current pulse , ib2 , must be associated with the translocation 
 of the unfolded dna strand derived from the internal hairpin . to support 
 this hypothesis ,
the previous control study with the dc87 strand ( similar length as the internal hairpin ) was compared to ib2 .
this comparison demonstrated that ib2 and the poly dc strand gave the same ion 
 current 13  1% io under these analysis 
 conditions ( 1.00 m kcl and 100 mv bias , figure 3f ) , and they showed similar translocation times ( figure 3e ) . as a further demonstration to identify ib2 as the translocation of the unfolded hairpin , 
 the mean event time vs voltage was plotted and compared to dc87 ( figure 3e ) .
comparison of these 
 event times gave similar values , as expected , because of the similarity 
 in their strand length , confirming that ib2 was the translocation of the unfolded hairpin .
in addition , the 
 duration of the ib2 pulses showed a gaussian 
 distribution similar to unimpeded translocation events , whereas the 
 entire event duration ( dominated by the much longer ia component ) displays an exponential distribution of . 
 in summary , the internal hairpin gave a unique pulse - like pattern 
 during the unzipping and translocation process that we identify as 
 threading of the poly dc tail , followed by unzipping of the hairpin 
 outside the vestibule that leads to translocation of the entire strand 
 through the protein channel ; this pulse - like pattern was not observed 
 for the fishhook hairpins .
previous studies have proposed that 
 fishhook hairpins are pulled 
 into the vestibule by the electrophoretic force , and unzip in the 
 confined context of the vestibule .
this 
 model is characterized by a single blockage current and long unzipping 
 time , due to spatial confinement of the hairpin during unzipping , 
 while the -barrel is occupied by the tail .
in contrast , the internal hairpin , as described above , 
 gave a short unzipping time with a unique pulse - like pattern in the 
 blockage current ( figure 3a ) , for which we 
 propose that the unzipping process occurs outside the vestibule .
to further investigate 
 the unzipping of internal hairpins through an -hl nanopore , 
 a group of internal hairpins was designed with the same dc30 tails and loop sequence , but the length of the hairpin stem was 
 increased from 9 to 15 base pairs ( bp s ) in 3 bp increments 
 ( figure 4a ) .
all of these internal hairpins 
 gave the same pulse - like pattern to the blocking current , as described 
 above .
the measured unzipping time constant (  ) for these hairpins 
 demonstrated that , as the stem length was increased , the unzipping 
 time increased , as expected ( figure 4b ) . in 
 these studies ,  showed an inverse correlation with voltage ; 
 this observation supports unzipping and translocation of the hairpins 
 through -hl ( figure s7 , supporting information ) . compared to the unzipping time of fishhook hairpins in the current 
 work and those previously reported by meller
s laboratory , the unzipping times of the internal hairpins 
 were dramatically shorter , by a factor of 20 .
furthermore , 
 the unzipping times of variable stem length internal hairpins were 
 sensitive to increased voltage that is consistent with a model of 
 hairpin unzipping followed by translocation ( figure 4b ) .
unzipping time vs stem length for internal hairpins studied at 
 different voltages ( trans vs cis ) .
experimental conditions : 
 1.00 m kcl , 10 mm pbs , ph 7.4 , 22.0  0.5 c .
recently , 
 simmel and gerland 
 explored the sequence dependence of unfolding fishhook dna hairpins 
 by -hl nanopores .
they reported 
 that it took much longer to unzip a hairpin rich in gc bp s 
 compared to one rich in at bp s and hypothesized that 
 the nature of the free energy landscapes for the unfolding process 
 caused the observed differences because a gc bp has one more 
 hydrogen bond than an at bp .
here we designed 
 a group of fishhook hairpins with a 3-dc30 tail 
 and an analogous group of internal hairpins to study the effect of 
 sequence dependence on hairpin unzipping ( figure 5a ) . in each group ,
the hairpins have the same tail , loop , 
 and stem length ( 12 bp s ) , in which the variable studied was 
 the sequence of base pairs in the stem .
the sequences chosen for study 
 were comprised of four different blocks of either three gc or 
 three at base pairs ( figure 5a ) .
the 
 nomenclature used for these hairpins has a number at the end to identify 
 the sequence ( i.e. , i - hp12 - 1 = internal hairpin with a 12-bp stem 
 comprised of sequence 1 ) .
( a ) sequence of 
 internal hairpins from i - hp12 - 1 to i - hp12 - 4 that are comprised of 
 four blocks of at or gc bp s ( color coded ) .
( b ) 
 unzipping times of internal and fishhook hairpins for hp1 to hp4 at 
 100 mv ( trans vs cis ) .
experimental conditions : 1.00 m kcl , 10 mm pbs , ph 7.4 , 
 22  0.5 c .
unzipping experiments were performed on the various sequences 
 of 
 internal hairpins and fishhook hairpins . as shown in figure 5b , internal hairpins and fishhook hairpins
had dramatically 
 different unzipping times even though they have exactly the same sequence 
 in the hairpin stem .
for the internal hairpin group , the experimental 
 thermal melting values ( tm ) were similar 
 ( table si 1 , supporting information ) .
in contrast to our expectation , it is demonstrated in figure 5b that these internal hairpins have different unzipping 
 time distributions ( figure s5 , supporting information ) .
for instance , i - hp12 - 1 is the most kinetically stable hairpin , 
 requiring 37  2 ms to unzip at 80 mv ( trans vs cis ) , whereas it only took 0.7  0.1 ms 
 to unzip i - hp12 - 4 at the same bias , a factor of about 50 times faster 
 than i - hp12 - 1 .
surprisingly , the fishhook hairpin group showed a similar 
 trend as that for the internal hairpin group , however , with much longer 
 unzipping times ( figure 5b and figure s8 , supporting information ) . at 100 mv ,
fishhook 
 f - hp12 - 4 was about 50 times faster to unravel than fishhook f - hp12 - 1 . 

additionally , the difference in blockage current patterns for the 
 fishhook and internal hairpins remained the same in these studies . 
 in short , there was a similar trend in terms of unzipping time in 
 both hairpin groups ; it took a much longer time to unzip the fishhook 
 hairpins than the internal hairpins even though they have identical 
 stem sequences .
the data collected in the sequence dependence 
 study indicated that 
 the location of the gc and at bp blocks within the stem 
 greatly affects the unzipping time of both internal and fishhook hairpins . 

the closer the gc bp block was to the tail , the longer it took 
 to unravel the hairpin .
once the unzipping process was initiated , 
 it was easier to unzip the rest of the hairpin , even if it contained 
 gc - rich bp blocks .
these data support the unzipping process 
 being initiated from the end of the hairpin , because those with the 
 more stable gc blocks at the ends took longer to unzip than 
 those with weaker at blocks at the ends ( figure 5b ) .
the 
 behavior of internal hairpins and fishhook hairpins is different in 
 terms of unzipping current and time , which led us to the question , 
 how many bases in the single - stranded tail are required to make the 
 hairpin behave as an internal hairpin ?
the answer to this question 
 will test our hypothesis that fishhook and internal hairpins unzip 
 at different locations , leading to different unzipping mechanisms . to address this question ,
these hairpins have the same 3 tail ( dc30 ) , loop , and a 12-base - pair stem ( figure 6a ) .
the 5 end was systematically changed to include 
 a 6- , 9- , or 12-mer dc tail .
experiments for these internal hairpins 
 with asymmetric tails were conducted , and the unzipping currents and 
 times were compared ( figure 6 ) .
these results 
 show that the tail length is crucial to the behavior of the internal 
 hairpins with asymmetric tails .
when the 5 tail was short 
 ( dc6 ) , it behaved like a fishhook hairpin ( figure 6b ) with the blocking current ( 11% of io ) being the same as f - hp12 - 1 and the unzipping time being 
 even longer than that of f - hp12 - 1 ( 590  50 ms vs 64  2 
 ms at 100 mv ) .
the longer unzipping time implies that the extra dc6 tail increased the difficulty for the hairpin to unzip inside 
 the vestibule . when the tail length was increased to 12 nucleotides 
 ( dc12 ) , it behaved like an internal hairpin ( figure 6d ) with both the blocking current giving the pulse - like 
 pattern described previously and the unzipping time being similar 
 to that of an i - hp12 - 1 ( 3.7  0.3 ms vs 2.6  0.1 ms at 
 100 mv ) . interestingly , when the 5 tail was dc9 , two current peaks were observed , one was centered at 11% of io , similar to the fishhook hairpin f - hp12 - 1 , 
 and had a remarkably slow unzipping time ( 920  60 ms at 100 
 mv , figure 6c ) .
the second population gave 
 the pulse - like current pattern similar to i - hp12 - 1 with a similar 
 unzipping time ( 3.0  0.4 ms at 100 mv , figure 5 and figure s19 , supporting information ) .
the unzipping time for the asymmetric internal hairpin with the 
 9-mer tail further suggests that it behaved either as a fishhook or 
 internal hairpin during unzipping depending on how it was captured 
 by -hl .
these results demonstrate that a tail length of 12 
 nucleotides or greater causes the hairpin to exclusively unzip outside 
 the vestibule of -hl , tail lengths of 6 nucleotides or smaller 
 unzip inside the vestibule , and tail lengths around 9 nucleotides 
 behave as both internal and fishhook hairpins with two distinct populations . 

additionally , the internal hairpin with short , asymmetric tails had 
 a significantly longer unzipping time than the fishhook hairpin , suggesting 
 that the short tail can enter the vestibule of the -hl and 
 therefore hinder the unzipping process in the confined space of the 
 protein cavity .
this data further supports our hypothesis that the 
 limited vestibule size causes slower unzipping for fishhook hairpins . unzipping 
 current blockage and time of internal hairpins with asymmetric 
 trails at 100 mv ( trans vs cis ) . 

these three hairpins have the same 3 tail , stem , and loop . 
 the numbers of bases at the 5 overhanging end are x = 6 ( b ) , 9 ( c ) , and 12 ( d ) .
the sequences of these hairpins 
 are shown in part a. experimental conditions : 1.00 m kcl , 10 mm pbs , 
 ph 7.4 , 22  0.5 c .
we have 
 demonstrated that unzipping of internal hairpins and fishhook hairpins 
 employs different mechanisms with the internal hairpins unzipping 
 much faster than the fishhook hairpins . in a previous study from our 
 laboratories , a 25-mer dna duplex ( similar to a fishhook hairpin that 
 unzips in the vestibule ) was not able to unzip and translocate under 
 similar conditions ( < 140 mv in 1.00 m kcl )
; therefore , we designed a long , internal hairpin with asymmetric 
 tails to determine if we could unzip an internal hairpin with a long 
 stem using the nanopore .
this hairpin was designed to have 54 bp s 
 in the stem , a 3 dc12 tail , and a 5 dc30 tail ( figure 7a ) . for this hairpin , 
 we preferred to bias the system toward 5 entry because it 
 provides more information about chemical modification than the 3 
 entry , based on our previous studies .
as shown in figure 7b , the unzipping 
 time  of this long hairpin displays strong voltage dependence 
 and showed the characteristic pulse - like current pattern ( figure s15 , supporting information ) previously observed for 
 internal hairpins .
these observations indicate the unzipping and translocation 
 of a long , internal hairpin with asymmetric tails can be rapid , supporting 
 our hypothesis that internal hairpins unzip outside of the vestibule . unzipping 
 of a long , asymmetric hairpin at 90 , 100 , and 110 mv 
 ( trans vs cis ) .
( b ) voltage - dependent unzipping of a long , asymmetric 
 hairpin ( 5-c30 agt tgc cac cta atg cgt cgt cgg 
 tct atc aag cct aca cag aat cag ttg tcg gtta cga caa ctg att ctg tgt 
 agg ctt gat aga ccg acg acg cat tag gtg gca act c12 - 3 ) . 

data were recorded at 80 mv , but the duration of unzipping was > 2 
 s ; therefore , a limited population of events were recorded that could 
 not be used for proper statistical analysis to provide a time constant .
the data presented above support 
 our hypothesis that internal hairpins 
 and fishhook hairpins unzip at different locations before they can 
 translocate through the -hl nanopore , representing different 
 unzipping mechanisms . here , two possible explanations for these results 
 are presented . in previous studies ,
long duplex dna or rna unzipping 
 was measured using optical tweezers and afm , in which the force for 
 dissociation was quantified . in these studies , the magnitude of the 
 applied force was dependent on the orientation of the base pairs . 
 a smaller force ( 1030 pn ) , depending on the sequence ,
was required to unfold or extend 
 the structure when a parallel force was applied relative to the base 
 pair axis , compared to the larger force being required when applied 
 perpendicular to the base pair axis ( 65300 pn ) .
our results demonstrate that 
 dna molecules are easier to unwind when the unzipping electrophertic 
 force , fe , is applied parallel to the 
 bp s ; the unzipping times for internal hairpins in the -hl 
 nanopore are much shorter than those for the fishhook hairpins ( figure 3 ) . in the process of unzipping of the fishhook hairpin , 
 the electrophoretic force fe is perpendicular 
 to the bp s ; therefore , it takes much longer to unzip and translocate 
 these hairpins . also ,
unlike internal hairpins , fishhook hairpins 
 can enter the vestibule , and consequently , the unfolding occurs in 
 this sterically demanding space ; hence , fishhook hairpins have less 
 freedom to change conformation to find the best state in which to 
 unzip .
however , for the internal hairpin remaining outside the protein 
 nanopore , there is no space restriction , and it is much easier for 
 it to find the conformation required to unzip .
relationship between 
 the direction of the unzipping force and the 
 base pairs in the hairpins studied .
( a ) the direction of unzipping 
 force is perpendicular to the direction of the bp s , representing 
 the unzipping of fishhook hairpins .
( b ) the direction of unzipping 
 force is parallel to the direction of the bp s , representing 
 the unzipping of internal hairpins .
in 
 summary , the unzipping processes of internal and fishhook hairpins 
 leading to translocation through the -hl nanopore were explored . 

the data presented here show that , similar to the unzipping of fishhook 
 hairpins , the unzipping of internal hairpins also depends on the stem 
 length and stem sequence ( figures 4 and 5 ) .
more importantly , the data indicate that fishhook 
 and internal hairpins unzip at different locations based on blockage 
 current level patterns and the unzipping times recorded .
fishhook 
 hairpins can enter the vestibule of -hl and unzip in the spatially 
 restricted context of the -hl , leading to long unzipping times 
 with one deep blockage current level .
in contrast , the internal hairpins 
 do not enter the vestibule and instead unzip outside the cis entrance of the -hl channel , leading to fast unzipping times 
 with a characteristic ion current pattern .
this observation of slow 
 unfolding inside the vestibule vs rapid unzipping outside the vestibule 
 was also recently observed for g - quadruplexes of different folds , some of which are small enough to enter the 
 protein cavity , and others are too large and therefore unfold rapidly 
 outside the vestibule under the influence of the applied electrophoretic 
 force . on the basis of the data presented above
, we propose that the 
 location of unzipping leads to a different unzipping mechanism . in 
 the case of the internal hairpin ,
the unzipping force is parallel 
 to the base pair plane that renders that structure easier to unzip 
 compared to the fishhook hairpin , in which the unzipping force is 
 perpendicular to the base pair plane ( figure 8) .
unzipping of the internal hairpins outside the vestibule gives 
 a pulse - like pattern to the ion current that is characteristic of 
 this process ( figure 3 ) .
the sequence - dependent 
 studies support this hypothesis ( figure 5 ) , 
 because there was a 520-fold difference in unzipping times 
 between fishhook hairpins and internal hairpins depending on the sequence 
 and the applied voltage bias ; additionally , the difference in ion 
 current for the fishhook and internal hairpins remained the same for 
 the sequences studied . on the basis of our unzipping model for 
 the hairpins ,
first , a set of sequence - dependent studies suggest the position 
 of cg and at bp s contribute to the unzipping time 
 of both fishhook and internal hairpins that is in agreement with experimental and theoretical work .
second , we identified 
 that tail lengths 12 dc nucleotides transform fishhook hairpins 
 to unzip like internal hairpins in the -hl nanopore .
third , 
 on the basis of our knowledge of the hairpin unzipping process , a 
 long , internal hairpin ( 54-bp stem ) with asymmetric tails was designed 
 to determine if a long duplex ( i.e. , stem ) could be unzipped outside 
 the vestibule .
these studies provide further insight into the process 
 of dna hairpin unwinding under an applied electrophoretic force and 
 expand the possible avenues for analyzing dna sequence and structure 
 using protein nanopores such as -hemolysin .
last but not least , 
 these studies were able to resolve a current difference of 6% 
 between hairpin dna immobilized in the nanopore waiting to unzip vs 
 the translocating unzipped dna , with the latter showing a deeper current 
 blockage level .
this demonstration of different currents for immobilized 
 and translocating dna has not been described previously .
 studies 
 on the interaction of hairpin dna with the -hemolysin 
 ( -hl ) nanopore have determined hairpin unzipping kinetics , 
 thermodynamics , and sequence - dependent dna / protein interactions . 
 missing 
 from these results is a systematic study comparing the unzipping process 
 for fishhook ( one - tail ) vs internal ( two - tail ) hairpins when they 
 are electrophoretically driven from the cis to the trans side of -hl via a 30-mer single - stranded tail . 
 in the current studies , fishhook hairpins showed long unzipping times 
 with one deep blockage current level . 
 in contrast , the internal hairpins 
 demonstrated relatively fast unzipping and a characteristic pulse - like 
 current pattern . 
 these differences were further explored with respect 
 to stem length and sequence context . 
 further , a series of internal 
 hairpins with asymmetric tails were studied , for which it was determined 
 that a second tail longer than 12 nucleotides results in internal 
 hairpin unzipping behavior , while tail lengths of 6 nucleotides behaved 
 like fishhook hairpins . 
 interestingly , these studies were able to 
 resolve a current difference of 6% between hairpin dna immobilized 
 in the nanopore waiting to unzip vs the translocating unzipped dna , 
 with the latter showing a deeper current blockage level . 
 this demonstration 
 of different currents for immobilized and translocating dna has not 
 been described previously . 
 these results were interpreted as fishhook 
 hairpins unzipping inside the vestibule , while the internal hairpins 
 unzip outside the vestibule of -hl . 
 lastly , we used this knowledge 
 to study the unzipping of a long double - stranded dna ( > 50 base 
 pairs ) 
 outside the vestibule of -hl . 
 the conclusions drawn from these 
 studies are anticipated to be beneficial in future application of 
 nanopore analysis of nucleic acids . 
Introduction
Experimental
Section
Results and Discussion
Conclusions