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Digestion of individual DNA molecules by l-exonuclease at liquid–solidinterface†

Seong Ho Kang,ab Seungah Leea and Edward S. Yeung*a

Received 15th March 2010, Accepted 13th April 2010

First published as an Advance Article on the web 30th April 2010

DOI: 10.1039/c0an00145g

Enzyme digestion of single DNA molecules was directly observed in real time by dual-color total internal

reflection fluorescence microscopy (TIRFM). Individual l-DNA molecules labeled with the fluorescent

dye, YOYO-1, were stretched in a laminar flow stream and immobilized on a bare fused-silica prism

surface based on hydrophobic and electrostatic interactions. Enzyme digestion was initiated by the influx

of l-exonuclease enzyme via capillary force. When the dye : bp ratio was higher than 1 : 20, the exact

digestion rate could not be measured because of induced photocleavage of the DNA molecules. At a dye :

bp ratio of 1 : 50, shortening of the DNA strand was recorded in real time. Unlike previous studies, the

length-based digestion rate of l-exonuclease showed 3 distinct values in the range of 0.173(�0.024) to

0.462(�0.152) mm s�1 at 37 �C. That is, different enzyme molecules exhibit different digestion dynamics.

Digestion was also monitored based on the decrease in fluorescence intensity, but uncertainties were

much larger due to the distance dependent excitation intensity in the TIRF mode.

1 Introduction

While stochastic events of individual molecules are masked in

conventional measurements through ensemble averaging, single-

molecule detection can be used to directly observe individual steps

or intermediates of biochemical reactions. Various scientific

phenomena related to enzymology, including restriction enzymes,

have also been explained at the single-molecule level.1–3 Molec-

ular motion has been examined in great detail to reveal changes in

the conformation or direction during enzymatic reactions. The

stretching of DNA molecules has been investigated in various

laboratories using a variety of forces, such as optical trapping,4,5

molecular combing,6–8 DNA electrostretching,9,10 micro-

chambers,11,12 microchannels,13 and nanochannels.14–16 In

particular, DNA molecular combing, using a receding meniscus,

has been evaluated as a relatively simple technique for straight-

ening individual DNA molecules on glass or modified substrates.1

Another DNA stretching method is physical–chemical binding

a DNA molecule that is undergoing adsorption and desorption at

liquid–solid interfaces in the presence of bulk flow.17 This method

is based on the principle that electrostatic and hydrophobic

interactions govern DNA adsorption and that adsorption

generally occurs only at the unpaired ends of certain DNAs.

l-Exonuclease is a highly processive 50 to 30 exodeoxy-

ribonuclease that selectively digests the phosphorylated region of

double-stranded DNA.18 Methods for straightening the DNA in

previous single-molecule digestion studies include: (i) the DNA

molecules were attached to beads or terminal biotin-labeled

aAmes Laboratory-USDOE and Department of Chemistry, Iowa StateUniversity, Ames, Iowa, 50011, USA. E-mail: yeung@ameslab.gov; Fax:+1 515 294 0105; Tel: +1 515 294 0105bDepartment of Chemistry and Research Institute of Physics and Chemistry(RINPAC), Chonbuk National University, Jeonju, 561-756, South Korea

† Electronic supplementary information (ESI) available: Optimumconditions for the digestion of DNA molecules by l-exonuclease andtheir TIRFM images. See DOI: 10.1039/c0an00145g

This journal is ª The Royal Society of Chemistry 2010

l-concatemer;19,20 (ii) the substrates were modified with polymers

or chemicals;18,21 or (iii) an electric field was applied to modified-

DNA molecules.22 However, these pretreatments and modifica-

tions affect the digestion rate or direction during the enzymatic

reaction. For example, when one end of the DNA molecule is

immobilized on an avidin-coated cover glass, the digestion can

only proceed from the free end, not both. The applied electric

field also influences the digestion rate. Above all, the digestion

modes and rates are not easily observed in real time using these

methods. In another study, the topological linkage between

l-exonuclease and partially digested double-stranded DNA was

investigated,18 but the mechanism was not well understood

despite the extensive experiments that were conducted.

In this study, various pathways for individual l-DNA digestion

and the associated digestion rates of l-exonuclease were observed

in real time in the evanescent field layer (<300 nm) on a fused-silica

prism surface by measuring the DNA length and the fluorescence

intensity using dual-color total internal reflection (TIR) geometry

to provide high contrast images by virtually eliminating all of the

background.23,24 The molecular conformation and the adsorption

behavior depend on both the pH and the buffer composition at the

water/fused silica interface.17 Individual l-DNA molecules that

were intercalated with YOYO-1 were stretched in a laminar flow

stream and immobilized on the prism surface through hydro-

phobic and electrostatic interactions at a pH of 4.2 without any

chemical modification of the substrate surface or the DNA

molecules. The enzymatic motion and the individual digestion

rates were also compared to the ensemble averaged data that was

obtained in the bulk solution using capillary electrophoresis.

2 Experimental

2.1 Preparation of samples

l-DNA (48 502 bp) was obtained from Promega (Madison, WI).

l-Exonuclease enzyme (28 kDa, pKa ¼ 5.29) was obtained from

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New England BioLabs (Ipswich, MA). The enzyme (50 mg mL�1)

was diluted with a 1� reaction buffer (pH 9.4, 2.5 mM MgCl2,

67 mM glycine–KOH, 50 mg mL�1 BSA). Prior to the enzyme

digestion reaction, the l-DNA sample was labeled with YOYO-1

(Molecular Probes, Eugene, OR) at various dye to nucleotide bp

ratios in a 10 mM Gly-Gly buffer solution (pH 8.2). For the

single-molecule imaging experiments, these DNA samples were

further diluted to 10 pM using a 2.5 mM acetate buffer solution

(pH 4.2) in order to promote the stretching of the DNA mole-

cules on the fused-silica surface. All of the buffer solutions were

filtered through a 0.2 mm membrane filter prior to use.

l-Exonuclease was fluorescently labeled with Alexa Fluor�532

using a Protein Labeling kit (A10236, Molecular Probes) and

purified according to the manufacturer’s instructions in order to

confirm the position of the enzyme on the DNA.25

Fig. 2 (A) Schematic diagram of the dual-color TIRFM system with

a temperature controller for the direct observation of single-molecule

digestion. Microscope, Axioskop50; exposure time, 10 ms (10 Hz);

objective lens, EC Plan-NEOFLUAR, 100�/1.3, oil; buffer solution,

2.5 mM sodium acetate (pH 4.2). Dye : bp ¼ 1 : 50; L, laser; P, pinhole;

SH, shutter; M, mirror; S, sample (l-DNA + l-exonuclease); CS, cover

slip; IO, immersion oil; OL, objective lens; F, filter; ICCD, intensified

charge-coupled device; TC, temperature controller. (B) Schematic of the

digestion of single-DNA molecule that was intercalated with YOYO-1

using l-exonuclease.

2.2 Digestion of single-DNA molecules

The stretching and immobilization method for the individual

l-DNA molecules on the prism surface was modified from

a previously reported technique.17 Briefly, 4 mL of the prepared

DNA sample in the 2.5 mM acetate buffer solution (pH 4.2) were

added to a corner of the cover slip (No.1 Corning, 22 mm

square). This sample was located at 0.4 cm along the x-axis and

0.4 cm along the y-axis from the edge (Fig. 1A). The cover slip

was turned and attached to the prism surface in order to create

a laminar flow stream between the cover slip and the right-angle

fused-silica prism surface (Melles Griot, Irvine, CA; A ¼ B ¼C ¼ 2.54 cm, refractive index, n ¼ 1.463). The l-DNA molecules

were immobilized on the prism surface at this pH and were

stretched through laminar flow (Fig. 1B). 4 mL of the l-exonu-

clease enzyme in the 1� reaction buffer were infused at the edge

of the cover slip, through capillary force, in order to initiate the

digestion reaction (circle in Fig. 2A). The area between the cover

slip and the objective lens was index-matched with an immersion

oil (Type FF, n ¼ 1.4850, The Microscope Depot, CA), and then

the digestion of single-DNA molecules was observed at 37 �C

using a dual-color TIRFM system (Fig. 2).

2.3 Dual-color TIRFM system for single-molecule detection

The basic dual-color TIRFM setup (Fig. 2A) used in this work

was similar to those described in previous reports.26,27 Two

different lasers, a 488 nm argon ion laser (8 mW; Model,

Fig. 1 (A) Laminar flow stream on the prism surface using a cover slip. (B) Sc

1760 | Analyst, 2010, 135, 1759–1764

2211-10SL-YIW, Cyonics Uniphase, San Jose, CA) and

a 532 nm laser (25 mW; Model, BWN-532-50E/56487, B&W

TEK, Newark, DE), were used as the excitation light sources. A

Pentamax 512-EFT/1EIA ICCD camera (Princeton Instruments,

Princeton, NJ) was mounted on top of a Zeiss Axioskop50

upright microscope (Zeiss, Germany), and a Zeiss 100�/1.3 N.A.

oil type EC Plan-NEOFLUAR microscope objective lens was

used. A filter cube set with 488 nm and 532 nm notch filters

(Semorck, Rochester, NY) was used to monitor the l-DNA

molecules that were labeled with YOYO-1 dye and the

l-exonuclease enzyme that was labeled with Alexa Fluor�532,

respectively. The laser beams were transmitted through an

optical pinhole in order to eliminate any extraneous light and to

hematics of the DNA stretching in the laminar flow on the prism surface.

This journal is ª The Royal Society of Chemistry 2010

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reduce the laser diameter. A Uniblitz mechanical shutter (model

LS2Z2, Vincent Associates, Rochester, NY) with a VMM-D1

shutter driver (Vincent Associates) was used to block the laser

beam when the camera was turned off in order to reduce the

photobleaching. A sampling frequency of 10 Hz was used for the

shutter driver set at an exposure of 10 ms and a delay of 90 ms.

The temperature of the prism was held constant at 37 �C using

a temperature controller (FRYER, A-50). All images were

collected using WinView/32� (version 2.5.14.1, Princeton

Instruments) and analyzed using MetaMorph 7.0 software

(Universal Imaging Co., Downingtown, PA).

Fig. 3 The effect of the fluorescent dye, YOYO-1, on the digestion of

single l-DNA at various molar ratios of DNA bp to YOYO-1 molecules.

2.4 Capillary electrophoresis for analysis of the bulk sample

The DNA digestion of l-exonuclease was characterized in the

bulk solution using a commercial capillary electrophoresis (CE)

system (P/ACE MDQ Beckman coulter, CA) that was equipped

with a 488 nm laser. A bare fused-silica capillary (Polymicro

Technologies, Phoenix, AZ) with a total length of 60 cm (effec-

tive length of 50 cm) and an I.D. of 75 mm was used for the

separation. 1� TBE buffer (0.089 M Tris, 0.089 M borate and

0.002 M EDTA, adjusted to a pH of 8.3 with 1 M NaOH) was

prepared by dissolving the pre-mixed powder (Amerosco, Solon,

OH) in ultrapure water ($15 MU). The dynamic coating matrix

of the capillary was created by dissolving 1.0% (w/v) of poly-

vinylpyrrolidone (PVP, Mr ¼ 1 000 000) (Polyscience, Warring-

ton, England) in 1� TBE buffer (pH 8.3). A cellulose derivative

solution containing 0.25% (w/v) of hydroethylcellulose (HEC,

Mr ¼ 250 000, Aldrich) in a 1� TBE buffer (pH 8.3) was selected

as the sieving matrix in order to resolve DNA over a wide range

of sizes. The coating and sieving matrixes were hydrodynamically

injected at a pressure of 20 psi for 5 min and 10 min, respectively.

The l-DNA samples were labeled with various molar ratios of

YOYO-1 dye (dye : bp ¼ 1 : 5 to 1 : 50) and incubated with

l-exonuclease (0.2 mg mL�1) for 0 min, 5 min, 10 min, 20 min,

and 30 min at 37 �C prior to the CE separation. The DNA

samples were electrokinetically injected at 9.5 kV for 5 s and

separated at an electric field strength of 217 V cm�1. The capillary

temperature was fixed at 18 �C in order to minimize the enzyme

digestion during the DNA separation. The CE data analysis was

carried out using the Beckman P/ACE MDQ Program version 2.3

and Origin 6.1 software (OriginLab Co., Northampton, MA).

Fig. 4 Changes in the digestion extent as a function of enzyme reaction

time in bulk solution that was determined using CE at 37 �C (A) and the

ratio of the peak areas of l-DNA that were labeled with a dye : bp ratio¼1 : 50 at different enzyme reaction times (B). The ordinate was the peak

area at the given time divided by the peak area at the initial point. The CE

conditions: applied separation voltage, 217 V cm�1; injection voltage,

9.5 kV for 5 s; running buffer, 1� TBE (pH 8.3); coating matrix, 1.0%

PVP (Mr ¼ 1 000 000); sieving matrix, 0.25% HEC (Mr ¼ 250 000);

capillary, 60 cm � 75 mm I.D. (effective length, 50 cm).

3 Results and discussion

3.1 The effects of fluorescent dye YOYO-1

The effect of the fluorescent dye YOYO-1 on the digestion of the

l-DNA was observed at various molar ratios of YOYO-1 to

DNA bp (i.e., 1 : 5, 1 : 10, 1 : 20, 1 : 30, 1 : 40, and 1 : 50). Fig. 3

shows the TIRFM images at various ratios of the l-DNA

molecules that were labeled with YOYO-1 dye. The relative

fluorescence intensity (RFI) increased with increasing YOYO-1

content, but a slightly longer migration time was required

because of the positive charge of YOYO-1. In the TIRFM

images, larger numbers of l-DNA molecules were adsorbed onto

the fused-silica prism surface because the higher ratio of posi-

tively charged YOYO-1 led to a stronger electrostatic interaction

with the silanoate ions (SiO�) on the prism surface at a pH of 4.2

This journal is ª The Royal Society of Chemistry 2010

(Fig. 3).17 However, images of the individual DNA molecules

could still be observed at the lowest YOYO-1 ratio of 1 : 50.

3.2 Enzyme digestion in bulk solution by CE

The enzyme reaction mixture was incubated at 37 �C in 200 mL

tube using the supplier’s instructions in order to facilitate the

l-exonuclease reaction. Quantitative l-DNA analysis was per-

formed in the bulk solution at 37 �C for various enzyme reaction

times ranging from 0 to 30 min using CE (Fig. 4). The peak area

of the l-DNA sample that was labeled with the dye : bp ratio of

1 : 50 decreased about 94% within 5 min (Fig. 4A) and leveled off

within 30 min at 37 �C (Fig. 4B). The rates are consistent with

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literature values of enzyme activity. This shows that low-level dye

labeling does not affect the digestion process.

Fig. 5 Different digestion modes based on the decrease in the length of

the single-DNA molecules by l-exonuclease at the interface of the buffer

solution and the fused-silica prism. (A) Type I¼ digestion at one end with

a small spot that still remained after the completion of the digestion

reaction. (B) Type II¼ complete digestion from both ends. (C) Type III¼digestion from both ends with a spot that still remained after the

completion of the digestion reaction. The ratio of dye : bp was 1 : 50. The

yellow arrows show the direction of DNA digestion. The conditions were

the same as in Fig. 2. Regions: a, the incubation time of l-exonuclease for

attachment to the individual DNA molecules; b, l-exonuclease digestion;

and c, digestion weakened or ceased because of the DNA nicks or

detachment of the enzyme.

3.3 Single-DNA molecule digestion by l-exonuclease

After the DNA molecules were stretched and immobilized on the

prism surface, the enzyme was injected. The l-exonuclease

digestion of the individual DNA molecules was observed using

TIRFM (Fig. S1 of ESI†) when 4 mL of a 1� enzyme reaction

buffer (pH 9.4) were injected at a position (0 cm x-axis and

0.5 cm y-axis) between the cover slip and the prism surface that

was earlier filled with 4 mL of the 2.5 mM sodium acetate buffer

(pH 4.2). The final pH of the enzyme digestion mixture solution

was about 9.21 (Table S1 of ESI†). Binding occurred when the

l-exonuclease enzyme was infused into the l-DNA sample on the

prism surface (Fig. S2 of ESI†). The dual-color TIRFM images

of the l-DNA molecules that were labeled with the fluorescent

dye YOYO-1 (Fig. S2A†, green) and the l-exonuclease enzyme

that was labeled with Alexa Fluor�532 (Fig. S2B†, red) were

observed at different excitation wavelengths of 488 and 532 nm,

respectively. The colocalization in each TIRFM image easily

confirmed that binding occurred (Fig. S2C†, yellow). The

l-exonuclease enzyme was preferentially bound at the end of the

l-DNA molecule.

When the labeled DNA was irradiated by the laser, photo-

cleavage was observed at levels of 100%, 45%, 37%, 22%, and 7%

at dye : bp ratios of 1 : 5, 1 : 10, 1 : 20, 1 : 30, and 1 : 40,

respectively. However, photocleavage was not observed at a ratio

of 1 : 50 for irradiation of 180 s at a laser power of 8 mW

(Fig. S3A of ESI†). Although the enzyme digestion of the DNA

molecules still took place at a high dye : bp ratio of 1 : 5, many

small fragments were observed because of the photocleavage.

These results were consistent with the fact that the phospho-

diester bonds of DNA are cleaved by activated YOYO-1 dye.28

Therefore, the actual ratio of dye : bp must be at or below 1 : 50

in order to clearly reveal the effect of the enzyme.

Fig. 5 shows the progression of single-DNA digestion by

l-exonuclease enzyme based on the decrease in the length of the

DNA molecule at a dye : bp ratio of 1 : 50. Side effects that would

hinder the digestion of l-DNA molecules were not detected for

the reaction under these conditions. These results agreed with

previous observations that the activity of l-exonuclease was not

altered at a pH of 7.5.29,30 Three different digestion modes existed

at 37 �C based on the decrease in the DNA length (Fig. 5). The

incubation time of the l-exonuclease reaction was at least 20 s

(region ‘‘a’’ in Fig. 5). This is because binding of the enzyme onto

the DNA strand must occur first. The DNA length rapidly

decreased after l-incubation (region ‘‘b’’ Fig. 5). The type I

digestion started from one end of the DNA molecule (Fig. 5A).

In contrast, the digestion reaction occurred at both ends of the

DNA molecule for types II and III digestion (Fig. 5B and C).

These observations differed from the results that were obtained

in previous single-DNA digestion studies, which showed just one

digestion pathway and one digestion rate.13,22 Previous

researchers specifically coupled one end of the DNA, such as

DNA–biotin, DNA–thiol, DNA–beads, in order to immobilize

the molecule. In these cases, the digestion of the DNA molecules

proceeded only at the free end of the DNA molecule. In this

study, only the electrostatic interaction between the DNA

1762 | Analyst, 2010, 135, 1759–1764

molecules and the fused-silica prism surface was controlled so

that the individual DNA molecules could be stretched on the

surface. The point of attachment did not necessarily have to be at

the end of the DNA but could be somewhere along the DNA

chain. The digestion process can thus be observed from both

ends of the DNA molecule in the absence of any pretreatments or

modifications of the DNA molecule and the substrate. Simulta-

neous digestion from both ends obviously involved 2 enzyme

molecules, one at each end of the DNA.

This journal is ª The Royal Society of Chemistry 2010

Table 1 Enzyme digestion rates of individual l-DNA molecules that were labeled with YOYO-1 (dye : bp ¼ 1 : 50) by l-exonuclease based on thedecrease in the DNA length for the different digestion modes

Type I II III

Fraction 0.30 (n ¼ 7) 0.22 (n ¼ 5) 0.48 (n ¼ 11)Digestion rate/bp s�1 (mm s�1) 1299 � 436a 508 � 70a 1357 � 447a

(0.442 � 0.148a) (0.173 � 0.024a) (0.462 � 0.152a)69 � 15b

(0.023 � 0.005b)

a The values indicate the rate at region ‘‘b’’ in Fig. 5A–C. b The values indicate the rate at region ‘‘c’’ in Fig. 5C.

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Among the digestion modes, type III digestion was observed

for the largest number of molecules, with a fraction of 0.48

compared to type I (0.30) and type II (0.22), as depicted in

Table 1. About 22% of the molecules exhibited a decrease in their

lengths from both ends. These molecules completely disappeared

within 137 � 13 s (type II, Fig. 5B). However, in the majority of

cases (type III, Fig. 5C), the lengths of the DNA molecules

rapidly decreased within about �40 s and then gradually

decreased up to�180 s. Occasionally, individual DNA molecules

remained in the form of a small spot (�6.5 mm in length) after

�60 s (type I, ‘‘b’’ region in Fig. 5A) or�40 s (type III, ‘‘b’’ region

in Fig. 5C). These results were similar to those obtained in the CE

experiment of the bulk solution (Fig. 4), where the l-DNA peak

did not completely disappear after an enzyme reaction time of

30 min. That is, the individual l-DNA molecules were either

partially digested (�60%, types I and III) or completely digested

(100% digestion, type II).21

The digestion rates were calculated using an estimated 1 kb

DNA molecule length of 0.34 mm. There appears to be 3 distinct

groups of digestion rates. From the fastest to the slowest, these

are region b for type I and type III, region b for type II, and

region c for type III. For all 3 types, digestion starts from left to

right, presumably each with the same enzyme molecule. It is

interesting and unexpected that in the second case the digestion

rate was clearly lower than in the other cases. A slower digestion

rate may be due to partial and temporary detachment of the

enzyme from the DNA strand. However, such detachment

cannot be observed at the spatial resolution of the present

experiment. For type I digestion, it appears that the enzyme

molecule detached from the DNA strand at 60 s so a section was

left for the remaining observation period. These results were

consistent with a previous report that showed the digestion of

l-DNA with l-exonuclease stopped halfway during direct

observation.22 l-Exonuclease is unable to digest DNA at the

nicks, even though it can bind to these nicks22 because the

enzyme was inactivated or released from the DNA at the position

of a nick.29 For type II digestion, before the enzyme was detached

(third image), a second enzyme molecule started to digest the

DNA strand from the right side. Careful examination of Fig. 5b

reveals that the rate increased briefly before complete digestion

(and disappearance) of the strand. Unfortunately only a few

frames were recorded for this second digestion regime so an

estimate of the second rate was not possible. For type III

digestion, region b corresponded to digestion from left to right

and region c corresponded to digestion from right to left,

presumably by another enzyme molecule. The second rate,

however, was substantially smaller than any of those observed

elsewhere. In all cases, the digestion rate appears to be constant

This journal is ª The Royal Society of Chemistry 2010

for a given enzyme during its active period. This is in contrast to

the digestion of surface bound proteins where the rate gradually

decreased as the enzyme approached the surface.31 The difference

here is that the DNA strand is much longer and is thus further

away from the surface for the enzyme to be affected by it.

The dependence of the relative fluorescence intensity (RFI,

integrated over the entire molecule) on the reaction time was also

measured (Fig. 6) in order to compare the calculated digestion

rates with respect to the decrease in the DNA length. Both linear

shaped (types i–iii, Fig. 6A–C) and non-linear shaped (type iv,

Fig. 6D) DNA molecules were selected. All of the fluorescence

intensities decreased with increasing digestion time for all of the

digestion types. However, the evanescent intensities depended on

the distance from the solution–prism interface, resulting in

a large scatter in the data points and preventing the reliable

calculation of the reaction rates. Therefore, the direct measure-

ment of the DNA lengths, as in Fig. 5, is important in monitoring

the digestion process.

4 Conclusions

The enzymatic digestion of individual DNA molecules with

l-exonuclease was observed in real-time using dual-color

TIRFM. The digestion was classified into three different types

based on the decrease in the length of the individual DNA

molecules. The individual molecules that were labeled with the

fluorescent dye YOYO-1 were easily stretched in a laminar flow

stream and immobilized on the prism surface without any

chemical modification of the DNA molecules or the bare fused-

silica prism. The digestion reaction of the immobilized DNA

molecules started after an incubation period following the influx

of the l-exonuclease enzyme. At a ratio of 1 : 50, the digestion

rates were measured based on decrease in the lengths of the DNA

molecules without interference from photocleavage and photo-

bleaching. These rates were in the range of 0.173� 0.024 to 0.462

� 0.152 mm s�1 (¼508 � 70 to 1357 � 447 bp s�1) at 37 �C for the

three digestion types. These results varied from previous reports

for fluorescently stained DNA after the DNA–bead complex was

trapped (digestion rate, 13–21 bp s�1),13 individual molecules of

unstained l-DNA that was attached to optically trapped beads

(digestion rate, 15–20 bp s�1),19 or individual DNA molecules

that are attached at one end to a glass surface through a biotin–

streptavidin linkage and at the opposite end to polystyrene beads

through a digoxigenin–antidigoxigenin linkage (digestion rate,

32 bp s�1),21 but were in the same range as digestion using

a fluorescence microscope after a stained l-DNA molecule with

one biotinylated terminal was fixed onto an avidin-coated cover

slip and straightened using a dc electric field (digestion rate,

Analyst, 2010, 135, 1759–1764 | 1763

Fig. 6 Real-time digestion of l-DNA molecules with l-exonuclease enzyme that was monitored by the decrease in the fluorescence intensities at the

solution–prism interface. (A) Type i ¼ digestion at one end with a small spot that still remained after the completion of the digestion reaction. (B)

Type ii¼ digestion from both ends. (C) Type iii¼ digestion from both ends with a spot that still remained after the completion of the digestion reaction.

(D) Type iv ¼ digestion from both ends of a non-linear shaped (incompletely stretched) DNA molecule. The ratio of dye : bp was 1 : 50. The conditions

were the same as in Fig. 5.

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�1000 bp s�1).22 Clearly the methods for immobilizing the DNA

strand here and in ref. 22 allow the DNA to be in an environment

closer to that in free solution.

Acknowledgements

The Ames Laboratory is operated for the US Department of

Energy by Iowa State University for the US Department of

Energy under Contract No. DE-AC02-07CH11358. This work

was supported by the Director of Science, Office of Basic Energy

Sciences, Division of Chemical Sciences and partially supported

by research funds of Chonbuk National University in 2008.

References

1 L. Lam, R. Iino, K. V. Tabata and H. Noji, Anal. Bioanal. Chem.,2008, 391, 2423–2432.

2 X. S. Xie and H. P. Lu, J. Biol. Chem., 1999, 274, 15967–15970.3 X. S. Xie, Single Mol., 2001, 2, 229–236.4 S. B. Smith, Y. Cui and C. Bustamante, Science, 1996, 271, 795–799.5 S. J. Koch, A. Shundrovsky, B. C. Jantzen and M. D. Wa, Biophys. J.,

2002, 83, 1098–1105.6 D. Bensimon, A. J. Simon, V. Croquette and A. Bensimon, Phys. Rev.

Lett., 1995, 74, 4754–4757.7 J. Herrick and A. Bensimon, Biochimie, 1999, 81, 859–871.8 X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra,

N. Hornigold, v. M. Slegtenhorst, J. Wolfe, S. Povey,J. S. Beckmann and A. Bensimon, Science, 1997, 277, 1518–1523.

9 M. Washizu and O. Kurosawa, IEEE Trans. Ind. Appl., 1990, 26,1165–1172.

10 M. Ueda, J. Biochem. Biophys. Methods, 1999, 41, 153–165.11 L. Lam, S. Sakakihara, K. Ishizuka, S. Takeuchi and H. Noji, Lab

Chip, 2007, 7, 1738–1745.

1764 | Analyst, 2010, 135, 1759–1764

12 L. Lam, K. Ishizuka, S. Sakakihara and H. Noji, Proceedings ofMicro Total Analysis Systems, 2006, 2, 1429–1431.

13 H. Kurita, K. Inaishi, K. Torii, M. Urisu, M. Nakano, S. Katsura andA. Mizuno, J. Biomol. Struct. Dyn., 2008, 25, 473–480.

14 J. O. Tegenfeldt, C. Prinz, H. Cao, S. Chou, W. W. Reisner, R. Riehn,Y. M. Wang, E. C. Cox, J. C. Sturm, P. Silberzan and R. H. Austin,Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 10979–10983.

15 R. Riehn, M. Lu, Y.-M. Wang, S. F. Lim, E. C. Cox andR. H. Austin, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 10012–10016.

16 W. Reisner, K. J. Morton, R. Riehn, Y. M. Wang, Z. Yu, M. Rosen,J. C. Sturm, S. Y. Chou, E. Frey and R. H. Austin, Phys. Rev. Lett.,2005, 94, 196101.

17 S. H. Kang, M. R. Shortreed and E. S. Yeung, Anal. Chem., 2001, 73,1091–1099.

18 K. Subramanian, W. Rutvisuttinunt, W. Scott and R. S. Myers,Nucleic Acids Res., 2003, 31, 1585–1596.

19 J. Dapprich, Cytometry, 1999, 36, 163–168.20 R. M. Zimmermann and E. C. Cox, Nucleic Acids Res., 1994, 22, 492–497.21 A. M. van Oijen, P. C. Blainey, D. J. Crampton, C. C. Richardson,

T. Ellenberger and X. S. Xie, Science, 2003, 301, 1235–1238.22 S. Matsuura, J. Komatsu, K. Hirano, H. Yasuda, K. Takashima,

S. Katsura and A. Mizuno, Nucleic Acids Res., 2001, 29, e79.23 T. Funatsu, Y. Harada, M. Tokunaga, K. Saito and T. Yanagida,

Nature, 1995, 374, 555–559.24 N. J. Harrick, Internal Reflection Spectroscopy, John Wiley & Sons,

New York, 1967, pp 1–65.25 http://probes.invitrogen.com/media/pis/mp10236.pdf.26 S. H. Kang, Y.-J. Kim and E. S. Yeung, Anal. Bioanal. Chem., 2007,

387, 2663–2671.27 S. Lee, B. H. Chung and S. H. Kang, Curr. Appl. Phys., 2008, 8, 700–705.28 B. Akerman and E. Tuite, Nucleic Acids Res., 1996, 24, 1080–1090.29 D. M. Carter and C. M. Radding, J. Biol. Chem., 1971, 246, 2502–

2512.30 C. M. Radding and D. M. Carter, J. Biol. Chem., 1971, 246, 2513–

2518.31 J. Li and E. S. Yeung, Anal. Chem., 2008, 80, 8509–8513.

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