NATURE METHODS | VOL.2 NO.2 | FEBRUARY 2005 | 127© Cold Spring Harbor Laboratory Press

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PUBLISHED IN ASSOCIATION WITH COLD SPRING HARBOR LABORATORY PRESS

Single-molecule DNA nanomanipulation: Improved resolution through use of shorter DNA fragmentsAndrey Revyakin1,2, Richard H Ebright2 & Terence R Strick1,3

1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA. 2Howard Hughes Medical Institute, Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA. 3Institut Jacques Monod, Centre National de la Recherche Scientifique UMR7592, Universités de Paris VI et VII, 2 Place Jussieu, 75251 Paris Cedex 05, France. Correspondence should be addressed to R.H.E. (ebright@waksman.rutgers.edu) or T.R.S. (strick@ijm.jussieu.fr).

Single-molecule nanomanipulation of supercoiled DNA permits measurement, in real time, of spatial and temporal parameters of protein-DNA interactions that affect DNA topology1–7. In this method, a double-stranded DNA molecule containing at least one target for the protein of interest is attached at one end to a magnetic bead and at the other end to a glass surface. The experimental setup and the monitoring of the end-to-end extension (l) of the stretched, supercoiled DNA molecule is diagramed in Figure 1a. The protein of interest is introduced into the system, and protein-dependent changes in DNA linking number (Lk) or DNA twist (Tw) are detected as changes in the number of plectonemic supercoils (changes in DNA writhe, Wr; Lk = Tw + Wr; ref. 8) and corresponding changes in l (Fig. 1b–d). This approach has been applied to analysis of supercoil formation and relaxation by topoisomerases1–5 and to promoter unwinding by bacterial RNA polymerase (RNAP)6,7. The spatial and temporal resolution of the method is expected to increase with decreasing length of the supercoiled DNA segment (equations in ref. 6). (Reducing the length of the supercoilable DNA segment should have no effect on the amplitude of changes in DNA extension resulting from protein-dependent changes in DNA topology (signal), but should reduce the amplitude of random fluctuations in DNA extension (noise), thereby resulting in improvement in the signal-to-noise ratio.) Previous work has involved supercoiled DNA segments 4–44 kilobases (kb) in length1–7. Here, we describe preparation of DNA molecules with 2-kb supercoilable DNA segments and document superior resolution in analysis of promoter unwinding and DNA compaction by bacterial RNAP.

MATERIALSREAGENTS

Anti-digoxigenin, sheep polyclonal (Roche)

Biotin-16-deoxyuridine triphosphate (dUTP) (1 mM; Roche)

BSA, fraction V (Roche; stock is 50 mg/ml in water)

Digoxigenin-11-dUTP, alkali stable (1 mM; Roche)

Magnetic beads, 0.8-µm diameter (4 mg/ml) (New England Biolabs; NEB)

Phosphate-buffered saline (PBS; Fluka)

Polyglutamic acid, Mw 1,500–3,000 (stock is 10 mg/ml in PBS; Sigma)

Polystyrene, Mw ∼280,000 (Sigma; stock is 0.1% in toluene)

Plasmid DNA for amplification (Fig. 2)

Primers for amplification (Table 1; Fig. 2)

QIAquick PCR Purification and Gel Extraction kits (Qiagen)

Restriction enzymes: MluI and NotI (NEB)

RNAP holoenzyme, σ-saturated (Epicentre)

Standard buffer (SB): 10 mM potassium phosphate buffer (pH 8), 0.1 mg/ml BSA, 0.1% Tween-20, 10 mM EDTA,

10 mM β-mercaptoethanol

T4 DNA ligase (10 U/µl) and 10× reaction buffer (NEB)

Thermostable DNA polymerase, high fidelity (3 U/µl; Roche)

dNTP mix (2.5 mM each; Roche)

Tosyl-activated beads, 3-µm diameter (10 mg/ml; Dynal)

Transcription buffer: 25 mM HEPES-NaCl pH 7.5, 75–150 mM NaCl, 10 mM MgCl2, 10 mM β-mercaptoethanol, 0.1 mg/ml BSA, 0.1% Tween-20

EQUIPMENT

Inverted videomicroscope with particle-tracking software

Motorized syringe pump

NdFeB (neodymium-iron-boron) magnets, grade 35, 0.25-inch diameter, 0.25-inch thick (MagnetSales)

Square glass capillary, 1 × 1 × 50 mm (Vitrocom)

Thermal cycler

Tygon tubing R3603, inner diameter 0.0402 inches, outer diameter 0.1082 inches (Kimberly-Clarke)

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128 | VOL.2 NO.2 | FEBRUARY 2005 | NATURE METHODS

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PROCEDURE1| Set up five amplification reactions, each as follows:

The procedure for preparation of DNA constructs is outlined in Fig. 2; sequences of primers are presented in Table 1. Plasmid pARTaqRPOC/N25 is analogous to pARTaqRPOC/rrnBP1 of ref. 6, but contains nucleotides from positions –78 to +16 of the bacteriophage T5 N25 promoter.▲CRITICAL STEP

2| Place the tubes in thermal cycler and incubate at 95 °C for 5 min.

3| To each tube, add 0.5 µl of thermostable DNA polymerase and pipet up and down to mix. The tubes should be left in the thermal cycler at 95 °C during delivery of the polymerase.

Preparation of DNA constructs for nanomanipulation

Primer NotRPOC2050 1.5 µl

Primer MluRPOC4050 1.5 µl

Plasmid pARTaqRPOC/N25 1 µl

DMSO 2.5 µl

dNTPs mix 4 µl

10× amplification buffer 5 µl

Water 34 µl

a b

c d

Figure 1 | Experimental approach. (a) Experimental setup. A double-stranded 2-kb DNA molecule containing a single promoter is tethered at one end, through multiple linkages, to a paramagnetic bead and at the other end, through multiple linkages, to the capillary floor. The DNA molecule is torsionally constrained and mechanically stretched between the bead and the glass surface by application of a pair of magnets above the DNA helix axis. The distance between the bead and the surface, which reflects the DNA end-to-end extension (l), is monitored in real time by videomicroscopy. The pair of magnets is rotated a specified number of counterclockwise or clockwise turns, and the bead is rotated in lock-step register. This rotation introduces a specified number of positive or negative superhelical turns into the DNA in lock-step register, plectonemic supercoils are formed, and, correspondingly, l is changed. (b) Calibration of l versus the number of superhelical turns. Over a range of positive and negative supercoiling, there is a linear relationship between l and the number of superhelical turns, with a change in l (δ) of 56 ± 5 nm per superhelical turn. (c,d) Detection of promoter unwinding. According to the relationship Lk = Tw + Wr (ref. 8), in a torsionally constrained DNA molecule with constant linking number (Lk), a change in twist (Tw; unwinding) must be compensated for by an equal, but opposite, change in writhe (Wr; number of supercoils). With positively supercoiled DNA, unwinding of about one turn of promoter DNA by RNAP must result in a compensatory gain of about one positive supercoil and, correspondingly, a decrease in l (∆lobs,pos). With negatively supercoiled DNA, unwinding of about one turn of promoter DNA by RNAP must result in a compensatory loss of about one negative supercoil and, correspondingly, an increase in l (∆lobs,neg).

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4| Amplify the 2-kb DNA fragment according to the following program:

5| Pool the reactions and purify the PCR product using the QIAquick PCR Purification kit.▲CRITICAL STEP

6| Digest the PCR product with restriction enzymes MluI and NotI according to the manufacturer’s instructions, at 37 °C for 1 h. Inactivate the enzymes by heating at 65 °C for 20 min.

7| Purify the 2-kb digestion product by electrophoresis through a 0.8% agarose gel containing 0.2 µg/ml ethidium bromide, and isolate the 2-kb product from the gel.Typical yields are on the order of 2 µg (∼2 pmol).▲CRITICAL STEP

8| Purify the DNA product from the gel using the QIAquick Gel Extraction kit.

9| Quantify the purified DNA and adjust concentration to 50 nM. Store the purified DNA at –20 °C.

10| Set up two separate amplification reactions to prepare prepare 1-kb biotin- and digoxigenin-labeled DNA fragments:

11| Repeat Steps 2 and 3.

12| Amplify each of the labeled products according to the following program:

13| Purify each of the amplified products using the QIAquick PCR Purification kit.

Cycle number Denaturation Annealing Polymerization1 30 s at 60 °C 1 min at 68 °C2–31 2 min at 94 °C 30 s at 60 °C 1 min at 68 °CLast 7 min at 68 °C

Reagents Biotin labeling reaction Digoxigenin labeling reactionPrimer MluRPOC4050 1.5 µlPrimer RPOC3140 1.5 µlPrimer NotRPOC50 1.5 µlPrimer RPOC820 1.5 µlPlasmid pARTaqRPOC/N25 1 µl 1 µlDMSO 2.5 µl 2.5 µldNTP mix 4 µl 4 µlBiotin-16-dUTP 1 µlDigoxigenin-11-dUTP 1 µl

10× amplification buffer 5 µl 5 µlWater 33 µl 33 µl

Cycle number Denaturation Annealing Polymerization1 30 s at 60 °C 2 min at 68 °C2–31 2 min at 94 °C 30 s at 60 °C 2 min at 68 °CLast 7 min at 68 °C

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14| Digest the product of the biotin labeling reaction with MluI and the product of the digoxigenin labeling reaction with NotI, according to the manufacturer’s instructions, at 37 °C for 1 h. Inactivate the enzymes by heating at 65 °C for 20 min.

15| Purify digestion products of the two reactions by electrophoresis through a 0.8% agarose gel containing 0.2 µg/ml ethidium bromide. Extract each of the products from the gel and purify the products using the QIAquick Gel Extraction kit.Typical yields are on the order of 2 µg (∼3 pmol).▲CRITICAL STEP

16| Quantify the DNA and adjust concentration to 200 nM. Aliquot the purified DNA and store at –80 °C to retard degradation of the biotin and digoxigenin residues.

17| Set up a ligation reaction by mixing the following:

Incubate the reaction at 25 °C for 3 h.▲CRITICAL STEP

18| Inactivate the ligase by adding EDTA to 10 mM and incubating at 65 °C for 10 min. Dispense the ligation products into 5-µl aliquots and store at –80 °C.

19| For binding to magnetic beads, dilute the DNA construct to ∼50 pM with 10 mM Tris (pH 8), 10 mM EDTA.The dilute solution can be stored at 4 °C for several months before the proportion of DNAs observed to be supercoiled under the microscope decays by about half.A good preparation of DNA should contain ∼50% supercoilable molecules.

2-kb DNA fragment (50 nM) from Step 9 4 µl1-kb digoxigenin-labeled fragment (200 nM) from Step 16 2.5 µl1-kb biotin-labeled fragment (200 nM) from Step 16 2.5 µl

10× T4 DNA ligase buffer 2 µl

T4 DNA Ligase (10 U/µl) 1 µlWater 8 µl

a

b

c

d

Figure 2 | Preparation of DNA fragment constructs. (a,b) Amplification by PCR is used to prepare a 1-kb multiply biotin-labeled DNA fragment, a 1-kb multiply digoxigenin-labeled DNA fragment and a 2-kb G/C-rich DNA fragment containing the bacteriophage T5 N25 promoter. (c,d) After restriction endonuclease digestion, the 1-kb biotin- and digoxigenin-labeled DNA fragments are ligated to the 2-kb promoter DNA fragment. The resulting DNA fragment can be bound to a streptavidin-coated magnetic bead, and the resulting bead-DNA assembly can be attached to a capillary floor coated with anti-digoxigenin.

Table 1 Preparation of DNA fragment. PCR primers. Primer Sequence (5′ to 3′) Concentration

MluRPOC4050 GAGAGAACGCGTGACCTTCTGGATCTCGTCCACCAGG 10 µMNotRPOC50 GAGAGAGCGGCCGCGAGAAGATCCGCTCCTGGAGCTACG 10 µMNotRPOC2050 GAGAGAGCGGCCGCGGACATCAAGGACGAGGTGTGG 10 µMRPOC820 TCCTGGCGCAGGTAGATGAG 10 µMRPOC3140 CTGATGCAAAAGCCCTCGGG 10 µM

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20| Clean capillary in a nitric acid bath for 4 h, rinse extensively with ultrapure water and dry with a stream of clean argon.▲CRITICAL STEP

21| Dip one end of the capillary into the polystyrene solution so as to draw in about 15 µl. Tip the capillary slowly at one end, and then at the other, to coat the entire inner surface with the polystyrene solution.This tipping of the tube should be done no more than two or three times, as a thick polystyrene layer will be more unstable than a thin one.➨ TROUBLESHOOTING▲CRITICAL STEP

22| Wick the excess solution out of the capillary, dry with a gentle stream of clean argon and fit the ends of the capillary with a 2-cm length of Tygon tubing.▲CRITICAL STEP

23| Inject into the capillary 100 µl of PBS containing 0.1 mg/ml polyclonal anti-digoxigenin. Place the capillary in a humid chamber and incubate at 37 °C overnight.▲CRITICAL STEP

24| Prepare a blocking solution of SB containing 10 mg/ml BSA, 3.3 mg/ml of polyglutamic acid and 3 mM sodium azide. Drain the capillary and inject 100 µl of the blocking solution into the capillary; place it in a humid chamber and incubate at 37 °C for at least 8 h.The resulting blocked capillary can be stored at 4 °C for up to 2 weeks.▲CRITICAL STEP➨ TROUBLESHOOTING

25| Before using the capillary, rinse it by flushing three times with 1 ml PBS. Inject 100 µl of 5 µg/ml 3-µm tosyl-activated beads in PBS into capillary and allow tosyl-activated beads to settle onto and bind to the surface (~ 30 min). Gently wash out unbound beads with 1 ml SB. Connect ends of capillary to input and output reservoirs, and place capillary onto microscope stage.▲CRITICAL STEP

26| Wash 10 µl of 0.8-µm magnetic beads in 200 µl PBS supplemented with 1 mg/ml BSA and resuspend beads in 10 µl PBS supplemented with 1 mg/ml BSA.

27| Deposit a 0.5-µl drop of the DNA fragment (50 pM) from Step 19 at the bottom of a small microcentrifuge tube.

28| Load a wide-bore pipette tip with 90 µl of SB.

29| Deposit 10 µl of beads onto the drop of DNA and immediately dilute the reaction with the 90 µl SB.This should be done by gently depositing the SB onto the bead plus DNA solution.

30| Resuspend the beads to homogeneity by tipping the tube upside down (without causing the liquid to drop) or by spinning the tube between the thumb and forefinger.

31| Before injecting bead-DNA mixture into the flow cell, move the magnets at least 2 cm away from the flow cell.

32| Inject 10 µl of the bead-DNA mixture from Step 29 into one of the plastic reservoirs connected to the capillary tube.

33| Inject another 20–50 µl of SB into the reservoir to ensure the majority of beads enter the capillary and are evenly distributed along its length.

Preparation of the flow cell and DNA tethering

Anchoring of DNA to magnetic beads

Anchoring of the DNA to the flow cell

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34| Allow the magnetic beads to sediment and incubate at 25 °C for ∼15 min.The majority of magnetic beads should move about on the surface and not appear immobile.➨ TROUBLESHOOTING

35| Establish a gentle flow of SB (150–200 µl/min, typically for 20 min) to remove unbound beads from the surface. (Buffer is injected into the input reservoir using a motorized syringe pump, and the output reservoir is drained by gravity feed.) Approximately every 2 min, slowly pass a rod with a small (2-mm diameter) magnet at the end just over the capillary, to lift unbound beads off the surface and into the flow field.▲CRITICAL STEP

36| Turn off flow and move the magnets as close as possible to the sample without allowing them to come into contact with it. This causes DNA molecules to extend away from the surface. Note the current rotational position of the magnets as the ‘magnet initial rotational state’.With the magnets positioned as close to the sample as possible, forces range from ∼0.5–1 pN, depending on the bead. At these forces (between 0.5 and 1 pN), a magnetic bead tethered by a single 2-kb DNA molecule hovers ∼0.6 µm above the surface and shows rapid, constrained, yet relatively isotropic Brownian motion.▲CRITICAL STEP

37| Rotate the magnets ten turns counterclockwise (in the direction of positive supercoiling). For a bead associated with a single supercoilable DNA molecule, the DNA molecule will form positive plectonemic supercoils and the bead will be observed to move toward the surface9. Rotating the magnets ten turns clockwise (in the direction of negative supercoiling) returns the system to the magnet initial rotational state.Beads tethered by a single supercoilable molecule can be identified rapidly by observing the behavior of beads as the magnets are rotated. (Changes in the vertical position can be judged by eye by focusing the image slightly above the bead; the bead image will grow larger as the bead moves away from the focal plane toward the surface.)

38| From the magnet initial rotational state, rotate the magnets again ten turns clockwise (in the direction of negative supercoiling). Bring the magnets back to the magnet initial rotational state.For a bead associated with a single supercoilable DNA molecule, the bead will not be observed to move toward the surface on generation of negative supercoiling, as under such levels of torsional strain DNA denatures and extends9,10.➨ TROUBLESHOOTING

39| Starting at the magnet initial rotational state, rotate the magnets ten turns counterclockwise (yielding positive supercoiling). Then, rotate the magnets by 20 turns clockwise, turn by turn, and track the DNA extension for at least 10 s after each turn. The position of the magnets at which DNA extension, l, is maximal is defined as the ‘rotational zero’ of the DNA molecule.Before the force calibration is initiated, it is necessary to find the rotational position of the magnets at which the DNA molecule contains no superhelical turns (rotational zero of the DNA; Fig. 1b). In most cases, the rotational zero of the DNA is within ±2 turns of the magnet initial rotational state.

40| Bring the DNA molecule to the rotational zero. Bring the magnets as close to the capillary as possible. Raise the magnets in 0.2-mm increments until they are 2 mm from the capillary. Determine the pulling force acting on the magnetic bead at each magnet position as described9. From the force calibration, determine the position of the magnets at which the pulling force acting on the bead is ∼0.3 pN.DNA nanomanipulation experiments addressing RNAP-dependent promoter unwinding should be performed at forces of ∼0.3 pN. Forces higher than ∼0.5 pN should be avoided as spontaneous denaturation of negatively supercoiled DNA occurs at such forces9,10 and as corresponding increases in DNA extension can be confused with RNAP-dependent changes in DNA extension.

Rapid selection of a single supercoilable

DNA

Calibration of the extension force acting on

the DNA molecule

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41| Bring the magnets as close as possible to the capillary. Pump at least 3 ml of transcription buffer containing 150 mM NaCl (150–200 µl/min) through the capillary. Set the temperature to 34 °C during the wash.

42| Set the magnetic force to 0.3 pN by bringing the magnets to the appropriate position, as determined in Step 40. Determine the new rotational zero of the DNA as described in Step 38. The rotational zero is salt- and temperature-dependent10; therefore, the rotational zero shifts by +1 to +2 superhelical turns upon transition from SB at 25 °C to transcription buffer at 34 °C.

43|Find the range of positive and negative supercoiling over which DNA extension changes linearly with number of negative or positive superhelical turns. For a 2-kb DNA molecule, the linear range is usually between three and seven superhelical turns, with a change in l (δ) of 56 ± 5 nm per superhelical turn (Fig. 1b).

44| Rotate the magnets four to five turns clockwise (in the direction of negative supercoiling, σ = –0.021 to –0.026) and monitor DNA extension for at least 20 min. No large spontaneous changes in the DNA extension should be observed. The Brownian fluctuations in the DNA extension should show a standard deviation of ∼40 nm. Return the DNA molecule to the rotational zero.➨ TROUBLESHOOTING

45| To detect promoter unwinding on positively supercoiled DNA, rotate the magnets five turns counterclockwise (in the direction of positive supercoiling, σ = 0.021 to 0.026). Initiate monitoring of DNA extension. Prepare 1 ml of 1 nM RNAP in transcription buffer containing 150 mM NaCl, and, using a pipet, inject 100 µl of RNAP solution into the input reservoir. Immediately withdraw 100 µl from the output reservoir. Repeat the injection-withdrawal cycle at least five times (about 500 µl of RNAP solution is required to equilibrate RNAP concentration in the capillary). A promoter unwinding event on a positively supercoiled DNA results in an abrupt decrease in the DNA extension by 40–80 nm.Under the given conditions (N25 promoter, 1 nM RNAP, 150 mM NaCl, five positive superhelical turns, 0.3 pN extending force, and 34 °C), the expected waiting time for promoter unwinding, Twait, is ∼1 min, and the expected lifetime of the unwound complex, Tunwound, is ∼1 min (Fig. 3a and ref. 7).➨ TROUBLESHOOTING▲CRITICAL STEP

46| To detect promoter unwinding on negatively supercoiled DNA, rotate the magnets five turns counterclockwise (in the direction of positive supercoiling). Prepare 1 ml of 0.1 nM RNAP in transcription buffer containing 150 mM NaCl, and inject at least 500 µl of RNAP solution into the system. Initiate monitoring of DNA extension. After ∼1 min of monitoring (no promoter unwinding events are expected within ∼1 min, as Twait is >10 min under these conditions), rotate the magnets by ten turns clockwise (in the direction of negative supercoiling) and continue monitoring of DNA extension. A promoter unwinding event on a negatively supercoiled DNA results in an abrupt increase in the DNA extension by 40–80 nm.Under the given conditions (N25 promoter, 0.1 nM RNAP, 150 mM NaCl, five negative superhelical turns, 0.3 pN extending force, and 34 °C), the expected waiting time for promoter unwinding, Twait, is ∼1 min, and the expected lifetime of the unwound complex, Tunwound, is >1 h (Fig. 3b and ref. 7).▲CRITICAL STEP

Detection of promoter unwinding events by RNAP

Table 2 Detection of RNAP-dependent promoter unwinding: improved resolution through use of shorter DNA fragment (N = 35; averaged data, 0.2-s window).

4-kb supercoilable DNA segment 2-kb DNA supercoilable DNA segment Mean ± s.e.m. Mean ± s.e.m.

∆lobs,pos 61 16 55 7

∆lobs,neg 53 16 52 7

Unwinding (bp) 11 2 10 1Compaction (nm) 4 11 2 5

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47| Initiate monitoring of DNA extension and detect promoter unwinding on negatively supercoiled DNA as described in Step 45 (0.1 nM RNAP, 150 mM NaCl, five negative superhelical turns, 0.3 pN extending force and 34 °C). Monitor the DNA extension in the unwound state for at least 1 min. Without stopping data acquisition, rotate the magnets by ten turns counterclockwise, to introduce five positive superhelical turns into the DNA. As the expected Tunwound under given conditions is ∼1 min (Step 45), a promoter rewinding event should be observed in ∼1 min. Monitor the DNA extension in the rewound state for at least 1 min. Without stopping data acquisition, rotate the magnets by ten turns clockwise to introduce five negative superhelical turns into the DNA. Monitor the DNA extension until a promoter unwinding event is observed and then monitor the DNA extension in the unwound state for an additional 1 min. Repeat the above procedure until a statistically significant number of promoter unwinding events (observed with negatively supercoiled DNA) and promoter rewinding events (observed with positively supercoiled DNA) is observed (at least 25 pairs of events).

Determination of the extent of DNA unwinding

and the extent of DNA compaction

a

b

Figure 3 | Detection of RNAP-dependent promoter unwinding: improved resolution through use of shorter DNA fragment. (a) Single-molecule traces of DNA extension versus time and histograms of observed changes in DNA extension for interaction of RNAP with the bacteriophage T5 N25 promoter, as assessed using positively supercoiled DNA (σ = 0.026, reversible promoter unwinding). Top row, data obtained with a 4-kb supercoilable DNA segment; bottom row, data obtained with a 2-kb supercoilable DNA segment. Green points, raw data obtained at a video rate of 30 frames per s; red points, averaged data (5-s window). (b) Single-molecule traces of DNA extension versus time and histograms of observed changes in DNA extension for interaction of RNAP with the bacteriophage T5 N25 promoter, as assessed using negatively supercoiled DNA (σ = –0.026, irreversible promoter unwinding). Top row, data obtained with a 4-kb supercoilable DNA segment; bottom row, data obtained with a 2-kb supercoilable DNA segment.

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Deconvolution of DNA unwinding (arising from RNAP-dependent promoter unwinding) and DNA compaction (arising from RNAP-dependent DNA wrapping and/or DNA bending) requires measurements on both positively supercoiled DNA and negatively supercoiled DNA6,7. DNA unwinding has opposite effects on DNA extension, l, in experiments with positively and negatively supercoiled DNA (increases l with positively supercoiled DNA; decreases l with negatively supercoiled DNA), whereas DNA compaction has equivalent effects on l in experiments with positively and negatively supercoiled DNA (decreases l with both positively and negatively supercoiled DNA). Therefore, effects of unwinding and compaction can be deconvoluted from data with positively and negatively supercoiled DNA by use of simple algebra.The procedure described allows large numbers of events on negatively and positively supercoiled DNA to be recorded in relatively short periods of time (with alternation between negatively and positively supercoiled DNA serving to recycle the DNA molecule between events and, in particular, to drive reversal of long-lived unwinding events on negatively supercoiled DNA). The procedure can be automated if prior knowledge is available concerning Twait on negatively supercoiled DNA and Tunwound on positively supercoiled DNA .➨ TROUBLESHOOTING

48| Examine the DNA-extension data. Visually identify promoter unwinding events under negative supercoiling (abrupt increases in the DNA extension by 40–80 nm) and promoter rewinding events under positive supercoiling (abrupt increases in the DNA extension by 40–80 nm). Pool the promoter unwinding events (observed with negative supercoiling) and promoter rewinding events (observed with positive supercoiling) into two sets. For each event in the two sets:

(i) Measure DNA extension before the event by averaging bead position over 5 s (discarding data obtained in the 2 s immediately preceding the event).

(ii) Measure DNA extension after the event by averaging bead position over 5 s (discarding data obtained in the 2 s immediately after the event)

(iii) Calculate the difference in DNA extension before and after the event.Construct histograms for events observed with negative supercoiling and for events observed with positive supercoiling, and determine mean amplitudes of events observed with negative supercoiling (∆lobs,neg) and events observed with positive supercoiling (∆lobs,pos).

49| Calculate the change in DNA extension attributable to DNA unwinding (∆lu) and the change in DNA extension attributable to DNA compaction arising from wrapping and/or bending (∆lc) as ∆lu = (∆lobs,pos + ∆lobs,neg) / 2 and ∆lc = (∆lobs,pos – ∆lobs,neg) / 2. Calculate the extent of unwinding (∆Tw) as ∆Tw = ∆lu / δ, where δ is the contour length of a plectonemic supercoil, determined as the change in DNA extension per change in number of superhelical turns in the linear region of Figure 1b (∼56 nm under the conditions here)7. Calculate the number of unwound base pairs by multiplying ∆Tw by 10.4, the number of base pairs per helical turn of DNA7. (See discussion in refs. 6 and 7.)

TROUBLESHOOTING TABLEPROBLEM SOLUTION

Step 21 The polystyrene layer appears to peel off the inner surface of the capillary.

If the polystyrene layer frequently becomes unstable and peels off, prepare a fresh solution of polystyrene with toluene and repeat the coating step.

Step 24 The capillary seems to be contaminated. Be sure to use sterile technique during the entire procedure. It is important to prevent bacterial contamination of the capillary during the course of its preparation and storage.

Step 34 The beads all appear immobile, that is, seem attached to the surface of the capillary.

Reblock the surface by incubating with BSA and polyglutamic acid for 1–2 h as described in Step 24. Other possible treatments for reducing nonspecific interactions include exposing the surface for 5–10 min either to a 1 M solution of sodium azide or to a 10% solution of SDS; the surface can also be exposed to a 10 mg/ml solution of polyglutamic acid in PBS.

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PROBLEM SOLUTION

Step 38 After clockwise rotation of the magnets (in the direction of negative supercoiling) with the magnets positioned as close to the sample as possible, the bead seems to move towards the surface.

The bead probably is tethered to the surface by two DNA molecules. Another bead, not showing this behavior, should be located in the capillary.

Step 44 In the absence of RNAP, at negative supercoiling, short-lived increases (lifetime ∼1 min) in DNA extension are observed.

Spontaneous changes in the extension of negatively supercoiled DNA arise from spontaneous denaturation of the DNA molecule at a high pulling force. Verify the calculations performed during the force calibration. Decrease the force by moving magnets away from the capillary in 0.5-mm increments. Monitor extension of negatively supercoiled DNA for at least 10 min at each magnet position to confirm that no further spontaneous denaturation events are observed.

High noise level in the absence of RNAP (standard deviation of DNA extension fluctuations >40 nm).

Increase the force by moving magnets toward the capillary in 0.5-mm increments. Monitor the extension of negatively supercoiled DNA for at least 1 min at each magnet position to verify that the noise level is within acceptable range. Alternatively, decrease the number of supercoils in increments of one turn. If this does not solve the problem, discard bead and repeat Steps 36–44.

Step 45 High noise in the presence of RNAP (standard deviation of DNA extension fluctuations > 40 nm), very short Twait (<10 s), or both.

Decrease RNAP concentration in 50% increments.

No unwinding events in the presence of RNAP, or very long Twait (>10 min).

Increase RNAP concentration in 50% increments. Check that the temperature has been set to 34 °C.

Very short Tunwound (<10 s). For positively supercoiled DNA, decrease force by moving the magnets away in 0.5-mm increments. For negatively supercoiled DNA, increase force by moving the magnets toward the capillary in 0.5-mm increments. Alternatively, decrease the concentration of NaCl in the transcription buffer in 25-mM increments.

Very long Tunwound (>10 min).Unless analysis of Tunwound is within the scope of work, long Tunwound should be avoided, since the time required to collect a significant number of promoter events increases proportionally to Tunwound.

For positively supercoiled DNA, increase force by moving the magnets toward the capillary in 0.5-mm increments. For negatively supercoiled DNA, decrease force by moving the magnets away in 0.5-mm increments. Alternatively, increase the NaCl concentration in 25-mM increments.

Magnetic bead becomes stuck to the surface during injections of reagents or data acquisition (observed as an abrupt decrease of the DNA extension to zero).

From the current rotational position of the magnets, rotate the magnets by one turn clockwise, then by one turn counterclockwise. If the DNA extension is not recovered, bring the magnets as close to the capillary as possible and repeat the rotation procedure. If the DNA extension still is not recovered, inject 200 µl polyglutamic acid in PBS (10 mg/ml) and repeat the rotation procedure. In nearly all cases, DNA extension can be recovered with this procedure. Once the DNA extension has been recovered, the rotational zero of the DNA must be determined again as described in Step 39. If the bead sticks to the surface frequently, incubate the system in the polyglutamic acid solution for at least 1 h.

Step 47 Light level decreases during data acquisition or fluctuates during magnet rotation, causing the bead tracking routine to lose track of the bead.

Raise magnets, rinse out free magnetic beads accumulated next to ceiling of capillary and return magnets to original position.

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NATURE METHODS | VOL.2 NO.2 | FEBRUARY 2005 | 137

PROTOCOL

CRITICAL STEPSStep 1 The target 2-kb DNA fragment should contain a centrally located promoter and should be otherwise G/C-rich (to avoid nonspecific melting of A/T-rich regions). For this work, the bacteriophage T5 N25 promoter was amplified from plasmid pSAN25 (gift of S. Adhya, National Institutes of Health) using the add-on PCR primers 5′-GAGAGAGGTACCGGTTGAATGTTGCGCGGTCAG-3′ and 5′-GAGAGAGGTACCGTTGTTCCGTGTCAGTGGTG-3′. The resulting 94 base pair DNA fragment (spanning positions –78 to +16, where +1 is the transcription start site) was then cloned into the unique KpnI site of the Thermus aquaticus rpoC gene (located at position 2889 downstream of the rpoC ORF)11. Analogous procedures can be used to prepare constructs for study of other DNA target sites.

Step 5 Use gel electrophoresis to verify that the expected 2-kb band is the dominant product of the amplification step.

Steps 7 and 15 Minimize the exposure time to the UV light to prevent nicking of the DNA.

Step 17 To reduce multimerization of the target 2-kb DNA, use at least a twofold excess of labeled DNA.

Steps 20–24 Glass surfaces should be cleaned, coated with polystyrene to allow adsorption of and functionalization by anti-digoxigenin, and blocked to reduce nonspecific interactions.

Step 25 Tosyl-activated beads serve as a position reference to correct for mechanical drift of the surface during experiments.

Step 35 It is important to rinse out as many unbound beads as possible, because otherwise they will tend to aggregate just under the magnets, blocking the light path and preventing measurements.

Step 36 Beads that are partially stuck on the surface will show a ‘rocking’ behavior, with an anisotropic Brownian motion oriented perpendicular to the direction of the magnetic field.

Steps 45 and 46 Unless the objective of the experiment is analysis of Twait and Tunwound, Twait and Tunwound >>1 min should be avoided, as the time required for collection of significant number of promoter unwinding events scales with Twait and Tunwound.

COMMENTSIn Figure 3, we compare detection of RNAP-dependent promoter unwinding using a 4-kb supercoilable DNA segment (as in previous work6,7) and using a 2-kb supercoilable DNA segment (as in this work). Inspection of the time traces in Figure 3, left panels, establishes that use of the shorter supercoilable DNA segment results in a significant reduction in noise: the standard deviation and time scale for DNA extension fluctuations in raw data are ~80 nm and 0.25 s using a 4-kb supercoilable DNA segment, but only ~40 nm and 0.13 s using a 2-kb supercoilable DNA segment. Inspection of the histograms of observed changes in DNA extension in Figure 3, right panels, and the error estimates in Table 2 establishes that, correspondingly, use of the shorter supercoilable DNA segment results in a significant reduction in distribution widths: the standard errors of mean values of ∆lobs,neg and ∆lobs,pos with 0.2-s averaging are ~16 nm using a 4-kb supercoilable DNA segment but only ~7 nm using a 2-kb supercoilable DNA segment (N = 35). As a result, when using the 2-kb supercoilable DNA segment, DNA unwinding can be determined to within ±1 bp, and DNA compaction can be determined to within ±5 nm, from real-time data averaged over a ~0.2-s window. The improved spatial and temporal resolution permits single base-pair, or near single-base-pair resolution, detection of RNAP-dependent changes in DNA unwinding and compaction on the 0.2-s time scale—a time scale relevant to key reaction steps in transcription, including promoter unwinding, promoter escape, transcription elongation and transcription termination.

ACKNOWLEDGMENTSWe thank S. Adhya for plasmid samples. This work was supported by funds from the Institut Jacques Monod, an Action Thématique et Incitative sur Programme grant from the Centre National de la Recherche Scientifique, the Universities of Paris VI and Paris VII, the Fondation pour la Recherche Médicale, and a Cold Spring Harbor Laboratory Fellowship to T.R.S., and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

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138 | VOL.2 NO.2 | FEBRUARY 2005 | NATURE METHODS

PROTOCOL

1. Strick, T.R., Croquette, V. & Bensimon, D. Single-molecule analysis of DNA uncoiling by a type II topoisomerase. Nature 404, 901–904 (2000).

2. Crisona, N.J., Strick, T.R., Bensimon, D., Croquette, V. & Cozzarelli, N.R. Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements. Genes Dev. 14, 2881–2892 (2000).

3. Stone, M.D. et al. Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases. Proc. Natl. Acad. Sci. USA 100, 8654–8659 (2003).

4. Dekker, N.H. et al. The mechanism of type IA topoisomerases. Proc. Natl. Acad. Sci. USA 99, 12126–12131 (2002).

5. Charvin, G., Bensimon, D. & Croquette, V. Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases. Proc. Natl. Acad. Sci. USA 100, 9820–9825 (2003).

6. Revyakin, A., Allemand, J., Croquette, V., Ebright, R.H.

& Strick, T.R. Single-molecule DNA nanomanipulation: detection of promoter unwinding events by RNA polymerase. Methods in Enzymology 370, 577–598 (2003).

7. Revyakin, A., Ebright, R.H. & Strick, T.R. Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation. Proc. Natl. Acad. Sci. USA 101, 4776–4780 (2004).

8. White, J.H. Self linking and the Gauss integral at higher dimensions. Am. J. Math. 91, 693–728 (1969).

9. Strick, T.R., Allemand, J.F., Bensimon, D. & Croquette, V. Behavior of supercoiled DNA. Biophys. J. 74, 2016–2028 (1998).

10. Strick, T.R., Croquette, V. & Bensimon, D. Homologous pairing in stretched supercoiled DNA. Proc. Natl. Acad. Sci. USA 95, 10579–10583 (1998).

11. Minakhin, L., Nechaev, S., Campbell, E.A. & Severinov, K. Recombinant Thermus aquaticus RNA polymerase, a new tool for structure-based analysis of transcription. J. Bacteriol. 183, 71–76 (2001).

SOURCEThis protocol was provided directly by the authors listed on the title page. For further details on construction of recombinant DNA molecules and standard cloning procedures, see Sambrook, J. & Russell, D.W., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

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