Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

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Abstract

We report a new approach to probing DNA–protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin–streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65 pN; access to this higher force regime is sufficient to probe the responses of protein–DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.

Highlights

► A hybrid system to combine DNA curtains and optical trap is developed. ► The rupture force of a single lipid out of lipid bilayer is estimated. ► Multiple biotin/dig handles enables DNA to be extend to B–S transition region. ► Promoter-bound RNAP is disrupted by stretching DNA at physiological salt condition.

Introduction

Single molecule measurements have proven to be powerful tools that provide unique insights into the underlying mechanisms of biological phenomena, many of which cannot be revealed through traditional ensemble biochemical or biophysical approaches [1], [2]. Most single molecule techniques fit into two classes: those based upon the detection of a fluorescence signal [3], [4], and those that rely upon force-based measurements [5], [6], [7]. There is a growing interest in combining these two types of different measurements [8], [9], [10], [11], [12].

To facilitate single molecule measurements, we have developed “DNA curtains” which utilize lipid bilayers, nano-fabricated barriers, and hydrodynamic flow to organize lipid-tethered DNA molecules into patterns on the surface of a microfluidic chamber [13], [14], [15]. These molecules can be visualized by total internal reflection fluorescence microscopy (TIRFM), allowing simultaneous observation of hundreds of individual molecules within a single field-of-view, and this experimental platform can be adapted to a number of biochemical problems related to protein–nucleic acid interactions [16], [17], [18], [19].

Here we developed a TIRFM with an integrated optical trap, and using this combined TIRF-trap microscope we measured the rupture force of single lipids within a supported bilayer by pulling individual DNA molecules from DNA curtains. Optical tweezers have proven to be powerful tools applying precise forces (0.1–100 pN) on individual molecules and have been used to interrogate various biological processes [20], [21], [22], [23], [24]. We show that DNA molecules anchored to single lipids are pulled free from the bilayer with the application of ∼20 pN. Increasing the number of attachment points on the bilayer allows the application of forces in excess of ∼65 pN, expanding the applicability of DNA curtains to combined fluorescence and forced-based measurements with a force regime relevant to most protein–DNA interactions.

Section snippets

DNA substrates

For single biotin or digoxigenin (dig) tags, λ-DNA (48,502-base pairs (bp); Invitrogen) was labeled at either end with oligonucleotides, as described [16]. For multiple tags, multiple biotin or dig tags were incorporated into a 1.4-kilobase λ-DNA fragment by PCR using low-fidelity Taq polymerase (Stratagene or Takara) along with biotin-dUTP (Roche) or dig-dUTP (Roche), respectively. The PCR products were then ligated to connector oligonucleotides, bearing a sequence complementary to the 4-nt

DNA molecules labeled with single tags detached from the bilayer at low force

To make force measurements within DNA curtains we constructed a TIRF microscope with an integrated infrared optical trap (Fig. 1A and B). An optical trap could be used to stretch a single DNA by attaching a fluorescent bead to one end of DNA (Fig. 1A). Initial experiments utilized substrates labeled with single biotin and digoxigenin tags (Fig. 2). When the DNA molecules were stretched under hydrodynamic flow, those molecules harboring an anti-dig bead were readily distinguished by the bright

Acknowledgments

This research was funded in part by the Initiatives in Science and Engineering (ISE; awarded to E.C.G. and S.W.) program through Columbia University, and by NIH grants (GM074739 and GM 082848) to E.C.G. In addition, E.C.G. is an Early Career Scientist with the Howard Hughes Medical Institute. This work was partially supported by the Nanoscale Science and Engineering Initiative of the National Science Foundation under NSF Award No. CHE-0641523 and by the New York State Office of Science,

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    Present address: Columbia Technology Ventures, 630 West 168th Street, New York, NY 10032, USA.

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