Biochemical and Biophysical Research Communications
Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains
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,
References (36)
Single-molecule approaches to characterizing kinetics of biomolecular interactions
Current Opinion in Biotechnology
(2011)- et al.
Force fluorescence spectroscopy at the single-molecule level, methods in enzymology
Vol 475: Single Molecule Tools Pt B
(2010) - et al.
Interaction of oxazole yellow dyes with DNA studied with hybrid optical tweezers and fluorescence microscopy
Biophysical Journal
(2009) - et al.
DNA curtains for high-throughput single-molecule optical imaging, methods in enzymology
Vol 472: Single Molecule Tools Pt A: Fluorescence Based Approaches
(2010) - et al.
Salt dependence of the elasticity and overstretching transition of single DNA molecules
Biophysical Journal
(2002) Single-molecule studies of viral DNA packing
Current Opinion in Virology
(2011)- et al.
Adhesion forces of lipids in a phospholipid membrane studied by molecular dynamics simulations
Biophysical Journal
(1998) - et al.
Detachment of agglutinin-bonded red blood cells. II. Mechanical energies to separate large contact areas
Biophysical Journal
(1991) - et al.
Biological mechanisms, one molecule at a time
Genes & Development
(2011) - et al.
Single-molecule approach to molecular biology in living bacterial cells
Annual Review of Biophysics
(2008)
Advances in single-molecule fluorescence methods for molecular biology
Annual Review of Biochemistry
Forcing a connection: impacts of single-molecule force spectroscopy on in vivo tension sensing
Biopolymers
Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy
Nature Methods
Stress-induced structural transitions in DNA and proteins
Annual Review of Biophysics and Biomolecular Structure
Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region
Nucleic Acids Research
Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers
Biophysical Journal
Parallel arrays of geometric nanowells for assembling curtains of DNA with controlled lateral dispersion
Langmuir
Cited by (0)
- 1
Present address: Columbia Technology Ventures, 630 West 168th Street, New York, NY 10032, USA.