Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA.

Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions.

Improved Free-Energy Landscape Quantification Illustrated with a Computationally Designed Protein-Ligand Interaction.

Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (koff ) and the distance to the transition state (Δx≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG≠ =6.3±0.2 kcal mol-1 ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [koff (=4±0.1×10-4  s-1 ) and Δx≠ (=8.3±0.1 Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay.

Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3'-to-5' DNA helicase.

A Surface-Coupled Optical Trap with 1-bp Precision via Active Stabilization.

Optical traps can measure bead motions with Å-scale precision. However, using this level of precision to infer 1-bp motion of molecular motors along DNA is difficult, since a variety of noise sources degrade instrumental stability. In this chapter, we detail how to improve instrumental stability by (1) minimizing laser pointing, mode, polarization, and intensity noise using an acousto-optical-modulator mediated feedback loop and (2) minimizing sample motion relative to the optical trap using a three-axis piezo-electric-stage mediated feedback loop. These active techniques play a critical role in achieving a surface stability of 1 Å in 3D over tens of seconds and a 1-bp stability and precision in a surface-coupled optical trap over a broad bandwidth (Δf = 0.03-2 Hz) at low force (6 pN). These active stabilization techniques can also aid other biophysical assays that would benefit from improved laser stability and/or Å-scale sample stability, such as atomic force microscopy and super-resolution imaging.