Simultaneous, coincident optical trapping and single-molecule fluorescence

444

556

Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a highresolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6 -30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.DNA hairpin ͉ energy landscape ͉ force clamp ͉ optical tweezers ͉ single molecule H airpins formed from self-complementary sequences supply a model system for studying folding and duplex formation, the most fundamental processes for generating structure in nucleic acids. Using hairpins, repeated measurements can be made on the same molecule, facilitating single-molecule studies. Furthermore, by simply altering the nucleotide sequence, physical properties such as folding energies, kinetic rates, and distances to transition states all can be changed systematically. Hairpins also play essential roles in vivo. DNA hairpins bind proteins to regulate transcription (1), and hairpin intermediates are involved in both replication and recombination (2, 3). RNA hairpins form tertiary contacts (4), bind to proteins (2), regulate transcription (5), and mediate RNA interference (6). Understanding the factors that influence hairpin folding should therefore not only elucidate principles of structure formation in nucleic acids but may also shed light on the biological roles played by these structures.Extensive calorimetric and melting studies have been carried out to generate predictive rules for the thermodynamic stability of arbitrary nucleic acid duplexes (7). The kinetic properties of duplex formation, however, remain less well understood, particularly those related to the nature of the transition state. Temperature-jump studies of annealing in short duplexes have been interpreted in terms of the nucleation of a transition state consisting of Ϸ1-3 bp, followed by zippering of the remaining stem (8). This interpretation, however, rests on the assumption that the enthalpy of activation arises ...

Section: Resultssupporting

confidence: 90%

Section: Dna Hairpin Constructsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Woodside

Behnke-Parks²,

Larizadeh³

et al. 2006

444

556

“…We are encouraged that both the experimental and theoretical tools (see, for example, refs. [45][46][47] are becoming available to address these important problems in the near future.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamic and kinetic modeling of transcriptional pausing

Tadigotla

Maoiléidigh

Sengupta

et al. 2006

106

185

We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA–DNA, DNA–RNA, and RNA–RNA base pairing associated with ( i ) the translocational and size fluctuations of the transcription bubble; ( ii ) changes in the associated DNA–RNA hybrid; and ( iii ) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process.

“…For the majority of our stretching measurements, we designed heterobifunctional phages (termed B-H 6 ), displaying H 6 epitopes at remote tips and biotin molecules linked through selenocysteine (Sec) residues at proximal tips. Guided by the established protocols for SM dsDNA studies (19), a procedure for suspending B-H 6 phage in solution between antibody-functionalized coverslips and streptavidin-coated polystyrene microspheres was developed (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Single M13 bacteriophage tethering and stretching

Khalil

Ferrer

Brau

et al. 2007

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution. With this system, we report the development of a SM assay based on M13 bacteriophage. We also report the quantitative characterization of the biopolymer's elasticity by using an optical trap with nanometerscale position resolution. Expanding the fluctuating rod limit of the wormlike chain to incorporate enthalpic polymer stretching yielded a model capable of accurately capturing the full range of extensions. Fits of the force-extension measurements gave a mean persistence length of Ϸ1,265 nm, lending SM support for a shorter filamentous bacteriophage persistence length than previously thought. Furthermore, a predicted stretching modulus roughly two times that of dsDNA, coupled with the system's linkage versatility and load-bearing capability, makes the M13 template an attractive candidate for use in tethered bead architectures.optical tweezers ͉ single molecule ͉ wormlike chain T he Ff class of filamentous bacteriophage, composed of the structurally akin species f1, fd, and M13, has elicited the interest of many wide-ranging scientific communities because of its selfassembling nature. Protected and transported within the highly organized, protein-based capsid is the structural and assembly information necessary for its own production. This structural feature provides a direct and accessible link between phenotype and genotype, which particularly in the case of M13 bacteriophage, has proven advantageous for numerous studies and applications. For instance, combinatorial libraries of polypeptides can be fused to M13 coat proteins, in a technique known as phage display, as a means of screening binding candidates against targets (1). Recently, targets have been extended beyond biologicals to a wide variety of inorganics, in efforts to discover biological systems capable of organizing and growing materials (2). In addition to serving as the vehicle for displaying these ligands, the unique structure of M13 itself has been exploited as a biological template for nanotechnology, such as in the directed synthesis of semiconducting/magnetic nanowires and lithium ion battery electrodes (3-5). Considering its utility as both a genetic blueprint and structural backbone for materials and device architecture, a better understanding of its mechanical behavior and a novel means of actively assembling M13 can greatly advance the design of future M13-based materials.M13 bacteriophage is a high-production rate virus composed of five different, modifiable proteins, the vast majority...