Weak tension accelerates hybridization and dehybridization of short oligonucleotides.

The hybridization and dehybridization of DNA subject to tension is relevant to fundamental genetic processes and to the design of DNA-based mechanobiology assays. While strong tension accelerates DNA melting and decelerates DNA annealing, the effects of tension weaker than 5 pN are less clear. In this study, we developed a DNA bow assay, which uses the bending rigidity of double-stranded DNA (dsDNA) to exert weak tension on a single-stranded DNA (ssDNA) target in the range of 2-6 pN. Combining this assay with single-molecule FRET, we measured the hybridization and dehybridization kinetics between a 15 nt ssDNA under tension and a 8-9 nt oligonucleotide, and found that both the hybridization and dehybridization rates monotonically increase with tension for various nucleotide sequences tested. These findings suggest that the nucleated duplex in its transition state is more extended than the pure dsDNA or ssDNA counterpart. Based on coarse-grained oxDNA simulations, we propose that this increased extension of the transition state is due to steric repulsion between the unpaired ssDNA segments in close proximity to one another. Using linear force-extension relations verified by simulations of short DNA segments, we derived analytical equations for force-to-rate conversion that are in good agreement with our measurements.

Determinants of cyclization-decyclization kinetics of short DNA with sticky ends.

Cyclization of DNA with sticky ends is commonly used to measure DNA bendability as a function of length and sequence, but how its kinetics depend on the rotational positioning of the sticky ends around the helical axis is less clear. Here, we measured cyclization (looping) and decyclization (unlooping) rates (kloop and kunloop) of DNA with sticky ends over three helical periods (100-130 bp) using single-molecule fluorescence resonance energy transfer (FRET). kloop showed a nontrivial undulation as a function of DNA length whereas kunloop showed a clear oscillation with a period close to the helical turn of DNA (∼10.5 bp). The oscillation of kunloop was almost completely suppressed in the presence of gaps around the sticky ends. We explain these findings by modeling double-helical DNA as a twisted wormlike chain with a finite width, intrinsic curvature, and stacking interaction between the end base pairs. We also discuss technical issues for converting the FRET-based cyclization/decyclization rates to an equilibrium quantity known as the J factor that is widely used to characterize DNA bending mechanics.

First passage time study of DNA strand displacement.

DNA strand displacement, in which a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule fluorescence resonance energy transfer approach termed the "fission" assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nucleotide displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence, except for T → U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25-1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of âˆ¼30-300 µs, depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

ERASE: a novel surface reconditioning strategy for single-molecule experiments.

While surface-based single-molecule experiments have revolutionized our understanding of biology and biomolecules, the workflow in preparing for such experiments, especially surface cleaning and functionalization, remains labor-intensive and time-consuming. Even worse, meticulously assembled flow channels can be used only once for most experiments. A reusable surface would thus dramatically increase productivity and efficiency of single-molecule experiments. In this paper, we report a novel surface reconditioning strategy termed ERASE (Epitaxial Removal Aided by Strand Exchange) that allows a single flow cell to be used for vast repetition of single-molecule experiments. In this method, biomolecules immobilized to the surface through a nucleic acid duplex are liberated when a competing DNA strand disrupts the duplex via toehold-mediated strand displacement. We demonstrate the wide-range applicability of this method with various common surface preparation techniques, fluorescent dyes, and biomolecules including the bacterial ribosome. Beyond time and cost savings, we also show ERASE can assort molecules based on a nucleic acid barcode sequence, thus allowing experiments on different molecules in parallel. Our method increases the utility of prepared surfaces and is a significant improvement to the current single-use paradigm.

Base-Pair Mismatch Can Destabilize Small DNA Loops through Cooperative Kinking.

Base-pair mismatch can relieve mechanical stress in highly strained DNA molecules, but how it affects their kinetic stability is not known. Using single-molecule fluorescence resonance energy transfer, we measured the lifetimes of tightly bent DNA loops with and without base-pair mismatch. Surprisingly, for loops captured by stackable sticky ends which leave single-stranded DNA breaks (or nicks) upon annealing, the mismatch decreased the loop lifetime despite reducing the overall bending stress, and the decrease was largest when the mismatch was placed at the DNA midpoint. These findings suggest that base-pair mismatch increases bending stress at the opposite side of the loop through an allosteric mechanism known as cooperative kinking. Based on this mechanism, we present a three-state model that explains the apparent dichotomy between thermodynamic and kinetic stability.

mRNA detection in budding yeast with single fluorophores.

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

Single-molecule fluorescence studies on DNA looping.

Structure and dynamics of DNA impact how the genetic code is processed and maintained. In addition to its biological importance, DNA has been utilized as building blocks of various nanomachines and nanostructures. Thus, understanding the physical properties of DNA is of fundamental importance to basic sciences and engineering applications. DNA can undergo various physical changes. Among them, DNA looping is unique in that it can bring two distal sites together, and thus can be used to mediate interactions over long distances. In this paper, we introduce a FRET-based experimental tool to study DNA looping at the single molecule level. We explain the connection between experimental measurables and a theoretical concept known as the J factor with the intent of raising awareness of subtle theoretical details that should be considered when drawing conclusions. We also explore DNA looping-assisted protein diffusion mechanism called intersegmental transfer using protein induced fluorescence enhancement (PIFE). We present some preliminary results and future outlooks.

The Effect of Basepair Mismatch on DNA Strand Displacement.

DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single basepair mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data indicate that a single base pair mismatch in the invader stalls branch migration and displacement occurs via direct dissociation of the destabilized incumbent strand from the substrate strand. We combined both branch migration and direct dissociation into a model, which we term the concurrent displacement model, and used the first passage time approach to quantitatively explain the salient features of the observed relationship. We also introduce the concept of splitting probabilities to justify that the concurrent model can be simplified into a three-step sequential model in the presence of an invader mismatch. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality.

Probing the elastic limit of DNA bending.

Sharp bending of double-stranded DNA (dsDNA) plays an essential role in genome structure and function. However, the elastic limit of dsDNA bending remains controversial. Here, we measured the opening rates of small dsDNA loops with contour lengths ranging between 40 and 200 bp using single-molecule Fluorescence Resonance Energy Transfer. The relationship of loop lifetime to loop size revealed a critical transition in bending stress. Above the critical loop size, the loop lifetime changed with loop size in a manner consistent with elastic bending stress, but below it, became less sensitive to loop size, indicative of softened dsDNA. The critical loop size increased from ∼ 60 bp to ∼ 100 bp with the addition of 5 mM magnesium. We show that our result is in quantitative agreement with the kinkable worm-like chain model, and furthermore, can reproduce previously reported looping probabilities of dsDNA over the range between 50 and 200 bp. Our findings shed new light on the energetics of sharply bent dsDNA.

Measuring shape-dependent looping probability of DNA.

Recently, several studies have shown that short doubled-stranded DNA (dsDNA) loops more readily than the wormlike chain model predicts. In most of these experiments, the intrinsic bendedness of dsDNA, which in theory can dramatically influence looping dynamics, was either avoided or unaccounted for. To investigate the effect of the shape of dsDNA on looping dynamics, we characterized the shapes of several synthetic dsDNA molecules of equal length but different sequences using gel electrophoresis. We then measured their looping rates using a FRET (Förster resonance energy transfer)-based assay and extracted the looping probability density known as the J factor (jM). We also used, for comparison, several dinucleotide angular parameter sets derived from the observed electrophoretic mobility to compute the jM predicted by the wormlike chain model. Although we found a strong correlation between curvature and jM, the measured jM was higher than most dinucleotide model predictions. This result suggests that it is difficult to reconcile the looping probability with the observed gel mobility within the wormlike chain model and underscores the importance of determining the intrinsic shape of dsDNA for proper theoretical analysis.

Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome.

The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence-a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNA(Phe), in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNA(Phe), in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNA(Phe), and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.

Toward a 3D physical model of diffusive polymer chains.

Recent studies in polymer physics have created macro-scale analogs to solute microscopic polymer chains like DNA by inducing diffusive motion on a chain of beads. These bead chains have persistence lengths of O(10) links and undergo diffusive motion under random fluctuations like vibration. We present a bead chain model within a new stochastic forcing system: an air fluidizing bed of granular media. A chain of spherical 6 mm resin beads crimped onto silk thread are buffeted randomly by the multiphase flow of grains and low density rising air "bubbles". We "thermalize" bead chains of various lengths at different fluidizing airflow rates, while X-ray imaging captures a projection of the chains' dynamics within the media. With modern 3D printing techniques, we can better represent complex polymers by geometrically varying bead connections and their relative strength, e.g., mimicking the variable stiffness between adjacent nucleotide pairs of DNA. We also develop Discrete Element Method (DEM) simulations to study the 3D motion of the bead chain, where the bead chain is represented by simulated spherical particles connected by linear and angular spring-like bonds. In experiment, we find that the velocity distributions of the beads follow exponential distributions rather than the Gaussian distributions expected from polymers in solution. Through use of the DEM simulation, we find that this difference can likely be attributed to the distributions of the forces imparted onto the chain from the fluidized bed environment. We anticipate expanding this study in the future to explore a wide range of chain composition and confinement geometry, which will provide insights into the physics of large biopolymers.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

FISHing on a Budget.

Sensitive quantification of RNA transcripts via fluorescence in situ hybridization (FISH) is a ubiquitous part of understanding quantitative gene expression in single cells. Many techniques exist to identify and localize transcripts inside the cell, but often they are costly and labor intensive. Here we present a method to use a singly labeled short DNA oligo probe to perform FISH in yeast cells. This method is effective for highly constrained FISH applications where the target length is limited (<200 nucleotides). This method can quantify different RNA isoforms or enable the use of fluorescence resonance energy transfer (FRET) to detect co-transcription of neighboring sequence blocks. Since this method relies on a single probe, it is also more cost-effective than a multiple probe labeling strategy.

Coarse-Grained Simulations of DNA Reveal Angular Dependence of Sticky-End Binding.

Annealing between sticky ends of DNA is an intermediate step in ligation. It can also be utilized to program specific binding sites for DNA tile and origami assembly. This reaction is generally understood as a bimolecular reaction dictated by the local concentration of the sticky ends. Its dependence on the relative orientation between the sticky ends, however, is less understood. Here we report on the interactions between DNA sticky ends using the coarse-grained oxDNA model; specifically, we consider how the orientational alignment of the double-stranded DNA (dsDNA) segments affects the time required for the sticky ends to bind, τb. We specify the orientation of the dsDNA segments with three parameters: θ, which measures the angle between the helical axes, and ϕ1 and ϕ2, which measure rotations of each strand around the helical axis. We find that the binding time depends strongly on both θ and ϕ2: ∼20-fold change with θ and 10-fold change with ϕ2. The binding time is the fastest when the helical axes of duplexes are pointing toward each other and the sticky ends protrude from the farthest two points. Our result is relevant for predicting hybridization efficiency of sticky ends that are rotationally restricted.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Stability and nuclear localization of yeast telomerase depend on protein components of RNase P/MRP.

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.

tRNA selection and kinetic proofreading in translation.

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.

Dual-probe RNA FRET-FISH in Yeast.

mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be used to unambiguously ascertain identity of the transcript without the use of a guide probe set used in other FISH techniques.