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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Single-probe RNA FISH in Yeast.

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

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.

Force distribution in a semiflexible loop.

Loops undergoing thermal fluctuations are prevalent in nature. Ringlike or cross-linked polymers, cyclic macromolecules, and protein-mediated DNA loops all belong to this category. Stability of these molecules are generally described in terms of free energy, an average quantity, but it may also be impacted by local fluctuating forces acting within these systems. The full distribution of these forces can thus give us insights into mechanochemistry beyond the predictive capability of thermodynamics. In this paper, we study the force exerted by an inextensible semiflexible polymer constrained in a looped state. By using a simulation method termed "phase-space sampling," we generate the equilibrium distribution of chain conformations in both position and momentum space. We compute the constraint forces between the two ends of the loop in this chain ensemble using Lagrangian mechanics, and show that the mean of these forces is equal to the thermodynamic force. By analyzing kinetic and potential contributions to the forces, we find that the mean force acts in the direction of increasing extension not because of bending stress, but in spite of it. Furthermore, we obtain a distribution of constraint forces as a function of chain length, extension, and stiffness. Notably, increasing contour length decreases the average force, but the additional freedom allows fluctuations in the constraint force to increase. The force distribution is asymmetric and falls off less sharply than a Gaussian distribution. Our work exemplifies a system where large-amplitude fluctuations occur in a way unforeseen by a purely thermodynamic framework, and offers computational tools useful for efficient, unbiased simulation of a constrained system.

DNA Scrunching in the Packaging of Viral Genomes.

The motors that drive double-stranded DNA (dsDNA) genomes into viral capsids are among the strongest of all biological motors for which forces have been measured, but it is not known how they generate force. We previously proposed that the DNA is not a passive substrate but that it plays an active role in force generation. This "scrunchworm hypothesis" holds that the motor proteins repeatedly dehydrate and rehydrate the DNA, which then undergoes cyclic shortening and lengthening motions. These are captured by a coupled protein-DNA grip-and-release cycle to rectify the motion and translocate the DNA into the capsid. In this study, we examined the interactions of dsDNA with the dodecameric connector protein of bacteriophage ϕ29, using molecular dynamics simulations on four different DNA sequences, starting from two different conformations (A-DNA and B-DNA). In all four simulations starting with the protein equilibrated with A-DNA in the channel, we observed transitions to a common, metastable, highly scrunched conformation, designated A*. This conformation is very similar to one recently reported by Kumar and Grubmüller in much longer MD simulations on B-DNA docked into the ϕ29 connector. These results are significant for four reasons. First, the scrunched conformations occur spontaneously, without requiring lever-like protein motions often believed to be necessary for DNA translocation. Second, the transition takes place within the connector, providing the location of the putative "dehydrator". Third, the protein has more contacts with one strand of the DNA than with the other; the former was identified in single-molecule laser tweezer experiments as the "load-bearing strand". Finally, the spontaneity of the DNA-protein interaction suggests that it may play a role in the initial docking of DNA in motors like that of T4 that can load and package any sequence.

Transitions of Double-Stranded DNA Between the A- and B-Forms.

The structure of double-stranded DNA (dsDNA) is sensitive to solvent conditions. In solution, B-DNA is the favored conformation under physiological conditions, while A-DNA is the form found under low water activity. The A-form is induced locally in some protein-DNA complexes, and repeated transitions between the B- and A-forms have been proposed to generate the forces used to drive dsDNA into viral capsids during genome packaging. Here, we report analyses on previous molecular dynamics (MD) simulations on B-DNA, along with new MD simulations on the transition from A-DNA to B-DNA in solution. We introduce the A-B Index (ABI), a new metric along the A-B continuum, to quantify our results. When A-DNA is placed in an equilibrated solution at physiological ionic strength, there is no energy barrier to the transition to the B-form, which begins within about 1 ns. The transition is essentially complete within 5 ns, although occasionally a stretch of a few base pairs will remain A-like for up to ∼10 ns. A comparison of four sequences with a range of predicted A-phobicities shows that more A-phobic sequences make the transition more rapidly than less A-phobic sequences. Simulations on dsDNA with a region of roughly one turn locked in the A-form allow us to characterize the A/B junction, which has an average bend angle of 20-30°. Fluctuations in this angle occur with characteristic times of about 10 ns.

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.

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.