Single Hairpins Research Articles

Hierarchical Mechanical Transduction of Precision-Engineered DNA Hydrogels with Sacrificial Bonds.

Engineering the response to external signals in mechanically switchable hydrogels is important to promote smart materials applications. However, comparably little attention has focused on embedded precision mechanisms for autonomous nonlinear response in mechanical profiles in hydrogels, and we lack understanding of how the behavior from the molecular scale transduces to the macroscale. Here, we design a nonlinear stress-strain response into hydrogels by engineering sacrificial DNA hairpin loops into model network hydrogels formed from star-shaped building blocks. We characterize the force-extension response of single DNA hairpins and are able to describe how the specific topology influences the nonlinear mechanical behavior at different length scales. For this purpose, we utilize force spectroscopy as well as microscopic and macroscopic deformation tests. This study contributes to a better understanding of designing nonlinear strain-adaptive features into hydrogel materials.

INTERPIN: A repository for intrinsic transcription termination hairpins in bacteria

The large-scale detection of putative intrinsic transcription terminators is limited to only a few bacteria currently. We discovered a group of hairpins, called cluster hairpins, present within 15 nucleotides from each other. These are expected to work in tandem to cause intrinsic transcription termination (ITT), while the single hairpin can do the same alone. Therefore, exploring these ITT sites and the hairpins across bacterial genomes becomes highly desirable. INTERPIN is the largest archived collection of in silico inferred ITT hairpins in bacteria, covering 12745 bacterial genomes and encompassing ten bacterial phyla for ∼25 million hairpins. Users can obtain details on operons, individual cluster, and single ITT hairpins that were screened therein. Integrated Genome Viewer (IGV) software interactively visualizes hairpin secondary and tertiary structures in the genomic context. We also discuss statistics for the occurrence of cluster or single hairpins and other termination alternatives while showing the validation of predicted hairpins against in vivo detected hairpins. The database is freely available at http://pallab.cds.iisc.ac.in/INTERPIN/.INTERPIN (database and software) can make predictions for both AT and GC-rich genomes, which has not been achieved by any other program so far. It can also be used to improve genome annotation as well as to get predictions to improve the understanding of the ITT pathway by further analysis.

Experimental Test of Sinai's Model in DNA Unzipping.

The experimental measurement of correlation functions and critical exponents in disordered systems is key to testing renormalization group (RG) predictions. We mechanically unzip single DNA hairpins with optical tweezers, an experimental realization of the diffusive motion of a particle in a one-dimensional random force field, known as the Sinai model. We measure the unzipping forces F_{w} as a function of the trap position w in equilibrium and calculate the force-force correlator Δ_{m}(w), its amplitude, and correlation length, finding agreement with theoretical predictions. We study the universal scaling properties since the effective trap stiffness m^{2} decreases upon unzipping. Fluctuations of the position of the base pair at the unzipping junction u scales as u∼m^{-ζ}, with a roughness exponent ζ=1.34±0.06, in agreement with the analytical prediction ζ=4/3. Our study provides a single-molecule test of the functional RG approach for disordered elastic systems in equilibrium.

Three-dimensional flow field measurements in the wake of a tethered sphere crossing the onset of vortex induced vibrations

The changing three-dimensional vortex shedding topology of a heavy, tethered sphere exposed to uniform flow crossing the onset of vortex induced vibrations (VIV) is reported together with simultaneously tracked sphere positions. Transient upstream flow conditions were imposed by increasing the reduced velocity from $U^{*} = 2.2$ to 4.5 (‘lock-in’, mode I). Three-dimensional vortex shedding under these transient conditions strongly resembled those at steady conditions at the same $U^{*}$ . Changes in the wake accompanying the onset of VIV indicated several well-defined stages associated with the changing instantaneous location of the velocity deficit centroid and the wake's symmetry plane's orientation. Before the onset of VIV, the appearance of induced vortices led to lock-in of streamwise sphere oscillations prior to lock-in in the transverse direction. Shortly after ( $U^{*} \approx 3.6$ ), preferential vortex shedding (wake symmetry plane aligned with tether) was lost. Upon reaching $U^{*} = 4.5$ , flow–structure interaction reorganised vortex shedding and sphere motion, resulting in steady state conditions after some delay. At this stage, the symmetry plane was aligned perpendicular to the tether and pairs of alternately shed, single hairpins exerted transverse forcing on the sphere. At $U^{*} = 7.2$ (mode II, steady upstream flow), pairs of double hairpins were shed per oscillation period with maximum instantaneous vortex force coefficients that were higher than at $U^{*} = 4.5$ . While the present Reynolds numbers ( $Re$ ) were chosen low, the different identified stages in the onset of VIV are also expected to be relevant at a higher $Re$ range.

Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy-landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

Clusters of hairpins induce intrinsic transcription termination in bacteria

Intrinsic transcription termination (ITT) sites are currently identified by locating single and double-adjacent RNA hairpins downstream of the stop codon. ITTs for a limited number of genes/operons in only a few bacterial genomes are currently known. This lack of coverage is a lacuna in the existing ITT inference methods. We have studied the inter-operon regions of 13 genomes covering all major phyla in bacteria, for which good quality public RNA-seq data exist. We identify ITT sites in 87% of cases by predicting hairpin(s) and validate against 81% of cases for which the RNA-seq derived sites could be calculated. We identify 72% of these sites correctly, with 98% of them located ≤ 80 bases downstream of the stop codon. The predicted hairpins form a cluster (when present < 15 bases) in two-thirds of the cases, the remaining being single hairpins. The largest number of clusters is formed by two hairpins, and the occurrence decreases exponentially with an increasing number of hairpins in the cluster. Our study reveals that hairpins form an effective ITT unit when they act in concert in a cluster. Their pervasiveness along with single hairpin terminators corroborates a wider utilization of ITT mechanisms for transcription control across bacteria.

Abstract B11: Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila

Abstract Colorectal cancer (CRC) is an extremely complex and genetically diverse disease with few available models that recapitulate the key features of heterogeneity. The Cancer Genome Atlas has identified 30 recurrent mutated genes as drivers of CRC. Individual tumors can carry any number of combinations of these mutations. A critical challenge in research has been the ability to model these genetically heterogenous tumors in animal systems. Highly sophisticated genetic tools in Drosophila make it an ideal model system to create and study different genome-based CRC tumors that well complement other animal models. Highly conserved signaling pathways and high similarity in gut homeostasis also make it a powerful and representative system to better understand CRC tumors and their progression. We have previously generated a panel of genetically complex Drosophila models that reflect the genomic landscape of sequenced colon tumors. The most complex model in this panel carried transgenic constructs to knock down Drosophila orthologs of tumor suppressors TP53, APC, SMAD4, and PTEN as well as to express oncogenic KRASG12V. However, creating more complex models using standard Drosophila crosses has been challenging. To address this, we have generated a multigenic vector that carries three different multiple cloning sites (MCSs) designed for cloning three separate transgenes. Two are designed for inducible protein expression to model oncogenic activation while the third is for a micro-RNA inspired synthetic multi-hairpin cluster to knock down multiple tumor suppressors. Here, we will report our efforts to further validate and expand this technology to build more complex cancer models. Using a series of test constructs, we show that transgenic flies carrying synthetic clusters of as many as 16 hairpins can be generated. Hairpins expressed in the context of synthetic clusters are as effective as single hairpins with no detectable positional effects. Finally, by introducing additional design elements such as intron-mediated hairpin cluster expression and bicistronic protein expression using a T2A self-cleaving peptide design, we have generated transgenes that express two proteins and up to 16 hairpins from a single transcript. We then used this updated platform to build a proof-of-concept colorectal cancer model that reflects IRS2 amplification, oncogenic KRASG12V, and loss of tumor suppressors TP53, APC, SMAD4, and SMAD2 from a single transcript designed for inducible expression. This new design frees up the remaining two cloning sites in our multigenic vector for additional genetic manipulations and pushes the limits of our technology to build a new generation of colorectal cancer models to study features of human tumors that have been traditionally challenging to capture in experimental models. Using this technology, we hope to better understand tumor development and find potential therapies that can specifically target these unique, personalized CRC genome-based models. Citation Format: Maria Quintero, Alexander Teague, Ross Cagan, Erdem Bangi. Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B11.

Structural Analysis of RNA by Small-Angle X-ray Scattering.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

Using a system’s equilibrium behavior to reduce its energy dissipation in nonequilibrium processes

Cells must operate far from equilibrium, utilizing and dissipating energy continuously to maintain their organization and to avoid stasis and death. However, they must also avoid unnecessary waste of energy. Recent studies have revealed that molecular machines are extremely efficient thermodynamically compared with their macroscopic counterparts. However, the principles governing the efficient out-of-equilibrium operation of molecular machines remain a mystery. A theoretical framework has been recently formulated in which a generalized friction coefficient quantifies the energetic efficiency in nonequilibrium processes. Moreover, it posits that, to minimize energy dissipation, external control should drive the system along the reaction coordinate with a speed inversely proportional to the square root of that friction coefficient. Here, we demonstrate the utility of this theory for designing and understanding energetically efficient nonequilibrium processes through the unfolding and folding of single DNA hairpins.

Using Equilibrium Behavior to Reduce Energy Dissipation in Non-Equilibrium Biomolecular Processes

Cells must operate far from equilibrium, utilizing and dissipating energy continuously to maintain their organization and to avoid stasis and death. However, they must also avoid unnecessary waste of energy. Recent studies have revealed that molecular machines are extremely efficient thermodynamically when compared to their macroscopic counterparts. However, the principles governing the efficient out-of-equilibrium operation of molecular machines remain a mystery. A theoretical framework has been recently formulated in which a generalized friction coefficient quantifies the energetic efficiency in non-equilibrium processes. Moreover, it posits that to minimize energy dissipation, external control should drive the system along the reaction coordinate with a speed inversely proportional to the square root of that friction coefficient. Here, we demonstrate the utility of this theory for designing and understanding energetically efficient non-equilibrium processes through the unfolding and folding of single DNA hairpins.

Expanded CUG repeats in DMPK transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3′-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3′-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3′-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation.

Free Energy of a Folded Polymer under Cylindrical Confinement

Monte Carlo computer simulations are used to study the conformational free energy of a folded polymer confined to a long cylindrical tube. The polymer is modeled as a hard-sphere chain. Its conformational free energy $F$ is measured as a function of $\lambda$, the end-to-end distance of the polymer. In the case of a flexible linear polymer, $F(\lambda)$ is a linear function in the folded regime with a gradient that scales as $f\equiv |dF/d\lambda| \sim N^0 D^{-1.20\pm 0.01}$ for a tube of diameter $D$ and a polymer of length $N$. This is close to the prediction $f \sim N^0 D^{-1}$ obtained from simple scaling arguments. The discrepancy is due in part to finite-size effects associated with the de-Gennes blob model. A similar discrepancy was observed for the folding of a single arm of a three-arm star polymer. We also examine backfolding of a semiflexible polymer of persistence length $P$ in the classic Odijk regime. In the overlap regime, the derivative scales $f \sim N^0 D^{-1.72\pm 0.02} P^{-0.35\pm 0.01}$, which is close to the prediction $f \sim N^0 D^{-5/3} P^{-1/3}$ obtained from a scaling argument that treats interactions between deflection segments at the second virial level. In addition, the measured free energy cost of forming a hairpin turn is quantitatively consistent with a recent theoretical calculation. Finally, we examine the scaling of $F(\lambda)$ for a confined semiflexible chain in the presence of an S-loop composed of two hairpins. While the predicted scaling of the free energy gradient is the same as that for a single hairpin, we observe a scaling of $f \sim D^{-1.91\pm 0.03} P^{-0.36\pm 0.01}$. Thus, the quantitative discrepancy between this measurement and the predicted scaling is somewhat greater for S-loops than for single hairpins.

Reversible Switching of Cooperating Replicators

How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

Mechanical Characterization of the HIV-1 RNA Hairpin using an Atomic Force Microscope

Pioneering single-molecule force spectroscopy (SMFS) studies of RNA structures used a custom-built optical trap, which continues to be the preferred measurement platform due to its exquisite stability and force precision. Here, we developed a rapid and precise SMFS assay to characterize the dynamics and energetics of RNA structures using a commercial atomic force microscope (AFM) equipped with a temperature-regulated closed-fluidic sample chamber. To do so, we integrated several technical improvements: (i) focused-ion beam modified cantilevers to provide sub-pN stability in conjunction with fast temporal response (∼40 µs); (ii) site-specific attachment to covalently couple RNA hairpins to PEG-coated coverslips and to reversibly attach such hairpins to PEG-coated AFM tips via a streptavidin-biotin bond; and (iii) an automated centering routine to achieve a vertical pulling geometry. By leveraging these improvements, we resolved the unfolding and refolding of single RNA hairpins in both dynamic and equilibrium assays, including repeatedly measuring the same individual molecule over >1 h. Initial studies focused on the HIV-1 RNA hairpin, which leads to a −1 programmed ribosomal frameshift. Dynamic force spectroscopy analysis of the HIV-1 hairpin yielded ΔG‡ (= 36 kBT), Δx‡ (= 7.1 nm), and k0 (= 1.3×10−9 s−1). Looking forward, the rapid data acquisition demonstrated here on a commercial AFM facilitates SMFS studies of macromolecular folding over a broader a range of physiochemical conditions (pH, temperature, denaturant).

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0 to 100%, we determined both the average time to cross the transition paths, τtp, and the distribution of individual transit times, PTP(t). We then estimated D from both τtp and PTP(t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. τtp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base pairs than for those with only G:C base pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0 to 100%. These results reveal that G:C base pairs form faster than A:T base pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding.

Crowding-Induced Hybridization of Single DNA Hairpins.

It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems.

Single-Molecule Stochastic Resonance

Stochastic resonance (SR) is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic (random or probabilistic) noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under the action of an oscillating mechanical force applied with optical tweezers. By varying the frequency of the force oscillation, we investigate the folding and unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measure several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that a good quantifier of the SR is the signal-to-noise ratio (SNR) of the spectral density of measured fluctuations in molecular extension of the DNA hairpins. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance-matching condition. Finally, we carry out experiments on short hairpins that show how SR might be useful for enhancing the detection of conformational molecular transitions of low SNR.Received 14 March 2012DOI:https://doi.org/10.1103/PhysRevX.2.031012This article is available under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.Published by the American Physical Society

In vivo RNAi screening identifies new mediators of p53 independent tumor suppressive functions of p19Arf in the liver

The sources of hepatocellular carcinoma are liver progenitor cells or hepatocytes. Interestingly, we observed differences between progenitor cell- and hepatocyte- derived hepatocellular carcinomas in mice. Oncogenic Nras (NrasG12V) efficiently triggers hepatocellular carcinomas derived from p53-/- liver progenitor cells, however almost no tumor growth is observed, when NrasG12V is delivered into p53-/- hepatocytes. Since aggressive hepatocellular carcinomas develop after short latency upon delivery of oncogenic Nras into p19Arf-/- hepatocytes, we suggest that p19Arf mediates a p53-independent tumor suppressive function in hepatocytes. To identify mediators of this p53-independent tumor suppressive function of p19Arf in the mouse liver, we set up an in vivo RNAi screen. As a proof of principle we could show that co-delivery of NrasG12V and p19Arf-shRNAs into p53-/- hepatocytes via transposable elements was able to phenocopy NrasG12V-driven hepatocarcinogenesis in p19Arf-/- livers. A focused shRNAmir library was compiled consisting of shRNAs targeting genes differenzially expressed in NrasG12V; p53-/- mouse livers compared to NrasG12V; p19Arf-/- livers. This shRNA library was divided into several low complexity pools and subjected to a positive selection screen in a p53-/- background. Via deep sequencing of gDNA derived from NrasG12V; p53-/- tumors, we identified several shRNAs knocking down new candidate genes mediating p53-independent tumor suppressive functions of p19Arf in the mouse liver. Among them are genes involved in the mitotic spindle assembly and the spindle checkpoint. Functional validation experiments using single hairpins have already been pursued for several candidates. ShRNA-mediated knockdown of those candidate genes allows for Ras driven tumorigenesis in a p53-deficient background.

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3'-half shares high sequence similarity with the TCV 3' end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3' end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot psi(3), which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.

Following translation by single ribosomes one codon at a time

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases--one codon-and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.