59 research outputs found
Single molecule experiments in biophysics: exploring the thermal behavior of nonequilibrium small systems
Biomolecules carry out very specialized tasks inside the cell where energies
involved are few tens of k_BT, small enough for thermal fluctuations to be
relevant in many biomolecular processes. In this paper I discuss a few concepts
and present some experimental results that show how the study of fluctuation
theorems applied to biomolecules contributes to our understanding of the
nonequilibrium thermal behavior of small systems.Comment: Proceedings of the 22nd Statphys Conference 2004 (Bangalore,India).
Invited contributio
Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results
Experimental variables of optical tweezers instrumentation that affect RNA
folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was
attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was
trapped by dual-beam lasers and the other was held by a micropipette. Several
experimental variables were changed while measuring the unfolding/refolding
kinetics, including handle lengths, trap stiffness, and modes of force applied
to the molecule. In constant-force mode where the tension applied to the RNA
was maintained through feedback control, the measured rate coefficients varied
within 40% when the handle lengths were changed by 10 fold (1.1 to 10.2 Kbp);
they increased by two- to three-fold when the trap stiffness was lowered to one
third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control
and where the force applied to the RNA varied in response to the end-to-end
distance change of the tether, the RNA hopped between a high-force folded-state
and a low-force unfolded-state. In this mode, the rates increased up to
two-fold with longer handles or softer traps. Overall, the measured rates
remained with the same order-of-magnitude over the wide range of conditions
studied. In the companion paper (1), we analyze how the measured kinetics
parameters differ from the intrinsic molecular rates of the RNA, and thus how
to obtain the molecular rates.Comment: PDF file, 30 pages, 7 figure
End-joining long nucleic acid polymers
Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two-step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 ± 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated examples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionanotechnology and in single-molecule experiments
Single-molecule experiments in biological physics: methods and applications
I review single-molecule experiments (SME) in biological physics. Recent
technological developments have provided the tools to design and build
scientific instruments of high enough sensitivity and precision to manipulate
and visualize individual molecules and measure microscopic forces. Using SME it
is possible to: manipulate molecules one at a time and measure distributions
describing molecular properties; characterize the kinetics of biomolecular
reactions and; detect molecular intermediates. SME provide the additional
information about thermodynamics and kinetics of biomolecular processes. This
complements information obtained in traditional bulk assays. In SME it is also
possible to measure small energies and detect large Brownian deviations in
biomolecular reactions, thereby offering new methods and systems to scrutinize
the basic foundations of statistical mechanics. This review is written at a
very introductory level emphasizing the importance of SME to scientists
interested in knowing the common playground of ideas and the interdisciplinary
topics accessible by these techniques. The review discusses SME from an
experimental perspective, first exposing the most common experimental
methodologies and later presenting various molecular systems where such
techniques have been applied. I briefly discuss experimental techniques such as
atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers
(MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I
then present several applications of SME to the study of nucleic acids (DNA,
RNA and DNA condensation), proteins (protein-protein interactions, protein
folding and molecular motors). Finally, I discuss applications of SME to the
study of the nonequilibrium thermodynamics of small systems and the
experimental verification of fluctuation theorems. I conclude with a discussion
of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond.
Matt
Transat—A Method for Detecting the Conserved Helices of Functional RNA Structures, Including Transient, Pseudo-Knotted and Alternative Structures
The prediction of functional RNA structures has attracted increased interest, as it allows us to study the potential functional roles of many genes. RNA structure prediction methods, however, assume that there is a unique functional RNA structure and also do not predict functional features required for in vivo folding. In order to understand how functional RNA structures form in vivo, we require sophisticated experiments or reliable prediction methods. So far, there exist only a few, experimentally validated transient RNA structures. On the computational side, there exist several computer programs which aim to predict the co-transcriptional folding pathway in vivo, but these make a range of simplifying assumptions and do not capture all features known to influence RNA folding in vivo. We want to investigate if evolutionarily related RNA genes fold in a similar way in vivo. To this end, we have developed a new computational method, Transat, which detects conserved helices of high statistical significance. We introduce the method, present a comprehensive performance evaluation and show that Transat is able to predict the structural features of known reference structures including pseudo-knotted ones as well as those of known alternative structural configurations. Transat can also identify unstructured sub-sequences bound by other molecules and provides evidence for new helices which may define folding pathways, supporting the notion that homologous RNA sequence not only assume a similar reference RNA structure, but also fold similarly. Finally, we show that the structural features predicted by Transat differ from those assuming thermodynamic equilibrium. Unlike the existing methods for predicting folding pathways, our method works in a comparative way. This has the disadvantage of not being able to predict features as function of time, but has the considerable advantage of highlighting conserved features and of not requiring a detailed knowledge of the cellular environment
Modulating RNA structure and catalysis: lessons from small cleaving ribozymes
RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field
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