174 research outputs found
Hachimoji DNA and RNA: A genetic system with eight building blocks
Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos
Salerno's model of DNA reanalysed: could solitons have biological significance?
We investigate the sequence-dependent behaviour of localised excitations in a
toy, nonlinear model of DNA base-pair opening originally proposed by Salerno.
Specifically we ask whether ``breather'' solitons could play a role in the
facilitated location of promoters by RNA polymerase. In an effective potential
formalism, we find excellent correlation between potential minima and {\em
Escherichia coli} promoter recognition sites in the T7 bacteriophage genome.
Evidence for a similar relationship between phage promoters and downstream
coding regions is found and alternative reasons for links between AT richness
and transcriptionally-significant sites are discussed. Consideration of the
soliton energy of translocation provides a novel dynamical picture of sliding:
steep potential gradients correspond to deterministic motion, while ``flat''
regions, corresponding to homogeneous AT or GC content, are governed by random,
thermal motion. Finally we demonstrate an interesting equivalence between
planar, breather solitons and the helical motion of a sliding protein
``particle'' about a bent DNA axis.Comment: Latex file 20 pages, 5 figures. Manuscript of paper to appear in J.
Biol. Phys., accepted 02/09/0
Accurate Estimates of Microarray Target Concentration from a Simple Sequence-Independent Langmuir Model
Background: Microarray technology is a commonly used tool for assessing global gene expression. Many models for estimation of target concentration based on observed microarray signal have been proposed, but, in general, these models have been complex and platform-dependent. Principal Findings: We introduce a universal Langmuir model for estimation of absolute target concentration from microarray experiments. We find that this sequence-independent model, characterized by only three free parameters, yields excellent predictions for four microarray platforms, including Affymetrix, Agilent, Illumina and a custom-printed microarray. The model also accurately predicts concentration for the MAQC data sets. This approach significantly reduces the computational complexity of quantitative target concentration estimates. Conclusions: Using a simple form of the Langmuir isotherm model, with a minimum of parameters and assumptions, and without explicit modeling of individual probe properties, we were able to recover absolute transcript concentrations with high R 2 on four different array platforms. The results obtained here suggest that with a ‘‘spiked-in’ ’ concentration serie
Application of Equilibrium Models of Solution Hybridization to Microarray Design and Analysis
Background: The probe percent bound value, calculated using multi-state equilibrium models of solution hybridization, is shown to be useful in understanding the hybridization behavior of microarray probes having 50 nucleotides, with and without mismatches. These longer oligonucleotides are in widespread use on microarrays, but there are few controlled studies of their interactions with mismatched targets compared to 25-mer based platforms. Principal Findings: 50-mer oligonucleotides with centrally placed single, double and triple mismatches were spotted on an array. Over a range of target concentrations it was possible to discriminate binding to perfect matches and mismatches, and the type of mismatch could be predicted accurately in the concentration midrange (100 pM to 200 pM) using solution hybridization modeling methods. These results have implications for microarray design, optimization and analysis methods. Conclusions: Our results highlight the importance of incorporating biophysical factors in both the design and the analysis of microarrays. Use of the probe ‘‘percent bound’ ’ value predicted by equilibrium models of hybridization is confirmed to be important for predicting and interpreting the behavior of long oligonucleotide arrays, as has been shown for shor
Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses
It is suspected that apart from tick-borne encephalitis virus several additional European Arboviruses such as the sandfly borne Toscana virus, sandfly fever Sicilian virus and sandfly fever Naples virus, mosquito-borne Tahyna virus, Inkoo virus, Batai virus and tick-borne Uukuniemi virus cause aseptic meningo-encephalitis or febrile disease in Europe. Currently, the microarray technology is developing rapidly and there are many efforts to apply it to infectious diseases diagnostics. In order to arrive at an assay system useful for high throughput analysis of samples from aseptic meningo-encephalitis cases the authors developed a combined multiplex ligation-dependent probe amplification and flow-through microarray assay for the detection of European Bunyaviruses. These results show that this combined assay indeed is highly sensitive, and specific for the accurate detection of multiple viruses
Single Molecule Statistics and the Polynucleotide Unzipping Transition
We present an extensive theoretical investigation of the mechanical unzipping
of double-stranded DNA under the influence of an applied force. In the limit of
long polymers, there is a thermodynamic unzipping transition at a critical
force value of order 10 pN, with different critical behavior for homopolymers
and for random heteropolymers. We extend results on the disorder-averaged
behavior of DNA's with random sequences to the more experimentally accessible
problem of unzipping a single DNA molecule. As the applied force approaches the
critical value, the double-stranded DNA unravels in a series of discrete,
sequence-dependent steps that allow it to reach successively deeper energy
minima. Plots of extension versus force thus take the striking form of a series
of plateaus separated by sharp jumps. Similar qualitative features should
reappear in micromanipulation experiments on proteins and on folded RNA
molecules. Despite their unusual form, the extension versus force curves for
single molecules still reveal remnants of the disorder-averaged critical
behavior. Above the transition, the dynamics of the unzipping fork is related
to that of a particle diffusing in a random force field; anomalous,
disorder-dominated behavior is expected until the applied force exceeds the
critical value for unzipping by roughly 5 pN.Comment: 40 pages, 18 figure
Large scale analysis of positional effects of single-base mismatches on microarray gene expression data
<p>Abstract</p> <p>Background</p> <p>Affymetrix GeneChips utilize 25-mer oligonucleotides probes linked to a silica surface to detect targets in solution. Mismatches due to single nucleotide polymorphisms (SNPs) can affect the hybridization between probes and targets. Previous research has indicated that binding between probes and targets strongly depends on the positions of these mismatches. However, there has been substantial variability in the effect of mismatch type across studies.</p> <p>Methods</p> <p>By taking advantage of naturally occurring mismatches between rhesus macaque transcripts and human probes from the Affymetrix U133 Plus 2 GeneChip, we collected the largest 25-mer probes dataset with single-base mismatches at each of the 25 positions on the probe ever used in this type of analysis.</p> <p>Results</p> <p>A mismatch at the center of a probe led to a greater loss in signal intensity than a mismatch at the ends of the probe, regardless of the mismatch type. There was a slight asymmetry between the ends of a probe: effects of mismatches at the 3' end of a probe were greater than those at the 5' end. A cross study comparison of the effect of mismatch types revealed that results were not in good agreement among different reports. However, if the mismatch types were consolidated to purine or pyrimidine mismatches, cross study conclusions could be generated.</p> <p>Conclusion</p> <p>The comprehensive assessment of the effects of single-base mismatches on microarrays provided in this report can be useful for improving future versions of microarray platform design and the corresponding data analysis algorithms.</p
Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies
The description of nonequilibrium processes in nano-sized objects, where the
typical energies involved are a few times, is increasingly becoming central to
disciplines as diverse as condensed-matter physics, materials science, and
biophysics. Major recent developments towards a unified treatment of
arbitrarily large fluctuations in small systems are described by fluctuation
theorems that relate the probabilities of a system absorbing from or releasing
to the bath a given amount of energy in a nonequilibrium process. Here we
experimentally verify the Crooks Fluctuation Theorem (CFT) under weak and
strong nonequilibrium conditions by using optical tweezers to measure the
irreversible mechanical work during the unfolding and refolding of a small RNA
hairpin and an RNA three-helix junction. We also show that the CFT provides a
powerful way to obtain folding free energies in biomolecules by determining the
crossing between the unfolding and refolding irreversible work distributions.
The method makes it possible to obtain folding free energies in nonequilibrium
processes that dissipate up to of the average total work exerted, thereby
paving the way for reconstructing free energy landscapes along reaction
coordinates in nonequilibrium single-molecule experiments.Comment: PDF file, 19 pages. Supplementary information available online at
www.nature.co
Experimental free energy measurements of kinetic molecular states using fluctuation theorems
Recent advances in non-equilibrium statistical mechanics and single molecule
technologies make it possible to extract free energy differences from
irreversible work measurements in pulling experiments. To date, free energy
recovery has been focused on native or equilibrium molecular states, whereas
free energy measurements of kinetic states (i.e. finite lifetime states that
are generated dynamically and are metastable) have remained unexplored. Kinetic
states can play an important role in various domains of physics, such as
nanotechnology or condensed matter physics. In biophysics, there are many
examples where they determine the fate of molecular reactions: protein and
peptide-nucleic acid binding, specific cation binding, antigen-antibody
interactions, transient states in enzymatic reactions or the formation of
transient intermediates and non-native structures in molecular folders. Here we
demonstrate that it is possible to obtain free energies of kinetic states by
applying extended fluctuation relations. This is shown by using optical
tweezers to mechanically unfold and refold DNA structures exhibiting
intermediate and misfolded kinetic states.Comment: main paper (16 pages, 5 figures) and supplementary information (22
pages, 14 figures
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