The prebiotic evolutionary advantage of transferring genetic information from RNA to DNA.

In the early 'RNA world' stage of life, RNA stored genetic information and catalyzed chemical reactions. However, the RNA world eventually gave rise to the DNA-RNA-protein world, and this transition included the 'genetic takeover' of information storage by DNA. We investigated evolutionary advantages for using DNA as the genetic material. The error rate of replication imposes a fundamental limit on the amount of information that can be stored in the genome, as mutations degrade information. We compared misincorporation rates of RNA and DNA in experimental non-enzymatic polymerization and calculated the lowest possible error rates from a thermodynamic model. Both analyses found that RNA replication was intrinsically error-prone compared to DNA, suggesting that total genomic information could increase after the transition to DNA. Analysis of the transitional RNA/DNA hybrid duplexes showed that copying RNA into DNA had similar fidelity to RNA replication, so information could be maintained during the genetic takeover. However, copying DNA into RNA was very error-prone, suggesting that attempts to return to the RNA world would result in a considerable loss of information. Therefore, the genetic takeover may have been driven by a combination of increased chemical stability, increased genome size and irreversibility

Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

Design of DNA origami

The generation of arbitrary patterns and shapes at very small scales is at the heart of our effort to miniaturize circuits and is fundamental to the development of nanotechnology. Here I review a recently developed method for folding long single strands of DNA into arbitrary two-dimensional shapes using a raster fill technique - 'scaffolded DNA origami'. Shapes up to 100 nanometers in diameter can be approximated with a resolution of 6 nanometers and decorated with patterns of roughly 200 binary pixels at the same resolution. Experimentally verified by the creation of a dozen shapes and patterns, the method is easy, high yield, and lends itself well to automated design and manufacture. So far, CAD tools for scaffolded DNA origami are simple, require hand-design of the folding path, and are restricted to two dimensional designs. If the method gains wide acceptance, better CAD tools will be required

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording

Periodic shock-emission from acoustically driven cavitation clouds:a source of the subharmonic signal

Single clouds of cavitation bubbles, driven by 254 kHz focused ultrasound at pressure amplitudes in the range of 0.48–1.22 MPa, have been observed via high-speed shadowgraphic imaging at 1 × 10⁶ frames per second. Clouds underwent repetitive growth, oscillation and collapse (GOC) cycles, with shock-waves emitted periodically at the instant of collapse during each cycle. The frequency of cloud collapse, and coincident shock-emission, was primarily dependent on the intensity of the focused ultrasound driving the activity. The lowest peak-to-peak pressure amplitude of 0.48 MPa generated shock-waves with an average period of 7.9 ± 0.5 μs, corresponding to a frequency of f₀/2, half-harmonic to the fundamental driving. Increasing the intensity gave rise to GOC cycles and shock-emission periods of 11.8 ± 0.3, 15.8 ± 0.3, 19.8 ± 0.2 μs, at pressure amplitudes of 0.64, 0.92 and 1.22 MPa, corresponding to the higher-order subharmonics of f₀/3, f₀/4 and f₀/5, respectively. Parallel passive acoustic detection, filtered for the fundamental driving, revealed features that correlated temporally to the shock-emissions observed via high-speed imaging, p(two-tailed) 200 μm diameter, at maximum inflation), that developed under insonations of peak-to-peak pressure amplitudes >1.0 MPa, emitted shock-waves with two or more fronts suggesting non-uniform collapse of the cloud. The observations indicate that periodic shock-emissions from acoustically driven cavitation clouds provide a source for the cavitation subharmonic signal, and that shock structure may be used to study intra-cloud dynamics at sub-microsecond timescales

DNA stabilized fluorescent metal nanoclusters for biosensor development

The final publication is available at Elsevier via https://doi.org/10.1016/j.trac.2013.12.014." © 2014. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Fluorescent silver, gold and copper nanoclusters (NCs) have emerged for biosensor development. Compared to semiconductor quantum dots, there is less concern about the toxicity of metal NCs, which can be more easily conjugated to biopolymers. These NCs need a stabilizing ligand. Many polymers, proteins and nucleic acids stabilize NCs, and many DNA sequences produce highly-fluorescent NCs. Coupling these DNA stabilizers with other sequences, such as aptamers, has generated a large number of biosensors. We summarize the synthesis of DNA and nucleotide-templated NCs; and, we discuss their chemical interactions. We briefly review properties of NCs, such as fluorescence quantum yield, emission wavelength and lifetime, structure and photostability. We categorize sensor-design strategies using these NCs into: (1) fluorescence de-quenching; (2) generation of templating DNA sequences to produce NCs; (3) change of nearby environment; and, (4) reacting with heavy metal ions or other quenchers. Finally, we discuss future trends.University of Waterloo || Canadian Foundation for Innovation || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201