Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.

Zooshikella harenae sp. nov., Isolated from Pacific Oyster Crassostrea gigas, and Establishment of Zooshikella ganghwensis subsp. marina subsp. nov. and Zooshikella ganghwensis subsp. ganghwensis subsp. nov.

Here, we describe the polyphasic taxonomy of a novel isolated strain WH53T from the genus Zooshikella isolated from the sand sediment located between the lumen of the Crassostrea gigas From Germany. Phylogenetic analysis determined that the strain WH53T had a high similarity to Zooshikella ganghwensis JC2044T (99.57%) and Zooshikella marina LMG 28823T (99.36%). Strain WH53T contained ubiquinone-9 (Q-9) as the predominant menaquinone, and the major fatty acids were C16:0, C16:1ω7c, and C18:1ω7c. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, amino phospholipids, and unidentified phospholipids were identified as their polar lipid composition. The DNA G+C content and genome size of strain WH53T were 40.08 mol% and 5,914,969 bp, respectively. Digital DNA–DNA hybridisation (dDDH) for strain WH53T against Z. ganghwensis JC2044T and Z. marina LMG 28823T showed low relatedness values of 26.3% and 26.1%, respectively. The extract of strain WH53T exhibited antimicrobial property. Strain WH53T represents a novel species in the genus Zooshikella. We propose the name of Zooshikella harenae sp. nov., with the type strain WH53T (= DSM 111628T = NCCB 100808T). Furthermore, the dDDH, average nucleotide identity (ANI), percentage of conserved proteins (POCP), and amino acid identity (AAI) value between Z. marina LGM 28823T and Z. ganghwensis DSM 15267T were 79.9%, 97.84%, 76.08%, and 87.01%, respectively, suggesting that both of them should be reclassified as Z. ganghwensis subsp. marina subsp. nov. and Z. ganghwensis subsp. ganghwensis DSM 15267 subsp. nov.

DNA hybridisation kinetics using single-molecule fluorescence imaging

Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

Hexaferrocenium Tri [Hexa(Isothiocyanato)Iron(III)] trihydroxonium Complex as a New DNA Intercalator for Electrochemical DNA Biosensor

Ferrocene or ferrocenium has been widely studied in the field of organometallic complexes because of its stable thermodynamic, kinetic and redox properties. Novel Hexaferrocenium Tri[Hexa(Isothiocyanato)Iron (III)]Trihydroxonium (HexaFc) complex was the product from the reaction of ferrocene, maleic acid and ammonium thiocyanate and it was confirmed by elemental analysis CHNS, FTIR and single crystal x-ray crystallography. In this study, HexaFc was used for the first time as an electroactive indicator for DNA hybridisation biosensor. The UV-Vis DNA titrations with this compound showed hypochromism and redshift at 250 nm with increasing DNA concentrations. The binding constant (Kb) for HexaFc complex towards CT-DNA (calf-thymus DNA) was 3.1 × 104 M−1. These results indicated intercalator behaviour of the complex. To test the usefulness of this complex for DNA biosensor application, a porcine DNA biosensor was constructed. The recognition probes were covalently immobilised onto silica nanospheres (SiNS) via glutaraldehyde linker on a screen-printed electrode (SPE). After intercalation with the complex, the biosensor response to complementary porcine DNA was measured using differential pulse voltammetry. The DNA biosensor demonstrated a linear response range to the complementary porcine DNA from 1 × 10−6 to 1 × 10−3 µM (R2 = 0.9642) with a limit detection of 4.83 × 10−8 µM. The results indicated that HexaFc complex is a feasible indicator for DNA hybridisation evaluation without the need of chemical attachment as a label.

Combining bacteriophage engineering and linear dichroism spectroscopy to produce a DNA hybridisation assay

A novel DNA sensing method based on LD spectroscopy and using bionanoparticle scaffolds is described, as demonstrated by the rapid detection of DNA strands associated with bacterial and viral pathogens.

Establishing a Field-Effect Transistor Sensor for the Detection of Mutations in the Tumour Protein 53 Gene (TP53)—An Electrochemical Optimisation Approach

We present a low-cost, sensitive and specific DNA field-effect transistor sensor for the rapid detection of a common mutation to the tumour protein 53 gene (TP53). The sensor consists of a commercially available, low-cost, field-effect transistor attached in series to a gold electrode sensing pad for DNA hybridisation. The sensor has been predominantly optimised electrochemically, particularly with respect to open-circuit potentiometry as a route towards understanding potential (voltage) changes upon DNA hybridisation using a transistor. The developed sensor responds sensitively to TP53 mutant DNA as low as 100 nM concentration. The sensor responds linearly as a function of DNA target concentration and is able to differentiate between complementary and noncomplementary DNA target sequences.

Possible association between Mycobacterium paratuberculosis infection and Crohn's disease in human

Crohn's disease is a granulomatous ileocolitis of humans, of unknown aetiology, which generally manifests itself during the prime of life. The chronic, progressive clinical course and histological findings are consistent wiht a mycobacterial aetiology. Evidence supporting a pathogenic role for a mycobacterium has become available only in the last decade with the isolation of this microorganism from Crohn's disease tissue. M. paratuberculosis, which is the causative agent of Johne's disease in animals, has been identified in patients with Crohn's disease by PCR and DNA hybridisation techniques. It has been shown that isolates of M. paratuberculosis from Crohn's disease are indentical with pathogenic strains in ruminants.