Development and testing of procedures for detecting HPV 6 and HPV 16 DNA in water samples

The present study aims to develop and test procedures for detecting the DNA of dangerous human papillomaviruses (HPV) types 6 and 16 in water samples. The conserved segments of HPV 6 L1 and HPV 16 L1 nucleic acid sequences were studied using bioinformatic methods with the help of the NCBI (National Center for Biotechnology Information) database and the BioEdit program. A total of 135 nucleic acid sequences of HPV6 L1 and 945 nucleic acid sequences of HPV16 L1 were examined. Five pairs of specific primers were developed for the identified conserved segments of nucleic acid sequences using specialized programs (PerlPrimer v.1.1.21, FastPCR 6.6, and Primer3Plus). In addition, several procedures for collecting samples from various water bodies located near Listvyanka settlement (Lake Baikal) were tested. The samples were subjected to comprehensive purification from insoluble particles and bacterial contamination to be tested for the presence of HPV DNA via PCR analysis using primers complementary to the nucleic acid sequences of HPV6 L1 and HPV16 L1. The conducted studies revealed HPV 6 and HPV 16 DNA in the water samples. Due to the use of the developed and tested procedures for collecting and examining samples from various water sources in the Baikal Natural Territory followed by a PCR analysis, it was possible to detect the presence of dangerous viruses. The proposed procedure of testing water samples for the presence of HPV can be useful in developing effective monitoring of water bodies and wastewater both in Baikal and other regions.

Tagging and Capturing of Lentiviral Vectors Using Short RNAs

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

A kinetic error filtering mechanism for enzyme-free copying of nucleic acid sequences

Accurate copying of nucleic acid sequences is essential for self-replicating systems. Modern cells achieve error ratios as low as 10-9 with sophisticated enzymes capable of kinetic proofreading. In contrast, experiments probing enzyme-free copying of RNA and DNA as potential prebiotic replication processes find error ratios on the order of 10%. Given this low intrinsic copying fidelity, plausible scenarios for the spontaneous emergence of molecular evolution require an accuracy-enhancing mechanism. Here, we study a 'kinetic error filtering' scenario that dramatically boosts the likelihood of producing exact copies of nucleic acid sequences. The mechanism exploits the observation that initial errors in template-directed polymerization of both DNA and RNA are likely to trigger a cascade of consecutive errors and significantly stall downstream extension. We incorporate these characteristics into a mathematical model with experimentally estimated parameters, and leverage this model to probe to what extent accurate and faulty polymerization products can be kinetically discriminated. While limiting the time window for polymerization prevents completion of erroneous strands, resulting in a pool in which full-length products show an enhanced accuracy, this comes at the price of a concomitant reduction in yield. We show that this fidelity-yield trade-off can be circumvented via repeated copying attempts in cyclically varying environments such as the temperature cycles occurring naturally in the vicinity of hydrothermal systems. This setting could produce exact copies of sequences as long as 50mers within their lifetime, facilitating the emergence and maintenance of catalytically active oligonucleotides.

Gold–Oligonucleotide Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold–oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10−7 M, ≥1.4 × 10−14 g/mL) initiates gold–oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold–oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (<60 s), and could be interpreted with a bespoke software and hardware electronic interface. We anticipate that our methodology will translate in silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave the way forward in the clinical evaluation of disease and complement existing strategies to overcome antimicrobial resistance.

Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

Nucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

Structural motifs and intramolecular interactions in non-canonical G-quadruplexes

G-rich nucleic acid sequences encompassing G-tracts of varying lengths can fold into different non-canonical G-quadruplexes with distinct structural features.

ACES: A co-evolution simulator generates co-varying protein and nucleic acid sequences

Sequence-specific and consequential interactions within or between proteins and/or RNAs can be predicted by identifying co-evolution of residues in these molecules. Different algorithms have been used to detect co-evolution, often using biological data to benchmark a methods ability to discriminate against indirect co-evolution. Such a benchmark is problematic, because not all the interactions and evolutionary constraints underlying real data can be known a priori. Instead, sequences generated in silico to simulate co-evolution would be preferable, and can be obtained using aCES, the software tool presented here. Conservation and co-evolution constraints can be specified for any residue across a number of molecules, allowing the user to capture a complex, realistic set of interactions. Resulting alignments were used to benchmark several co-evolution detection tools for their ability to separate signal from background as well as discriminating direct from indirect signals. This approach can aid in refinement of these algorithms. In addition, systematic tuning of these constraints sheds new light on how they drive co-evolution between residues. Better understanding how to detect co-evolution and the residue interactions they predict can lead to a wide range of insights important for synthetic biologists interested in engineering new, orthogonal interactions between two macromolecules.

Genomic characterization of a diazotrophic microbiota associated with maize aerial root mucilage

A geographically isolated maize landrace cultivated on nitrogen-depleted fields without synthetic fertilizer in the Sierra Mixe region of Oaxaca, Mexico utilizes nitrogen derived from the atmosphere and develops an extensive network of mucilage-secreting aerial roots that harbors a diazotrophic microbiota. Targeting these diazotrophs, we selected nearly 600 microbes from a collection isolated from these plants and confirmed their ability to incorporate heavy nitrogen (15N2) metabolites in vitro. Sequencing their genomes and conducting comparative bioinformatic analyses showed that these genomes had substantial phylogenetic diversity. We examined each diazotroph genome for the presence of nif genes essential to nitrogen fixation (nifHDKENB) and carbohydrate utilization genes relevant to the mucilage polysaccharide digestion. These analyses identified diazotrophs that possessed canonical nif gene operons, as well as many other operon configurations with concomitant fixation and release of >700 different 15N labeled metabolites. We further demonstrated that many diazotrophs possessed alternative nif gene operons and confirmed their genomic potential to derive chemical energy from mucilage polysaccharide to fuel nitrogen fixation. These results confirm that some diazotrophic bacteria associated with Sierra Mixe maize were capable of incorporating atmospheric nitrogen into their small molecule extracellular metabolites through multiple nif gene configurations while others were able to fix nitrogen without the canonical (nifHDKENB) genes.Data SummaryGenetic resources, including biological materials and nucleic acid sequences, were accessed under an Access and Benefit Sharing (ABS) Agreement between the Sierra Mixe community and the Mars Corporation, and with authorization from the Mexican government. An internationally recognized certificate of compliance has been issued by the Mexican government under the Nagoya Protocol for such activities (ABSCH-IRCC-MX-207343-3). Any party seeking access to the nucleic acid sequences underlying the analysis reported here is subject to the full terms and obligations of the ABS agreement and the authorization from the government of Mexico. Individuals wishing to access nucleic acid sequence data for scientific research activities should contact Mars Incorporated Chief Science Officer at [email protected].