Short DNA Sequences Research Articles

Exploiting G-Quadruplex-DNA Damage as a Tool to Quantify Singlet Oxygen Production.

G-Quadruplexes (G4s) are highly dynamic and polymorphic nucleic acid structures that can adopt a variety of conformations. When exposed to oxidative conditions, more specifically singlet oxygen, the guanosine nucleobases can be oxidized, which in turn can affect the conformation and folding of the G4. Based on this peculiar phenomenon, it is rationalized that G4s can serve as quantification sensors for the production of singlet oxygen. Here, a method for determining the quantum yield of singlet oxygen generation for visible as well as UV-light excited photosensitizers, using a short G4 DNA sequence, readily available from common DNA companies, as a biological and water-soluble probe, is presented.

Molecular Identification of the Diversity of Insects, Spiders, Lizards, Birds and Mammals of Tuensang District, Nagaland, India

ABSTRACT: DNA barcoding is a method of species identification that revolutionized the way we study and understand biodiversity. With advancements in molecular genetics and DNA sequence archives, it has become possible to use short DNA sequences to identify species, even when the specimens are difficult to distinguish by traditional morphological methods. In this article, we explore the utility of DNA barcoding in the Tuensang ecosystem of Nagaland, India and evaluate its effectiveness for species identification, informing ongoing conservation of populations and species. A total of 62 species, which included insects, spiders, lizards, birds and mammals, were collected from Tuensang areas of northeast India and identified using DNA barcodes. DNA was extracted from muscle tissue and PCR was done with two pairs of primers targeting the mitochondrial COI gene. Sanger sequencing was employed and the obtained sequences were analysed to identify the species and reconstruct the evolutionary relationships amongst them. Our results provided molecular characterization of species from Tuensang areas of Nagaland for the first time.

Review—Aptamers and Biomimetic Receptors in Biosensing: Innovations and Applications

The fields of biosensing have been transformed by the discovery of extraordinary molecular recognition components, such as aptamers and biomimetic receptors. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method used to select aptamers, or short sequences of single-stranded DNA (ssDNA) or RNA (ssRNA), based on their unique binding affinity to target molecules. Molecularly imprinted polymers (MIPs) are a type of biomimetic receptor that mimics the selectivity of natural receptors inside a synthetic matrix. They make it possible to identify pathogens, and illness biomarkers with accuracy. Aptamers and biomimetic receptors play crucial roles in various fields including diagnostics, therapeutics, and biosensing. Their high specificity, versatility, and adaptability enable targeted detection, drug delivery, and biomolecule manipulation, thereby contributing to advancements in personalized medicine, biotechnology, and disease diagnosis. Aptamers and biomimetic receptors have been combined with cutting-edge technologies, like nanotechnology and lab-on-a-chip systems, to create biosensors that are quick, portable, and extremely sensitive. These recognition features are anticipated to become more important as technology develops, helping to address global issues, advance biosensing capabilities, and raise people’s standard of living everywhere. Recent advancements and innovation on Aptamers and Biomimetic Receptors in Biosensing have been discussed in this review article.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

A blueprint most wonderful, the homeobox discovery.

This is a personal, non-linear summary of the discovery of the homeobox, a short DNA sequence encoding a DNA-binding domain conserved in developmental control genes. It is based on our recollections, a few decaying lab notebooks and letters, the early research papers we published, and conversations with a few colleagues who were in Basel at the time. It presents a simple story, when the research we did was anything but, with failed experiments, blind alleys and dumb ideas. Homeobox DNA sequences were independently discovered by Matt Scott and Amy Weiner in Thomas Kaufmann's lab at Indiana University ( Scott and Weiner, 1984). The accompanying Perspective from Scott (2024), provides their fascinating story.

A Brief Introduction to Microhaplotypes and their Uses

Microhaplotypes are genetic markers that are short DNA sequences, typically consisting of two or more single nucleotide polymorphisms (SNPs) sufficiently close molecularly (<300 basepairs) that their alleles are inherited together from parent to child. Microhaplotypes can be considered a special class of haplotypes, “micro” because they extend for only a few dozens of basepairs, not thousands of basepairs. The advantage of microhaplotypes is that, with massively parallel sequencing of a small segment, the phase is known. Microhaps have been shown to be highly informative in demonstrating uniqueness of DNA profiles and in determining the biogeographic ancestry of an individual. Tests of biologic relationships, such as paternity tests, can be done with microhaplotypes. Deconvolution of DNA mixtures is an area in which microhaplotypes appear to be especially promising. This brief introductory review presents the scientific background for the development of microhaplotypes as a new type of genetic marker and examples of some of the recent applications.

A Study of Computational Genome Assembly by Graph Theory

Abstract The assembly of billions of short sequencing reads into a contiguous genome is a daunting task. The foundation knowledge of current DNA assembly models is concentrated among a select group, where the solution to the genome assembly challenge lies in proper ordering the genomic data. This contribution’s objective is to provide an overview of the original graph models used in DNA sequencing by hybridization. With the updated analytical approach based on the bidirectional bipartite graph class, the theoretical basic structure of the DNA assembly model has been described in new perspective by incorporating few short hypothetical DNA sequences. On the Galaxy platform, by using Spades assembler and Velvet assembler, the comparative outcomes of an experiment are presented, and we also identify their working schemes. Here, the working principle of de Bruijn graph has been discussed in broader point of view.

Generating 2D Barcode for DNA Barcode Sequences.

DNA barcode sequence is a short DNA sequence representing a sample from a particular species. The commonly used DNA barcodes are at least 200bps long. This large number of characters cannot be encoded in two-dimensional codes for sample recognition and tracking. In the present study, we described a method that can be used to compress the DNA sequences and then generate the corresponding QR code. With the large numbers of software and hardware, the QR code can be used efficiently for printing, labeling, and scanning.

Evaluating Metabarcoding Markers for Identifying Zooplankton and Ichthyoplankton Communities to Species in the Salish Sea: Morphological Comparisons and Rare, Threatened or Invasive Species

Zooplankton and ichthyoplankton community assessments depend on species diagnostics, yet morphological identifications are time-consuming, require taxonomic expertise, and are hampered by a lack of diagnostic characters, particularly for larval stages. Metabarcoding can identify multiple species in communities from short DNA sequences in comparison to reference databases. To evaluate species resolution across phylogenetic groups and food webs of zooplankton and ichthyoplankton, we compare five metabarcode mitochondrial (mt)DNA markers from gene regions of (a) cytochrome c oxidase subunit I, (b) cytochrome b, (c) 16S ribosomal RNA, and (d) 12S ribosomal RNA for DNA extracted from net tows in the Northeastern Pacific Ocean’s Salish Sea across seven sites and two seasons. Species resolved by metabarcoding are compared to invertebrate morphological identifications and biomass estimates. Results indicate that species resolution for different zooplankton and ichthyoplankton taxa can markedly vary among gene regions and markers in comparison to morphological identifications. Thus, researchers seeking “universal” metabarcoding should take caution that several markers and gene regions likely will be needed; all will miss some taxa and yield incomplete overlap. Species resolution requires careful attention to taxon marker selection and coverage in reference sequence repositories. In summary, combined multi-marker metabarcoding and morphological approaches improve broadscale zooplankton diagnostics.

Global mitochondrial and chloroplast genome diversity in the threatened aquatic carnivorous plant Aldrovanda vesiculosa

The submerged aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) is threatened by rapid deterioration of wetlands and oligotrophic lake habitats. Its native distribution spans four continents, but many historic populations are now extinct. Previous genetic studies found distinction between populations from Australia and those from the rest of the world, but due to limited genetic markers, neither detailed phylogenetic relationships nor the migration routes of A. vesiculosa populations were revealed. We used a de novo assembly of the A. vesiculosa mitochondrial genome and a previously published plastid genome as references for mapping short DNA sequence reads from 17 globally distributed populations. Phylogenetic trees were constructed based on detected polymorphisms. Genetic diversity of both the mitochondrial and plastid genome was low (Pi 0.55 × 10−4 and 0.7 × 10−4, respectively). Greater polymorphisms were found in the mitochondrial compared with the plastid genome, owing to its larger size (1.27 Mb). Australian populations formed a monophyletic clade in both plastid and mitochondrial trees, while the mitochondrial tree also distinguished populations from southern and northern Europe. Aldrovanda vesiculosa likely migrated to Australia and Africa from a southern European refuge during the last interglacial period∼100,000 years ago. When the last glaciation started, some populations could have survived in eastern Europe and moved north, when the continental glacier retreated. Aldrovanda vesiculosa experienced repeated population bottlenecks that reduced its genetic diversity.

Telomere length is not a useful tool for chronological age estimation in animals.

Telomeres are short repetitive DNA sequences capping the ends of chromosomes. Telomere shortening occurs during cell division and may be accelerated by oxidative damage or ameliorated by telomere maintenance mechanisms. Consequently, telomere length changes with age, which was recently confirmed in a large meta-analysis across vertebrates. However, based on the correlation between telomere length and age, it was concluded that telomere length can be used as a tool for chronological age estimation in animals. Correlation should not be confused with predictability, and the current data and studies suggest that telomeres cannot be used to reliably predict individual chronological age. There are biological reasons for why there is large individual variation in telomere dynamics, which is mainly due to high susceptibility to a wide range of environmental, but also genetic factors, rendering telomeres unfeasible as a tool for age estimation. The use of telomeres for chronological age estimation is largely a misguided effort, but its occasional reappearance in the literature raises concerns that it will mislead resources in wildlife conservation.

Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

Technique for the identification of insect species in processed foods based on three short DNA sequences

For pest control, it is necessary to identify insect species in food. However, it is difficult to identify the insect species in processed food based on their morphological characteristics owing to deformation or fragmentation. DNA sequence-based identification is useful not only to corroborate morphological identification, but also as an alternative identification approach. Therefore, markers are required to obtain the target specific amplicons from insect DNA sequences fragmented by food processing. We developed a DNA sequence-based insect species identification technique that combined two markers in the cytochrome oxidase subunit I (COI) region and one marker in the 16S ribosomal RNA (rRNA) region, with amplicons up to 200 bp in size. The identification results from this technique were consistent with those from the morphological identification of 89% of the 38 insect samples selected from seven orders. This technique identified the insect species in insect samples recovered from model food that were heat-treated at 118 °C for 18 min.

Altering under-represented DNA sequences elevates bacterial transformation efficiency.

Manipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium's genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in "stealth" mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

Sequence self-selection by cyclic phase separation.

The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the dense phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the previously most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for auto-catalytic self-replicating oligonucleotides.

Extensive natural Agrobacterium-induced transformation in the genus Camellia.

The genus Camellia underwent extensive natural transformation by Agrobacterium. Over a period of 15 million years, at least 12 different inserts accumulated in 72 investigated Camellia species. Like a wide variety of other wild and cultivated plants, Camellia species carry cellular T-DNA sequences (cT-DNAs) in their nuclear genomes, resulting from natural Agrobacterium-mediated transformation. Short and long DNA sequencing reads of 435 accessions belonging to 72 Camellia species (representing 12 out of 14 sections) were investigated for the occurrence of cT-DNA insertions. In all, 12 different cT-DNAs were recovered, either completely or partially, called CaTA to CaTL. Divergence analysis of internal cT-DNA repeats revealed that the insertion events span a period from 0.075 to 15 Mio years ago, and yielded an average transformation frequency of one event per 1.25 Mio years. The two oldest inserts, CaTA and CaTD, have been modified by spontaneous deletions and inversions, and by insertion of various plant sequences. In those cases where enough accessions were available (C. japonica, C. oleifera, C. chekiangoleosa, C. sasanqua and C. pitardii), the younger cT-DNA inserts showed a patchy distribution among different accessions of each species, indicating that they are not genetically fixed. It could be shown that Camellia breeding has led to intersectional transfer of cT-DNAs. Altogether, the cT-DNAs cover 374kb, and carry 47 open reading frames (ORFs). Two Camellia cT-DNA genes, CaTH-orf358 and CaTK-orf8, represent new types of T-DNA genes. With its large number of cT-DNA sequences, the genus Camellia constitutes an interesting model for the study of natural Agrobacterium transformants.

Prospecting the cancer therapeutic edge of chitosan-based gold nanoparticles through conformation selective binding to the parallel G-quadruplex formed by short telomeric DNA sequence: A multi-spectroscopic approach

The biological relevance of G4 structures formed in telomere & oncogenes promoters make them extremely crucial therapeutic target for cancer treatment. Herein, we have synthesized chitosan-based gold nanoparticles (CH-Au NPs) through green method and have investigated their interaction with G4 structures formed by short telomeric sequences to evaluate their potential for targeting G4 structures. Firstly, we have characterized morphological/physical attributes of synthesized CH-Au NPs and salt dependent structural aspects of model G-rich DNA sequence, 12-mer d(T2G4)2 [TETRA] using spectroscopic and biophysical techniques. The molecular interactions between CH-Au NPs and parallel/antiparallel TETRA G4 structures were evaluated using UV–Visible, CD, Fluorescence, CD melting, DLS and Zeta potential studies. The experimental data indicated that CH-Au NPs showed strong binding interactions with Parallel TETRA G4 and provided thermal stabilization to the structure, whereas their interactions with Antiparallel TETRA G4 DNA and Ct-DNA (DNA duplex) were found to be negligible. Further, CH-Au NPs were also investigated for their selectivity aptitude for different G4 structures formed by human telomeric sequences; d(T2AG3)3 [HUM-12] and d(T2AG3)4T [HUM-25]. Our findings suggested that CH-Au NPs exhibited topology specific binding aptitude towards G4 structure, which can be utilized to inhibit/modulate crucial biological functions for potential anticancer activity.

Whole-genome detection using multivalent DNA-coated colloids

To minimize the incorrect use of antibiotics, there is a great need for rapid and inexpensive tests to identify the pathogens that cause an infection. The gold standard of pathogen identification is based on the recognition of DNA sequences that are unique for a given pathogen. Here, we propose and test a strategy to develop simple, fast, and highly sensitive biosensors that make use of multivalency. Our approach uses DNA-functionalized polystyrene colloids that distinguish pathogens on the basis of the frequency of selected short DNA sequences in their genome. Importantly, our method uses entire genomes and does not require nucleic acid amplification. Polystyrene colloids grafted with specially designed surface DNA probes can bind cooperatively to frequently repeated sequences along the entire genome of the target bacteria, resulting in the formation of large and easily detectable colloidal aggregates. Our detection strategy allows "mix and read" detection of the target analyte; it is robust and highly sensitive over a wide concentration range covering, in the case of our test target genome Escherichia coli bl21-de3, 10 orders of magnitude from [Formula: see text] to [Formula: see text] copies/mL. The sensitivity compares well with state-of-the-art sensing techniques and has excellent specificity against nontarget bacteria. When applied to real samples, the proposed technique shows an excellent recovery rate. Our detection strategy opens the way to developing a robust platform for pathogen detection in the fields of food safety, disease control, and environmental monitoring.

Programmable RNA detection with CRISPR-Cas12a

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores.

Single-molecule quantification of the strength and sequence specificity of interactions between proteins and nucleic acids would facilitate the probing of protein-DNA binding. Here we show that binding events between the catalytically inactive Cas9 ribonucleoprotein and any pre-defined short sequence of double-stranded DNA can be identified by sensing changes in ionic current as suitably designed barcoded linear DNA nanostructures with Cas9-binding double-stranded DNA overhangs translocate through solid-state nanopores. We designed barcoded DNA nanostructures to study the relationships between DNA sequence and the DNA-binding specificity, DNA-binding efficiency and DNA-mismatch tolerance of Cas9 at the single-nucleotide level. Nanopore-based sensing of DNA-barcoded nanostructures may help to improve the design of efficient and specific ribonucleoproteins for biomedical applications, and could be developed into sensitive protein-sensing assays.