Short DNA Research Articles

Methylome of circulating cell free DNA as a novel biomarker tool: from acute coronary syndrome to broader risk stratification of cardiovascular disease

Abstract Background Cell death during tissue injury leads to release of chromosomal DNA which is promptly degraded. However, short DNA fragments wrapped around nucleosomes survive longer in the bloodstream and can be isolated as circulating cell-free DNA (ccfDNA). Oncology field pioneered the use of cfDNA as clinical biomarker by identifying sequence modifications induced in cancer cells. Importantly, ccfDNA also retains the same epigenetic information as the tissue of origin. Despite major advances for disease diagnosis and therapy, coronary artery diseases (CAD) and associated conditions such acute myocardial infarction (AMI) or heart failure (HF) are responsible for leading number of deaths worldwide. Preventive and surveillance strategies with non-invasive testing are needed. Purpose Evaluation of ccfDNA methylation profiles as prognostic non-invasive biomarker for risk stratification of acute coronary syndromes and their potential application in preventive and surveillance strategies in other cardiovascular disorders. Methods CcfDNA isolated from citrate-plasma collected from our discovery cohort, comprising healthy controls, STEMI, NSTEMI and unstable angina (UA) patients, was analyzed applying Whole Genome Bisulfite Sequencing (WGBS) using a low-input BS-seq (PBAT) protocol. Computational analysis generated differentially methylated regions (DMRs) by tiling CpG areas and setting a threshold of minimal 25% methylation difference compared to the healthy group. To assess the validity of our findings, a selection of disease-specific DMRs were tested using targeted methylation sequencing protocol in a new set of patients. This procedure consisted in enzymatic conversion of ccfDNA methylated sites followed by probe-specific enrichment libraries and sequencing at a Novaseq 6000 platform. Results The study identified a total of 1941 DMRs and from those 486 were STEMI, 223 NSTEMI and 684 UA specific. From the identified DMRs, 74.13% from STEMI, 72.68% from NSTEMI and 61.62% from UA annotated with heart-related diseases using DisGeNET gene-disease associations. Interestingly, most of identified DMRs located within intronic regions (~60%) and also annotated as enhancers and lncRNAs. The independent validation with targeted methylation sequencing identified 566, 306 and 248 DMRs, respectively, and reduced the number of disease-specific DMRs to 171 in STEMI, 115 in NSTEMI and 82 in UA. Conclusion and Future Approaches Methylation profiles of ccfDNA showed potential as a biomarker to stratify the severity of acute coronary syndromes, a novel non-invasive detection methodology that could be extrapolated to risk stratification in other cardiovascular disease such as heart failure or coronary artery disease.

Environmental niche models improve species identification in DNA barcoding

Abstract Recent advances in DNA barcoding have immeasurably advanced global biodiversity research in the last two decades. However, inherent limitations in barcode sequences, such as hybridization, introgression or incomplete lineage sorting can lead to misidentifications when relying solely on barcode sequences. Here, we propose a new Niche‐model‐Based Species Identification (NBSI) method based on the idea that species distribution information is a potential complement to DNA barcoding species identifications. NBSI performs species membership inference by incorporating niche modelling predictions and traditional DNA barcoding identifications. Systematic tests across diverse scenarios show significant improvements in species identification success rates under the newly proposed NBSI framework, where the largest increase is from 4.7% (95% CI: 3.51%–6.25%) to 94.8% (95% CI: 93.19%–96.06%). Additionally, obvious improvements were observed when using NBSI on potentially ambiguous sequences whose genetic nearest neighbours belongs to another species or more than two species, which occurs commonly with species represented by single or short DNA barcodes. These results support our assertion that environmental factors/variables are valuable complements to DNA sequence data for species identification by avoiding potential misidentifications inferred from genetic information alone. The NBSI framework is currently implemented as a new R package, ‘NicheBarcoding’, that is open source under GNU General Public Licence and freely available from https://CRAN.R‐project.org/package=NicheBarcoding.

Genome resource announcement for emerging pathogen Bipolaris gigantea, isolated from Microstegium vimineum

Bipolaris gigantea is a pathogen of the invasive grass Microstegium vimineum and an emerging pathogen of other hosts such as hemp and barley, causing characteristic eyespot foliar lesions. The fungus is characterized by long pale hyaline conidia measuring about 300 to 350 μm. Previously, this pathogen was classified in the Drechslera genus, and recently reassigned as Bipolaris based on molecular identification. Here, we generated a high-quality draft genome sequence of B. gigantea isolate BGF using long and short read DNA sequencing. The assembled genome, 30.23 Mb in size with a BUSCO completeness score of 98.4%, exhibited synteny with the common grass pathogen B. sorokiniana isolate 10943. Average nucleotide identity (ANI) and single nucleotide polymorphisms (SNP) analyses revealed high sequence identity with B. gigantea isolates from Cannabis sativa fields in Kentucky. This research provides a genomic resource that will contribute to future studies to understand the host range evolution and pathogenicity of this novel pathogen.

DNA-Based Chemical Unclonable Functions for Cryptographic Anticounterfeit Tagging of Pharmaceuticals.

Counterfeit products are a problem known across many industries. Chemical products such as pharmaceuticals belong to the most targeted markets, with harmful consequences for consumer health and safety. However, many of the currently used anticounterfeit measures are associated with the packaging, with the readout method and level of security varying between different solutions. Identifiers that can be directly and safely mixed into the product to securely authenticate a batch would be desirable. For this purpose, we propose the use of chemical unclonable functions based on pools of short random DNA oligos, which allow the integration of a cryptographic authentication system into chemical products. We demonstrate and characterize a simplified workflow for readout, showing that results are robust and clearly differentiate between the correct tag and a counterfeit. As a proof of concept, we demonstrate the labeling of an acetaminophen formulation with a chemical unclonable function. The acetaminophen was successfully authenticated from a subsample of the product at a DNA admixing concentration of below 50 ng/g. Stability tests revealed that the readout is stable at room temperature for several years, exceeding the shelf life of most drug products. Our work thus shows that chemical unclonable functions are a valid alternative to state-of-the-art anticounterfeit methods, enabling a secure authentication scheme that is physically linked to the product and safe for consumption. The method is widely applicable beyond pharmaceuticals, allowing for more secure product tracing across industries.

A preliminary study on detecting human DNA in aquatic environments: Potential of eDNA in forensics

Human environmental DNA (eDNA) application have not been fully applied or adequately considered in the fields of eDNA and forensics. Nonetheless, this technique holds great potential as a complementary tool for detecting human DNA in aquatic environments, particularly in cases involving crimes connect to such environments. However, the detectability or stability of eDNA can vary depending on several factors. Therefore, this preliminary study investigates the detection and degradation rates of human eDNA, as well as the recovery of nuclear short tandem repeat (STR) profiles and mitochondrial DNA (mtDNA) sequencing, using water samples from both saltwater and freshwater sources. To conduct the experiment, whole human blood was spiked into the water samples. Water samples were then filtered using a 5 µm pore size filter, and samples were collected at various time intervals up to 23 days. A human specific qPCR assay targeting HV1 region of human mtDNA was used to detect human eDNA. Results demonstrated that human eDNA remains detectable for up to 36 hours in freshwater samples and up 84 hours in saltwater samples. The limit of detection (LOD) of human eDNA, (205 copies/µl), was achieved after 60 hours in freshwater and 180 hours in saltwater samples. Partial STR profiles could be recovered up to 24 hours for freshwater and saltwater. Results from mtDNA sequencing indicate that full mtDNA profiles could be recovered from freshwater samples up to 48 hours and remained detectable up to 72 hours in saltwater. Overall, the findings of this study underscore the importance of considering and incorporating human eDNA analysis as a valuable tool in forensic practice. By harnessing the power of eDNA, law enforcement agencies can enhance their investigation capabilities, improve the accuracy of forensic reconstructions, and ultimately contribute to the resolution of cases involving aquatic environments. Further research and validation are needed to optimize and expand the utilization of eDNA techniques in forensic investigations.

High Temperature, Isothermal Growth Promotes Close Packing and Thermal Stability in DNA-Engineered Colloidal Crystals.

We report a strategy to accelerate the synthesis and increase the crystallinity of colloidal crystals (CCs) engineered with DNA. Specifically, by holding the DNA-modified Au particle building blocks above the Tm of the DNA bonding elements (i.e., free from the particles), but slightly below the Tm of the anticipated CC during the assembly process, crystallinity is increased, and enthalpically favored phases with high degrees of facet registration are observed. We studied the utility of this approach with systems for which the commonly adopted slow-cooling approach yielded primarily amorphous aggregates. In particular, we used it to synthesize high-volume fraction CCs from large (80 nm) anisotropic nanoparticles (cubes and rhombic dodecahedra) with short (<14 nm) DNA designed to restrict the degrees of freedom for the DNA bonds and maintain the anisotropy of the particle building block. Small-angle X-ray scattering and electron microscopy studies show that the crystalline phases synthesized via this method are more thermally stable than their corresponding aggregate phases, likely due to an increased number of DNA-DNA bonds between particles. Crystal size tunability (between 0.5 and 15 μm edge lengths) and epitaxial growth were demonstrated using this strategy by modulating the NaCl concentration in tandem with previously synthesized CC nuclei. Taken together, this isothermal strategy demonstrates how to deliberately crystallize a wide variety of anisotropic colloidal materials and expands the phase space accessible to nanoparticles modified with DNA.

An electrochemical proximity assay (ECPA) for antibody detection incorporating flexible spacers for improved performance.

A clever approach for biosensing is to leverage the concept of the proximity effect, where analyte binding to probes can be coupled to a second, controlled binding event such as short DNA strands. This analyte-dependent effect has been exploited in various sensors with optical or electrochemical readouts. Electrochemical proximity assays (ECPA) are more amenable to miniaturization and adaptation to the point-of-care, yet ECPA has been generally targeted toward protein sensing with antibody-oligonucleotide probes. Antibodies themselves are also important as biomarkers, since they are produced in bodily fluids in response to various diseases or infections, often in low amounts. In this work, by using antigen-DNA conjugates, we targeted an ECPA method for antibody sensing and showed that the assay performance can be greatly enhanced using flexible spacers in the DNA conjugates. After adding flexible polyethylene glycol (PEG) spacers at two distinct positions, the spacers ultimately increased the antibody-dependent current by a factor of 4.0 without significant background increases, similar to our recent work using thermofluorimetric analysis (TFA). The optimized ECPA was applied to anti-digoxigenin antibody quantification at concentrations ranging over two orders of magnitude, from the limit of detection of 300pM up to 50nM. The assay was functional in 90% human serum, where increased ionic strength was used to counteract double-layer repulsion effects at the electrode. This flexible-probe ECPA methodology should be useful for sensing other antibodies in the future with high sensitivity, and the mechanism for signal improvement with probe flexibility may be applicable to other DNA-based electrochemical sensor platforms.

Single-Molecule adenosine detection and chiral selectivity by DNA aptamer conformational changes using α-Hemolysin nanopore

Nucleic acid aptamers, which are short single-stranded RNA or DNA molecules, undergo conformational changes upon recognizing small molecules. However, their structural characterization is challenging due to the flexibility of single-stranded oligonucleotides. Nanopores provide a sensitive method for single-molecule analysis of molecular conformational changes as they transport through the pore. In this study, we employed α-hemolysin (α-HL) nanopore with high spatiotemporal resolution to investigate the conformational changes of DNA aptamer (ADO-23) upon binding to adenosine (Ado) molecules. Unlike the events generated by the aptamer alone, the DNA-Ado complex produced two distinct new characteristic events, indicating the formation of two unique secondary structures. By analyzing current events, the structures formed by the DNA-Ado complex were speculated to be hairpin DNA and G-quadruplex (G4) DNA. Furthermore, we demonstrated the potential of the aptamer as a chiral selector within the nanopore. The results showed that the aptamer specifically bound to D-Ado and not to L-Ado. Finally, we realized specific and sensitive detection of small molecule Ado by α-HL pore, with a detection limit as low as 10.5 nM, and successfully applied it to the detection of real human serum samples. The nanopore platform provides a powerful tool for studying small molecule-biomolecule conformational changes, and we constructed sensors for detecting adenosine through the aptamer. This work also offers a promising new approach to the recognition of enantiomers at the single-molecule level.

AptamerRunner: An accessible aptamer structure prediction and clustering algorithm for visualization of selected aptamers

Aptamers are short single-stranded DNA or RNA molecules with high affinity and specificity for targets and are generated using the iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Next-generation sequencing (NGS) revolutionized aptamer selections by allowing a more comprehensive analysis of SELEX-enriched aptamers as compared to Sanger sequencing. The current challenge with aptamer NGS datasets is identifying a diverse cohort of candidate aptamers with the highest likelihood of successful experimental validation. Herein we present AptamerRunner, an aptamer sequence and/or structure clustering algorithm that synergistically integrates computational analysis with visualization and expertise-directed decision making. The visual integration of networked aptamers with ranking data, such as fold enrichment or scoring algorithm results, represents a significant advancement over existing clustering tools by providing a natural context to depict groups of aptamers from which ranked or scored candidates can be chosen for experimental validation. The inherent flexibility, user-friendly design, and prospects for future enhancements with AptamerRunner has broad-reaching implications for aptamer researchers across a wide range of disciplines.

Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments

Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available in silico. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 μg (14–98 μg) after 5 days in 2 mL cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.

Human 8-oxoguanine glycosylase OGG1 binds nucleosome at the dsDNA ends and the super-helical locations

The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme’s function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites.

In-silico identification of simple sequence repeat (SSR) markers and phylogenetic analysis from chloroplast genomes of the genus Bambusa

Microsatellites or simple sequence repeats (SSRs), are short DNA sequences composed of repetitive units typically 1 to 6 base pairs long. These highly variable sequences are found in both protein-coding and noncoding DNA regions and exhibit a high degree of polymorphism. In the present study, chloroplast genomes of five species of Bambusa viz. B. bambos, B. multiplex, B. teres, B. vulgaris, and B. tulda, along with three outgroup species viz. Sorghum timorense, Hordeum vulgare, and Triticum aestivum were retrieved from NCBI in FASTA and GenBank formats and screened for chloroplast SSRs (cpSSRs) using MISA Perl script. The sequence analysis of the chloroplast genomes of different species of Bambusa confirmed the presence of 144 cpSSRs. The number of cpSSRs varied among the five bamboo species, with B. vulgaris exhibiting the highest count (31) and B. multiplex showing the lowest (27). Tetranucleotides were the most observed repeats in Bambusa, followed by mono-, di- and trinucleotides. Of the 144 SSR markers identified, only 16 (11.11 %) were suitable for primer design since the remaining sequences failed to produce acceptable primer pairs. An unweighted pair-group method with an arithmetic mean (UPGMA) dendrogram was constructed to analyze the genetic relationships among the genotypes. This analysis revealed three distinct clusters among the genotypes. SSR markers were found to be informative in differentiating the species of Bambusa and were likely to find potential applications in genetic fingerprinting, population genetics, conservation, and phylogenetics.

Potent and selective ‘genetic zipper’ method for DNA-programmable plant protection: innovative oligonucleotide insecticides against Trioza alacris Flor

Chemical insecticides increased the chemical burden on natural ecosystems posing environmental health risk factor. The urgent need for a more sustainable and ecological approach has produced many innovative ideas, including eco-friendly ‘genetic zipper’ method (or CUAD platform) based on contact oligonucleotide insecticides. Oligonucleotide insecticides have enjoyed success recently on many sternorrhynchans showing highly adaptable structure for distinct insect pest species and selective mode of action. In this article, we describe the efficiency of the oligonucleotide insecticides (briefly, olinscides or DNA insecticides) Alacris-11 and Laura-11, as well as their combined use in mixture (1:1), designed for control of bay sucker (Trioza alacris Flor), an important psyllid pest of noble laurel (Laurus nobilis L.). These olinscides are based on short unmodified antisense DNA oligonucleotides that target ITS2 between 5.8S rRNA and 28S rRNA in pre-rRNA (Laura-11) and 28S rRNA region in mature 28S rRNA and pre-rRNA (Alacris-11). The maximum pest mortality, observed on the 14th day of the experiment, comprised 95.01 ± 4.42% for Alacris-11, 97.16 ± 2.48% for Laura-11, and 98.72 ± 1.14% for their mixture (1:1). The control oligonucleotide CTGA-11 did not cause any significant mortality (9.38 ± 0.57%), emphasizing selectivity in the action of oligonucleotide insecticides. The results show potent and specific nature of oligonucleotide insecticides for pest control and open up new frontiers in control of economically important psyllids in agriculture and forestry, including Asian citrus psyllid (Diaphorina citri Kuwayama) and many others. Scientists can easily adopt ‘genetic zipper’ method for plethora of insect pests because DNA is a programmable molecule and provides game-changing characteristics for plant protection.Graphical

Quantum Dot Reporters Designed for CRISPR-Based Detection of Viral Nucleic Acids.

Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.

Unveiling the multifaceted interactions of antitumor drug mitoxantrone with ct-DNA through biophysical and in silico studies

Mitoxantrone, an anthraquinone derivative, is a widely used anticancer drug with its well-known ability to engage in complex interactions with DNA. Although known for its intercalating ability, the enigma surrounding its binding modes with DNA persists. The existing corpus of literature primarily focuses on mitoxantrone-DNA interactions with short DNA sequences, thereby yielding insights into its interactive nature is limited to this specific sequence. This study aims to elucidate the diverse modes with which mitoxantrone interacts with calf thymus DNA using a combination of spectroscopy, calorimetry and in silico studies. The findings from spectroscopic, calorimetric and molecular dynamic results in correlation with existing literature, unveil a fascinating narrative: mitoxantrone intercalates at lower concentrations but promotes condensation at higher concentrations. Although intercalation with side chains positioned in the minor/major groove is the major binding mode in GC-rich sequences, molecular modelling studies hint at an alternative binding mode in AT-rich sequences where it exclusively displays pure electrostatic interaction. These findings underscore the pivotal role of both drug structure and base sequence in dictating binding mode and affinity. Such insights not only deepen the understanding of structure-activity relationships but also hold promise for guiding future drug design strategies.

Use of high-resolution fluorescence in situ hybridization for fast and robust detection of SARS-CoV-2 RNAs

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (−) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Separation of telomere protection from length regulation by two different point mutations at amino acid 492 of RTEL1.

RTEL1 is an essential DNA helicase that plays multiple roles in genome stability and telomere length regulation. A variant of RTEL1 with a lysine at position 492 is associated with short telomeres in Mus spretus , while a conserved methionine at this position is found in M. musculus , which has ultra-long telomeres. In humans, a missense mutation at this position ( Rtel1 M492I ) causes a fatal telomere biology disease termed Hoyeraal-Hreidarsson syndrome (HHS). Introducing the Rtel1 M492K mutation into M. musculus shortened the telomeres of the resulting strain, termed 'Telomouse', to the length of human telomeres. Here, we report on a mouse strain carrying the Rtel1 M492I mutation, termed 'HHS mouse'. The HHS mouse telomeres are not as short as those of Telomice but nevertheless they display higher levels of telomeric DNA damage, fragility and recombination, associated with anaphase bridges and micronuclei. These observations indicate that the two mutations separate critical functions of RTEL1: M492K mainly reduces the telomere length setpoint, while M492I predominantly disrupts telomere protection. The two mouse models enable dissecting the mechanistic roles of RTEL1 and the different contributions of short telomeres and DNA damage to telomere biology diseases, genomic instability, cancer, and aging.

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Engineering the apparent quantum yield and emission rate of fluorophore molecules by coupling fluorophore dipoles with plasmon modes of gold using low frequency electric fields

Abstract Localized surface plasmons produced by gold and silver nanostructures have been utilized to enhance the intensity of fluorophore molecules. The issue with using nanostructure plasmons for fluorescence enhancement is their short-range nature (5–50 nm from the nanostructures), which limits accessibility to a few molecules. In addition, fluorophore dipoles needed to be aligned with the plasmon electric fields to maximize the fluorescence enhancement. To address these issues, we used low-frequency electric fields (&lt;5 MHz) and commercially available nanorod and nanosphere samples and studied their effectiveness in enhancing the fluorescence of fluorophore-labeled short single-stranded DNA molecules (22 bases). We demonstrated that DNA molecules and nanorod particles can effectively be manipulated around the charging frequency of DNA molecules (∼3 MHz). Nanorod particles enhanced the fluorescence emission rate by ∼50-fold. When the 3 MHz electric field was introduced, the emission rate increased to over 700-fold. We also found that the introduction of a 3 MHz electric field aided the enhancement of the intrinsic quantum yield fluorophore molecules, which resulted in over a 1000-fold fluorescence enhancement. This enhancement was due to the very high electric produced by polarized DNA dipoles at 3 MHz, which resulted in a torque on fluorophore dipoles and subsequently aligning the fluorophore dipole axis with the plasmon electric field. At a fundamental level, our results demonstrate the role of the low-frequency electric field in the fluorophore–plasmon coupling. These findings can directly be applied to many fluorescence detection systems, including the development of biosensors.