DNA Labeling Research Articles

Yolk-shell nanospheres based silane-functionalized carbon dots: Designed synthesis and application in highly selective determination of aflatoxin biosynthesis-related genes nor-1 in cereal crops

This study presented advanced monitoring approaches for the aflatoxin gene cluster in Aspergillus flavus and Aspergillus parasiticus, which are effective tools for controlling aflatoxin proliferation in the food chain. A novel approach involved a ratiometric fluorescence probe based on yolk-shell nanospheres (bCDs@SiO2@gQDs). The probe's fluorescence was activated by DNA hybridization with the modified bCDs@SiO2@gQDs and gold nanoparticles (AuNPs), functioning through donor–acceptor pairs. The exceptional properties of the gQDs and the high FRET efficiency of this system established a functionalized platform for DNA labeling. When the nor-1 gene was present, FRET between the gQDs and AuNPs triggered fluorescence quenching, enabling nor-1 gene detection. The yolk-shell nanostructure of the fluorescent nanoprobes provided an internal reference, reducing experimental errors due to background noise, instrumentation factors, and probe concentration. This allowed for accurate and highly sensitive quantitative determination of the nor-1 gene. The ratiometric fluorescence assay demonstrated a broad linear range (0.1–100 nM) and a low detection limit (12.83 pM) for the nor-1 gene. Tests on non-contaminated maize samples spiked with various concentrations of the nor-1 gene achieved recovery rates between 95.43 % and 101.46 %. This biosensor represents a significant advancement in food safety, offering a simple, rapid, and sensitive method for monitoring agricultural products.

Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

Analytical modeling and numerical simulation of graded JAM Split Gate-All-Around (GJAM-SGAA) Bio-FET for label free Avian Influenza antibody and DNA detection

This manuscript presents the analytical model of a novel biosensor called Graded JAM Split Gate-All-Around (GJAM-SGAA) Bio-FET for the detection of Avian Influenza antibody and DNA. The GJAM-SGAA Bio-FET utilizes a silicon Gate-All-Around FET which operates in the Junctionless Accumulation Mode (JAM), with a graded doping in the channel. This Bio-FET also features a gate underlap double sided cavity that enables detection of biomolecules without the use of labels. Gate underlap cavities overcome the fabrication complexity of nanocavities and provides structural stability. A comparative analysis between the GJAM-SGAA Bio-FET and non-graded JAM Split Gate-All-Around FET (SGAA-FET) demonstrates that the proposed BioFET has 5.72 times higher ION current sensitivity, 5.3 times increase in threshold voltage sensitivity (SVth), and 2.13 × 102 times improvement in switching ratio sensitivity for avian influenza biomolecule. The SVth of proposed biosensor is compared with the existing biosensors and found that a triple-metal engineered gate and graded doped channel substantially boosts the sensitivity of proposed biosensor.

Chemical Expansion of the Methyltransferase Reaction: Tools for DNA Labeling and Epigenome Analysis.

DNA is the genetic matter of life composed of four major nucleotides which can be further furnished with biologically important covalent modifications. Among the variety of enzymes involved in DNA metabolism, AdoMet-dependent methyltransferases (MTases) combine the recognition of specific sequences and covalent methylation of a target nucleotide. The naturally transferred methyl groups play important roles in biological signaling, but they are poor physical reporters and largely resistant to chemical derivatization. Therefore, an obvious strategy to unlock the practical utility of the methyltransferase reactions is to enable the transfer of "prederivatized" (extended) versions of the methyl group.However, previous enzymatic studies of extended AdoMet analogs indicated that the transalkylation reactions are drastically impaired as the size of the carbon chain increases. In collaborative efforts, we proposed that, akin to enhanced SN2 reactivity of allylic and propargylic systems, addition of a π orbital next to the transferable carbon atom might confer the needed activation of the reaction. Indeed, we found that MTase-catalyzed transalkylations of DNA with cofactors containing a double or a triple C-C bond in the β position occurred in a robust and sequence-specific manner. Altogether, this breakthrough approach named mTAG (methyltransferase-directed transfer of activated groups) has proven instrumental for targeted labeling of DNA and other types of biomolecules (using appropriate MTases) including RNA and proteins.Our further work focused on the propargylic cofactors and their reactions with DNA cytosine-5 MTases, a class of MTases common for both prokaryotes and eukaryotes. Here, we learned that the 4-X-but-2-yn-1-yl (X = polar group) cofactors suffered from a rapid loss of activity in aqueous buffers due to susceptibility of the triple bond to hydration. This problem was remedied by synthetically increasing the separation between X and the triple bond from one to three carbon units (6-X-hex-2-ynyl cofactors). To further optimize the transfer of the bulkier groups, we performed structure-guided engineering of the MTase cofactor pocket. Alanine replacements of two conserved residues conferred substantial improvements of the transalkylation activity with M.HhaI and three other engineered bacterial C5-MTases. Of particular interest were CpG-specific DNA MTases (M.SssI), which proved valuable tools for studies of mammalian methylomes and chemical probing of DNA function.Inspired by the successful repurposing of bacterial enzymes, we turned to more complex mammalian C5-MTases (Dnmt1, Dnmt3A, and Dnmt3B) and asked if they could ultimately lead to mTAG labeling inside mammalian cells. Our efforts to engineer mouse Dnmt1 produced a variant (Dnmt1*) that enabled efficient Dnmt1-directed deposition of 6-azide-hexynyl groups on DNA in vitro. CRISPR-Cas9 editing of the corresponding codons in the genomic Dnmt1 alleles established endogenous expression of Dnmt1* in mouse embryonic stem cells. To circumvent the poor cellular uptake of AdoMet and its analogs, we elaborated their efficient internalization by electroporation, which has finally enabled selective catalysis-dependent azide tagging of natural Dnmt1 targets in live mammalian cells. The deposited chemical groups were then exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. These findings offer unprecedented inroads into studies of DNA methylation in a wide range of eukaryotic model systems.

Glass Nanopipette-Based Plasmonic SERS Platform for Single-Cell MicroRNA-21 Sensing during Apoptosis.

As one of the most widely distributed microRNAs, microRNA-21 (miRNA-21) significantly regulates target genes' expression levels and participates in many cellular and intercellular activities, and its abnormal expression is always related to some diseases, especially cancer. Hence, detecting miRNA-21, as a biomarker, at the single-cell level helps us to reveal cell heterogeneity and expression level variation during the state change of cells. In this study, we constructed a gold nanoparticles nanomembrane (AuNPs-NM)-modified plasmonic glass nanopipette (P-nanopipette) surface-enhanced Raman scattering (SERS) sensing platform to sensitively detect content variation of the intracellular miRNA-21 during the electrostimulus (ES)-induced apoptosis process. The cytoplasm-located miRNA-21 was first extracted by using the extraction DNA (HP1)-modified P-nanopipette through a hybridization chain reaction (HCR). The nanopipette was then incubated with a labeling DNA (HP2) and reporter 4-MBA-modified Raman tag. The Raman signal (collected from the tip area near the orifice within 1 μm) showed a good response to the content variation of intracellular miRNA-21 under ES, and the proposed single-cell SERS detection platform provides a simple way to study intracellular substance change and evaluate cancer treatment outcomes.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

A flow cytometry method for safe detection of bacterial viability.

Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris-Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non-CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability-DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram+ , Gram- ) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization.

Evaluation of domestic animal sperm head morphology via flow cytometric DNA labelling and pulse shape analysis using bull and stallion spermatozoa as model species.

The aim of the present study was to test a rapid, robust flow cytometric technique for the detection of sperm head abnormalities of domestic bulls and stallions. The so-called PulSA approach detects the pulse profiles of propidium-iodide labelled spermatozoa. In the first experiment, species-specific threshold values were established on sperm samples that were tested for sperm head abnormalities with a classic visual morphology analysis. In the second experiment, serial mixtures of bull and stallion spermatozoa mimicking different percentages of sperm head abnormalities were analysed. Non-metric multidimensional scaling showed a clear separation between the normal and mixed samples. The PulSA approach may be a useful tool in identifying sub- or infertile breeding males as well as in studying the evolutionary aspects of sperm morphology and morphometry.

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

A Triphosphate Pronucleotide (TriPPPro)Reporter with Optimized Cell-Permeable Dyes for Metabolic Labeling of Cellular and Viral DNA in Living Cells.

The metabolic labeling of nucleic acids in living cells is highly desirable to track the dynamics of nucleic acid metabolism in real-time and has the potential to provide novel insights into cellular biology as well as pathogen-host interactions. Catalyst-free inverse electron demand Diels-Alder reactions (iEDDA) with nucleosides carrying highly reactive moieties such as axial 2-trans-cyclooctene (2TCOa) would be an ideal tool to allow intracellular labeling of DNA. However, cellular kinase phosphorylation of the modified nucleosides is needed after cellular uptake as triphosphates are not membrane permeable. Unfortunately, the narrow substrate window of most endogenous kinases limits the use of highly reactive moieties. Here, we apply our TriPPPro (triphosphate pronucleotide) approach to directly deliver a highly reactive 2TCOa-modified 2'-deoxycytidine triphosphate reporter into living cells. We show that this nucleoside triphosphate is metabolically incorporated into de novo synthesized cellular and viral DNA and can be labeled with highly reactive and cell-permeable fluorescent dye-tetrazine conjugates via iEDDA to visualize DNA in living cells directly. Thus, we present the first comprehensive method for live-cell imaging of cellular and viral nucleic acids using a two-step labeling approach.

Triphosphat‐Pronucleotid (TriPPPro)‐Reporter mit Optimierten Zellpermeablen Farbstoffen zum Metabolischen Labeling von Zellulärer und Viraler DNA in Lebenden Zellen

AbstractDas metabolische Labeling von Nucleinsäuren in lebenden Zellen ist von äußerstem Interesse, um die Dynamik des Nucleinsäure‐Stoffwechsels in Echtzeit zu verfolgen und hat somit das Potenzial, neue Einblicke in die Zellbiologie sowie in Pathogen‐Wirtszell‐Interaktionen zu liefern. Eine katalysatorfreie Diels–Alder‐Reaktion mit inversem Elektronenbedarf (iEDDA) mit Nucleosiden, die eine hochreaktive Gruppe wie das axiale 2‐trans‐Cycloocten (2TCOa) tragen, ist ideal geeignet, um die intrazelluläre Markierung von DNA zu ermöglichen. Da Triphosphate von Nucleosiden nicht membranpermeabel sind, ist, nach der Aufnahme in die Zelle, die Phosphorylierung der modifizierten Nucleoside durch zelluläre Kinasen erforderlich. Leider schränkt dabei das enge Substratfenster der meisten endogenen Kinasen die Verwendung von Nucleosid‐Analoga mit hochreaktiven Gruppen ein. Hier wenden wir unseren TriPPPro‐ (Triphosphat‐Pronucleotid) Ansatz an, um einen hoch reaktiven 2TCOa‐modifizierten 2′‐Desoxycytidintriphosphat‐Reporter direkt in lebenden Zellen freizusetzen. Wir zeigen, dass dieses Nucleosidtriphosphat metabolisch in de novo synthetisierte zelluläre und virale DNA eingebaut wird und mit hochreaktiven und zellpermeablen Fluoreszenzfarbstoff‐Tetrazin‐Konjugaten via iEDDA markiert werden kann, um DNA in lebenden Zellen direkt sichtbar zu machen. Damit stellen wir die erste umfassende Methode zur Visualisierung zellulärer und viraler Nucleinsäuren in lebenden Zellen vor.

Quantifying cellular dynamics in mice using a novel fluorescent division reporter system.

The dynamics of cell populations are frequently studied in vivo using pulse-chase DNA labeling techniques. When combined with mathematical models, the kinetic of label uptake and loss within a population of interest then allows one to estimate rates of cell production and turnover through death or onward differentiation. Here we explore an alternative method of quantifying cellular dynamics, using a cell fate-mapping mouse model in which dividing cells can be induced to constitutively express a fluorescent protein, using a Ki67 reporter construct. We use a pulse-chase approach with this reporter mouse system to measure the lifespans and division rates of naive CD4 and CD8 T cells using a variety of modeling approaches, and show that they are all consistent with estimates derived from other published methods. However we propose that to obtain unbiased parameter estimates and full measures of their uncertainty one should simultaneously model the timecourses of the frequencies of labeled cells within both the population of interest and its precursor. We conclude that Ki67 reporter mice provide a promising system for modeling cellular dynamics.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Artificial DNA in hydrology

AbstractThe use of artificial DNA (artDNA) in hydrological applications is becoming increasingly popular, either in dissolved form (dissolved artDNA) or encapsulated and protected by a layer (encDNA). DNA can be detected even at low concentrations and offers the ability to create numerous uniquely identifiable DNA labels, making it ideal for a wide range of multi‐tracer applications. A literature review revealed that in streams, the breakthrough curve of artDNA is visually similar to that of a conservative tracer in terms of time to rise, time to peak, and dispersion coefficient. In saturated porous or fractured media, the time of first arrivals and time to peak of artDNA are usually earlier than that of a conservative tracer, indicating size exclusion of both dissolved artDNA and encDNA. Transport in subsurface media can be described by one‐site or two‐site kinetic attachment. The recovery of artDNA in environmental systems is always less than 100% due to adsorption and decay. Although the processes responsible for both are known, yet they cannot be quantified or predicted in mass balance approaches. Despite these limitations, artDNA can be used in various hydrological applications in environmental studies and engineering. Finally, attention should focus on the use of rapid detection of DNA tracers in the field, on upscaling of DNA production, and on increasing the efficiency of the DNA encapsulation process.This article is categorized under: Science of Water > Hydrological Processes Science of Water > Water Quality Science of Water > Methods

Watson-Crick Base Pairs with Protecting Groups: The 2-Amino Groups of Purine- and 7-Deazapurine-2,6-Diamine as Target Sites for DNA Functionalization by Selective Nucleobase Acylation.

The recognition of Watson-Crick base pairs carrying nucleobase protecting groups is reported as a new approach for DNA functionalization. The 2-amino groups of purine- and 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides served as molecular targets for this functionalization. The 2-amino group withstands oligonucleotide deprotection with ammonia, whereas all other protecting groups are released after chemical DNA synthesis. On this basis, a method was developed for the selective functionalization of oligonucleotides at the 2-position of purines and 7-deazapurines. Melting experiments and Tm values obtained from hybridization studies revealed that duplexes with protected (2-amino-dA) and (2-amino-7-deaza-dA)-dT base pairs are as stable as their nonprotected counterparts. Mismatch discrimination of protected purine- and 7-deazapurine-2,6-diamine DNA was superior to that of nonprotected DNA. Click functionalization in the minor groove of the DNA double helix became accessible via introduction of heptynoyl protecting groups bearing a terminal triple bond. Click reactions with pyrene azide validated the usability. DNA conjugates with bulky pyrene residues at the 2-position (minor groove) developed the same high stability as those functionalized at the 7-position (major groove). This demonstrates the potential of our new method using protected base pairs for DNA functionalization and paves the way for new DNA labeling strategies.

Rapid and Sensitive Detection of Antibiotic Resistance Genes by Utilizing TALEs as a Diagnostic Probe with 2D-Nanosheet Graphene Oxide.

As antibiotic resistance has risen as one of the major health concerns associated with infectious diseases due to the reduced efficacy of antibiotics, rapid and sensitive detection of antibiotic resistance genes is critical for more effective and faster treatment of infectious diseases. A class of programmable DNA-binding domains called transcriptional activator-like effectors (TALEs) provides a novel scaffold for designing versatile DNA-binding proteins due to their modularity and predictability. Here, we developed a simple, rapid, and sensitive system for detecting antibiotic resistance genes by exploring the potential of TALE proteins for the creation of a sequence-specific DNA diagnostic along with 2D-nanosheet graphene oxide (GO). TALEs were engineered to directly recognize the specific double-stranded (ds) DNA sequences present in the tetracycline resistance gene (tetM), avoiding the need for dsDNA denaturation and renaturation. We take advantage of the GO as an effective signal quencher to quantum dot (QD)-labeled TALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbed on the GO surface, which will bring QDs in close proximity to GO. Due to the fluorescence quenching ability of GO, QDs are expected to be quenched by GO via fluorescence resonance energy transfer (FRET). QD-labeled TALE binding to the target dsDNA would lead to the conformational change, which would result in dissociation from the GO surface, thereby restoring the fluorescence signal. Our sensing system was able to detect low concentrations of dsDNA sequences in the tetM gene after only 10-minute incubation with the DNA, providing a limit of detection as low as 1 fM of Staphylococcus aureus genomic DNA. This study demonstrated that our approach of utilizing TALEs as a new diagnostic probe along with GO as a sensing platform can provide a highly sensitive and rapid method for direct detection of the antibiotic resistance gene without requiring DNA amplification or labeling.

Fluorescent labeling of RNA and DNA on the Hoogsteen edge using sulfinate chemistry.

We have devised a single pot, low-cost method to add azide groups to unmodified nucleic acids without the need for enzymes or chemically modified nucleoside triphosphates. This involves reacting an azide-containing sulfinate salt with the nucleic acid, leading to replacement of C-H bonds on the nucleobase aromatic rings with C-R, where R is the azide-containing linker derived from the original sulfinate salt. With the addition of azide functional groups, the modified nucleic acid can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the fluorescent labeling of a wide variety of nucleic acids, including natively folded RNAs, under mild conditions with minimal effects upon biochemical function and ribozyme catalysis. To demonstrate this, we show that a pair of labeled complementary ssDNA oligonucleotides (oligos) can hybridize to form dsDNA, even when labeled with multiple fluorophores per oligo. In addition, we also demonstrate that two different group II introns can splice when prelabeled internally with fluorophores, using our method. Broadly, this demonstrates that sulfinate modification of RNA is compatible with ribozyme function and Watson-Crick pairing, while preserving the labile backbone.

Synthesis of S-Adenosyl-L-Methionine Analogs with Extended Transferable Groups for Methyltransferase-Directed Labeling of DNA and RNA.

S-Adenosyl-L-methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium-bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases, enabling covalent derivatization and subsequent labeling of their cognate target sites in DNA or RNA. Although AdoMet analogs with saturated aliphatic chains are less popular than propargylic ones, they can be useful for dedicated studies that require certain chemical derivatization. Here we describe synthetic procedures for the preparation of two AdoMet analogs, one with a transferable 6-azidohex-2-ynyl group (carrying an activating C≡C triple bond and a terminal azide functionality), and the other one with a transferable ethyl-2,2,2-d3 group (an isotope-labeled aliphatic moiety). Our synthetic approach is based on direct chemoselective alkylation of S-adenosyl-L-homocysteine at sulfur with a corresponding nosylate or triflate, respectively, under acidic conditions. We also describe synthetic routes to 6-azidohex-2-yn-1-ol and conversion of the alcohols to corresponding nosylate and triflate alkylators. Using these protocols, the synthetic AdoMet analogs can be prepared within 1 to 2 weeks. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 6-azidohex-2-yn-1-ol Basic Protocol 2: Synthesis of 4-nitrobenzenesulfonate Basic Protocol 3: Synthesis of trifluoromethanesulfonates Basic Protocol 4: S-Alkylation of AdoHcy with sulfonates Basic Protocol 5: Purification and characterization of AdoMet analogs.

Highly Fluorescent 2‐Aminopurine Derivatives: Synthesis, Photo‐physical Characterization, and Preliminary Cytotoxicity Evaluation

AbstractNew fluorescent nucleobase analogues (FBAs) are emerging as extraordinarily useful tools for DNA labelling technologies. The highly fluorescent adenine analogue 2‐aminopurine (2AP) is still the most used within the few hundreds of newly FBAs synthesized, but its excitation in the UV region demands for high energy sources endangering living cells. New and highly fluorescent 2AP derivatives, 2‐amino‐6‐cyanopurines, were obtained using simpler but efficient synthesis method. All the new compounds exhibit advantageous photophysical properties over 2AP, showing absorption and emission bands ranging the visible region (blue‐green region), high fluorescence quantum yields and Stokes’ shifts, especially in non‐protic organic solvents. Density Functional Theory calculations (DFT) of electronic and vibrational structure were performed, allowing to predict absorption and emission spectra. In addition, these 2‐amino‐6‐cyanopurines exhibit little to no toxicity in assays using yeast cells.