Adenine Analogue Research Articles

A DNA Aptamer for 2-Aminopurine: Binding-Induced Fluorescence Quenching.

2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various biochemical and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because stacking with the surrounding bases quench its fluorescence. 2AP-labeled DNA or RNA sequences have been used for the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases. In this study, we isolated aptamers for 2AP using the library immobilization capture-SELEX technique. A dominating aptamer family was isolated after 15 rounds of selection. The Kd values for the most abundant 2AP1 aptamer are 209 nM in a fluorescence assay and 72 nM in an isothermal titration calorimetry test. A 32 nM 2AP limit of detection was tested based on its intrinsic fluorescence change upon aptamer binding. Additionally, we conducted some mutation analysis. Furthermore, we tested the selectivity of this aptamer and discovered that it can bind adenine and adenosine with approximately 100-fold lower affinity than 2AP.

Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues.

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e.it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.

An exonuclease I-assisted quencher-free 2-aminopurine aptasensor based on a multipath paper-based device for ultrasensitive detection of kanamycin

A quencher-free multipath microfluidic paper-based analytical device (µPAD) was constructed for ultrasensitive detection of kanamycin based on exonuclease I (Exo I)-assisted signal enhancement of 2-aminopurine (2-AP). Here, Exo I, a single-stranded DNA-specific nuclease, was introduced to fully liberate 2-AP mononucleotides to greatly enhance biosensing sensitivity. 2-AP, a fluorescent adenine analogue embedded in single-stranded DNA (ssDNA), was employed as the detection signal source. The fluorescence of 2-AP is strong in the mononucleotide state, while it can have low fluorescence and even no fluorescence in ssDNA and dsDNA, respectively. The 2-AP fluorescence probe included 2-AP DNA and kanamycin aptamer. When kanamycin was present, binding occurred between kanamycin and the aptamer, leading to ssDNA, which was further digested by Exo I. In this case, free 2-AP mononucleotides were liberated, indicating strong fluorescence. In addition, the captured kanamycin was released for binding with the new aptamer, which resulted in the formation of a binding-hydrolysis-release cycle with the aid of Exo I. Under optimal conditions, this µPAD exhibited sensitive and multipath detection of kanamycin at concentrations as low as 1.26 × 10−14 M with a wide range of 10−13–10−7 M. Furthermore, satisfactory results were achieved for analysing spiked kanamycin in milk and honey samples. This strategy is a very promising tool for monitoring antibiotics and evaluating the safety of animal-derived foods.

Probing and perturbing riboswitch folding using a fluorescent base analogue.

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ1 . We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.

The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis

ABSTRACT Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.

Investigation of hydrogen bonding in small nucleobases using DFT, AIM, NCI and NBO technique

In this work hydrogen bonded base pairing properties of two adenine analogue nucleobases and thymine were investigated. wB97XD/6-311++G(2d,2p) level of theory was employed to optimize the monomers and the base pairs in different orientations. Atoms In Molecule, AIM analysis was carried out to analyse the individual hydrogen bonds in the studied complexes. NH…N and OH…N were found to be major contributors to the overall interaction energy than O…HC and N…HC bonds. Non Covalent Interaction, NCI and Reduced Density Gradient, RDG plots revealed that interactions are mainly weak and intermediate in nature. Finally Natural Bond Orbital, NBO calculations were performed to calculate the second order perturbation energy E(2) which helped to understand the interaction between electron donor and acceptors in the individual complexes.

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.

Raman Spectroscopic and DFT Study of COA-Cl and Its Analogues.

COA-Cl is a newly synthesized adenosine analogue that exhibits various physiological activities. Its angiogenic, neurotropic, and neuroprotective potencies make it promising for the development of medicines. In this study, we show Raman spectroscopic study of COA-Cl to elucidate molecular vibrations and related chemical properties. Density functional theory calculations were combined with the Raman spectroscopic data to understand the details of each vibrational mode. Comparative analysis with adenine, adenosine, and other nucleic acid analogues enabled identification of unique Raman peaks originating from the cyclobutane moiety and chloro group of COA-Cl. This study provides fundamental knowledge and crucial insights for further development of COA-Cl and related chemical species.

Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA.

Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.

Preliminary Computational Analysis of Pyrazinamide-Based Derivatives Reveals Possible Inhibition of SARS-CoV-2 RNA-Dependent RNA Polymerase, and Their Possible Use as Antiviral Agents

Pyrazinamide is a pyrazine analog currently used to treat tuberculosis. It shares a core structure similar to that of favipiravir, which is a promising drug candidate that may inhibit the SARS-CoV-2 RNA-dependent RNA polymerase. This feature could be an opportunity for further drug development of anti-COVID-19 medication starting from a pyrazinamide core structure. This study aimed to determine and predict the most effective pyrazinamide-based analogs against the SARS-CoV-2 RNA-dependent RNA polymerase by using combined ligand- and structure-based computational analysis. This study performed a rational in silico study to screen pyrazinamide-like molecules from a commercially available ZINC database, with a similarity score higher than 0.40, and then these screened for acceptable pharmacokinetic properties, and then to further conduct molecular docking analysis with SARS-CoV-2 RNA-dependent RNA polymerase. The results showed that compound 12, having a dichloropyrimidine core structure had a similarity score of 0.446. It exerted the most binding affinity with RNA-dependent RNA polymerase, with estimated docking scores of −5.72, −5.25, −7.06, −7.00 and −4.63 kcal/mol in intact, ribosylated, mono-phosphoribosylated, di-phosphoribosylated and tri-phosphoribosylated forms, respectively. Watson-Crick base-pairing of compound 12 indicated that it favored binding with the uracil nucleoside of the RNA template. Compound 12 was confirmed as the lead compound, being a pyrazinamide-like molecule, and so might be a most promising candidate molecule, as an adenine analog RNA-dependent RNA polymerase inhibitor. It is suggested that the antiviral effect of this lead compound should be studied further as part of a drug discovery and development process. HIGHLIGHTS In silico analysis was performed to screen pyrazinamide-like compounds against the RNA-dependent RNA polymerase Pharmacokinetics and drug-likeliness properties of fourteen preselected compounds were assessed by using SwissADME with favorable results Among these preselected compounds, compound 12 showed the best docking score in almost all of its phosphoribosylated forms GRAPHICAL ABSTRACT

Base-Stacking Heterogeneity in RNA Resolved by Fluorescence-Detected Circular Dichroism Spectroscopy.

RNA plays a critical role in many biological processes, and the structures it adopts are intimately linked to those functions. Among many factors that contribute to RNA folding, van der Waals interactions between adjacent nucleobases stabilize structures in which the bases are stacked on top of one another. Here, we utilize fluorescence-detected circular dichroism spectroscopy (FDCD) to investigate base-stacking heterogeneity in RNA labeled with the fluorescent adenine analogue 2-aminopurine (2-AP). Comparison of standard (transmission-detected) CD and FDCD spectra reveals that in dinucleotides, 2-AP fluorescence is emitted almost exclusively by unstacked molecules. In a trinucleotide, some fluorescence is emitted by a population of stacked and highly quenched molecules, but more than half originates from a minor ∼10% population of unstacked molecules. The combination of FDCD and standard CD measurements reveals the prevalence of stacked and unstacked conformational subpopulations as well as their relative fluorescence quantum yields.

Fluorescent adenine analogues with ESPT characteristic utilized for real-time detecting DNA adduct

The 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG) is the representative damaged nucleoside that may increase the risk of developing diseases. Accordingly, the selective detection of 8-oxoG in DNA with minimal disturbance to the native structure is important to have an in-depth understanding of the formation mechanism and becomes an attractive tool for genomic research. To identify the DNA adduct in real-time efficiently, a series of quasi-intrinsic optical probes are performed based on the natural adenine, which has preference to form a stable base pair with 8-oxoG in the syn conformation. The calculations revealed that the A-analogues in solution could bring red-shifted absorption spectra and bright photoluminescence arisen from the additional π-conjugation by means of fluorophore modification and the ring expansion. Especially, A1 possesses large Stokes shifts and the highest fluorescence intensity in emission, which is proposed as the biosensor to monitor the optical changes in the presence and absence of the considered 8-oxoG. It is found that the fluorescence is insensitive to base pairing with thymine, while the excited state intermolecular proton transfer (ESPT) induced efficient fluorescence quenching is observed upon pairing with the 8-oxoG. To evaluate the direct usefulness of the bright adenine analogues in biological environment, we further examined the influences of linking deoxyribose on the absorption and emission, which are consistent with the experimental data.

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription. Our results showed that Z exhibited no mutagenic yet substantial inhibitory effects on transcription mediated by purified T7 RNA polymerase and by human RNA polymerase II in HeLa nuclear extracts and in human cells. A structurally related adenine analog, 2-aminopurine (2AP), strongly blocked T7 RNA polymerase but did not impede human RNA polymerase II in vitro or in human cells, where no mutant transcript could be detected. The lack of mutagenic consequence and the presence of a strong blockage effect of Z on transcription suggest a role of Z in transcriptional regulation. Z is also subjected to removal by transcription-coupled nucleotide-excision repair (TC-NER), but not global-genome NER in human cells. Our findings provide new insight into the effects of Z on transcription and its potential biological functions.

Quinazoline-Based Analog of Adenine As an Antidote Against MLL-rearranged Leukemia Cells: Synthesis, Inhibition Assays and Docking Studies

Background: Post-translational modifications of histones constitute a dynamic process impacting gene expression. A well-studied modification is lysine methylation. Among the lysine histone methyltransferases, DOT1L is implicated in various diseases, making it a very interesting target for drug discovery. DOT1L has two substrates, the SAM cofactor that gives the methyl group and the lysine H3K79 substrate. Results: Using molecular docking, the authors explored new bisubstrate analogs to enlarge the chemical landscape of DOT1L inhibitors. The authors showed that quinazoline can successfully replace the adenine in the design of bisubstrate inhibitors of DOT1L, showing similar activity compared with the adenine derivative but with diminished cytotoxicity. Conclusion: The docking model is validated together with the use of quinazoline in the design of bisubstrate inhibitors.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers’ RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.

EdU Incorporation To Assess Cell Proliferation and Drug Susceptibility in Naegleria fowleri.

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri, and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study, we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2'-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor-linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy, and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (7-deaza-2'-deoxy-7-ethynyladenosine), an adenine analog, also was incorporated by N. fowleri but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

The oxidation of guanine by photoionized 2-aminopurine

2-Aminopurine (2AP) is a fluorescent nucleobase analogue of the DNA nucleobase adenine and can be inserted in place of adenine into nucleic acids with minimal structural perturbation. The inserted 2-aminopurine can be selectively excited, and the fluorescence observed used as a sensitive probe for local structure, in addition selective ionization of 2-aminopurine within a nucleic acid is possible and once formed the ionized 2-aminopurine will oxidize nearby guanine bases, providing a route to study charge transfer within nucleic acids. Time resolved infrared spectroscopy is a powerful tool to study oxidation processes on picosecond, and longer timescales. There is a lack of availability of high quality IR spectra in the literature of the individual, short lived, photoproducts, required to fully elucidate the mechanism of the interaction of photoionized 2-aminopurine with guanine. Density functional theory (DFT) methods (B3LYP/6–31G+(d) and EDF1/6–31G+(d)) have been used to calculate the energies and infrared spectra of 2-aminopurine, guanine and potential photoproducts that may result from photoionization reactions between 2-aminopurine and guanine. Direct comparison can therefore be made between areas of bleaching of bands in the IR spectra of 2-aminopurine or guanine and the appearance of new transient bands of potential photoproducts. These potential photoproducts could include deprotonated neutral radical species, deprotonated anion species, radical cations and radical anions. It was found in the current work that the key intermediates in the oxidation reaction between photoionized 2-aminopurine and guanine were: 2AP•(− H ,N2) (formed from deprotonation from the 2AP •+ amino group); G•(− H ) (formed from deprotonation from the N1 nitrogen, or possibly the amino group, of the initially formed G •+ ), and 2AP − (− H ,N2) (formed from the reduction of 2AP•(− H ,N2) by guanine).

Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

Fluorescent base analogues (FBAs) are widely used as spectroscopic tools to probe RNA and DNA structure. Because of their chemical similarity with the naturally occurring bases, they are typically assumed to exert minimal perturbations on the systems they are used to study. However, this cannot be taken for granted. We have systematically investigated the effects of FBA substitution on the folding of the preQ1 riboswitch, which regulates gene expression in response to the ligand preQ1. We placed a popular FBA, the adenine analogue 2-aminopurine (2-AP), at different locations within a stretch of six adenine residues termed “L3”.

Control of Intermolecular Photoinduced Electron Transfer in Deoxyadenosine-Based Fluorescent Probes.

In this work, we report on the Photoinduced Electron Transfer (PET) reaction between a donor (adenine analogue) and an acceptor (3-methoxychromone dye, 3MC) in the context of designing efficient fluorescent probes as DNA sensors. Firstly, Gibbs energy was investigated in disconnected donor-acceptor systems by Rehm-Weller equation. The oxidation potential of the adenine derivative was responsible for exergonicity of the PET reaction in separated combinations. Then, the PET reaction in donor-π-acceptor conjugates was investigated using steady-state fluorescence spectroscopy, acid-mediated PET inhibition and transient absorption techniques. In conjugated systems, PET is a favorable pathway of fluorescent quenching when an electron-rich adenine analogue (d7A) was connected to the fluorophore (3MC). We found that formation of ground-state complexes even at nm concentration range dominated the dye photophysics and generated poorly emissive species likely through intermolecular PET from d7A to 3MC. On the other hand, solution acidification disrupts complexation and turns on the dye emission. Bridging an electron-poor adenine analogue with high oxidation potential (8 d7A) to 3MC presenting low reduction potential is another alternative to prevent complex formation and produce highly emissive monomer conjugates.

Reactions of Adenine and Its N-Exo Substituted Analogues with Phenyl Glycidyl Ether

The features of reactions of adenine with phenyl glycidyl ether depending on the solvent nature were studied. In DMF in the presence of K2CO3, an N9-alkyl derivative, an experimental antiviral drug 9-(2-hydroxy-3-phenoxypropyl)adenine, was formed predominantly. During alkylation in acetic acid, besides N9-, N3-, and N7-alkylation products were also isolated. Alkylation of 6-[alkyl(dialkyl)amino]purines with phenyl glycidyl ether in DMF produced N-exo substituted 9-(2-hydroxy-3-phenoxypropyl)adenine analogues.