Fluorescent Nucleobase Analogues Research Articles

Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.

BODIPY-fused uracil: synthesis, photophysical properties, and applications.

Fluorescent nucleobase and nucleic acid analogs are important tools in chemical and molecular biology as fluorescent labelling of nucleobases has applications in cellular imaging and anti-tumor activity. Boron-dipyrromethene (BODIPY) dyes exhibiting high brightness and good photostability are extensively used as fluorescent labelling agents and as type II photosensitizers for photodynamic therapy. Thus, the combination of nucleobases and BODIPY to obtain new compounds with both anti-tumor activity and fluorescent imaging functions is the focus of our research. We synthesized two new nucleobase analogs 1 and 2 by fusing the BODIPY core directly with uracil which resulted in favorable photophysical properties and high emission quantum efficiencies particularly in organic solvents. Further, we explored the newly synthesized derivatives, which possessed good singlet oxygen generation efficiencies and bio-compatibility, as potential PDT agents and our results show that they exhibit in vitro anti-tumor activities.

Screening for Novel Fluorescent Nucleobase Analogues Using Computational and Experimental Methods: 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) as a Case Study.

Novel fluorescent nucleic acid base analogues (FBAs) with improved optical properties are needed in a variety of biological applications. 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) is structural analogue of two existing highly fluorescent FBAs, 2-aminopurine (2AP) and 8-vinyladenine (8VA), and can therefore be expected to have similar base pairing as well as better optical properties compared to its counterparts. In order to determine the absorption and fluorescence properties of 2A6Cl8VP, as a first step, we used TD-DFT calculations and the polarizable continuum model for simulating the solvents and computationally predicted absorption and fluorescence maxima. To test the computational predictions, we also synthesized 2A6Cl8VP and measured its UV/vis absorbance, fluorescence emission, and fluorescence lifetime. The computationally predicted absorbance and fluorescence maxima of 2A6Cl8VP are in reasonable agreement to the experimental values and are significantly redshifted compared to 2AP and 8VA, allowing for its specific excitation. The fluorescence quantum yield of 2A6Cl8VP, however, is significantly lower than those of 2AP and 8VA. Overall, 2A6Cl8VP is a novel fluorescent nucleobase analogue, which can be useful in studying structural, biophysical, and biochemical applications.

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.

Evaluation by Experimentation and Simulation of a FRET Pair Comprising Fluorescent Nucleobase Analogs in Nucleosomes.

Förster resonance energy transfer (FRET) is an attractive tool for understanding biomolecular dynamics. FRET-based analysis of nucleosomes has the potential to fill the knowledge gaps between static structures and dynamic cellular behaviors. Compared with typical FRET pairs using bulky fluorophores introduced by flexible linkers, fluorescent nucleoside-based FRET pair has great potential since it can be fitted within the helical structures of nucleic acids. Herein we report on the construction of nucleosomes containing a nucleobase FRET pair and the investigation of experimental and theoretical FRET efficiencies through steady-state fluorescence spectroscopy and calculation based on molecular dynamics simulations, respectively. Distinguishable experimental FRET efficiencies were observed depending on the positions of FRET pairs in nucleosomal DNA. The tendency could be supported by theoretical study. This work suggests the possibility of our approach to analyze structural changes of nucleosomes by epigenetic modifications or internucleosomal interactions.

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.

Pulse-shaped broadband multiphoton excitation for single-molecule fluorescence detection in the far field

Multiphoton excitation of fluorescence has many potential advantages over resonant (one-photon) excitation, but the method has not found widespread use for ultrasensitive applications. We recently described an approach to the multiphoton excitation of single molecules that uses a pulse shaper to compress and tailor pulses from an ultrafast broadband laser in order to optimise the brightness and signal-to-background ratio following non-linear excitation. Here we provide a detailed description of the setup and illustrate its use and potential by optimising two-photon fluorescence of a common fluorophore, rhodamine 110, at the single-molecule level. We also show that a DNA oligonucleotide labelled with a fluorescent nucleobase analogue, tC, can be detected using two-photon FCS, whereas one-photon excitation causes rapid photobleaching. The ability to improve the signal-to-background ratio and to reduce the incident power required to attain a given brightness can be applied to the multiphoton excitation of any fluorescent species, from small molecules with low multiphoton cross sections to the brightest nanoparticles.

Screening for novel fluorescent nucleobase analogs (FBAs) using computational and experimental methods ‐ 2‐amino‐8‐vinylpurine (2A8VP), as a Case study

Fluorescent nucleobase analogs (FBAs) are molecular reporters that are structurally similar to naturally occurring nucleotides but that have significantly higher emission quantum yields and red‐shifted absorption and emission maxima. These reporter molecules play an important role in the study of structure and dynamics of nucleic acids and proteins by providing deeper insight into the mechanisms of various biological processes. The experimental synthesis and characterization of FBAs usually precedes their computational characterization. Here, we start from computation to screen for fluorescent emission wavelength and fluorescence intensity thus reducing the time and material needed in developing new novel fluorescent reporters. The photophysical properties of a putative fluorescent analog 2‐amino‐8‐vinylpurine (2A8VP) have been predicted using TD‐DFT theory. Based on these results, we have attempted its synthesis starting from 2‐amino‐6‐chloro‐purine nucleobase. Several blue‐emissive molecules have been obtained, one with a particularly high quantum yield. These reaction products have been characterized by NMR, LC‐MS, and HPLC to obtain their structural, absorbance, and fluorescence properties to compare them with the computed properties of the 2A8VP target molecule.

Fluorescent nucleobase analogs constructed by aldol‐type condensation: Design, properties, and synthetic optimization for fluorogenic labeling of 5‐formyluracil

AbstractFluorescent nucleosides play essential roles in designing functional nucleic acid systems for various biological applications. Therefore, expanding the scope of fluorescent nucleosides with unique properties has become an important challenge. Herein, we report novel fluorescent nucleosides constructed by aldol‐type condensation between 5‐formylpyrimidine and a fluorogenic dye. Two new fluorescent nucleobase analogs (4a and 4c), derived from 2‐dicyanomethylene‐3‐cyano‐4,5,5‐trimethyl‐2,5‐dihydrofuran and a quinolinium moiety, respectively, were determined through reaction screening, and their photophysical properties and formation kinetics were investigated. Among the two, 4a exhibits a unique fluorescence featuring a large Stokes shift (67–117 nm) and high environmental sensitivity, as evidenced by its viscosity‐induced fluorescence and aggregation‐induced emission behavior. The kinetic study revealed that 4a was readily synthesized even in aqueous solutions with pH 7 (kobs = 6.4 × 10−5/s). The overall results indicate that 4a is a promising molecule for use in fluorescent nucleic acid systems, particularly for selective fluorogenic labeling of 5‐formyluracil.

Determination of two-photon absorption in nucleobase analogues: a QR-DFT perspective

With the prevalence of fluorescence spectroscopy in biological systems, and the benefits of two-photon absorption techniques, presented here is an assessment of the two-photon accessibility of modern fluorescent nucleobase analogues utilising quadratic response DFT. Due to the complex environment experienced by these nucleobases, the two-photon spectra of each analogue has been assessed in the presence of both pi -stacked and hydrogen-bonding interactions involving the canonical nucleobases. Findings suggest that the pi -stacking environment provides a more significant effect on the spectra of the analogues studies than a hydrogen-bonding environment; analogue structures presenting high two-photon cross-section values for one or more states coincide with polycyclic extensions to preserved canonical base structure, as observed in the qA family of analogues, while analogue structures more closely resembling the structure of the base in question present a much more muted spectra in comparison. Results from this investigation have also allowed for the derivation of a number of design rules for the development of potential, two-photon specific, analogues for future use in both imaging and potential photochemical activation.Graphical abstract

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).

Single-molecule fluorescence detection of a tricyclic nucleoside analogue.

Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε442 = 20 000 M−1 cm−1 and Φem,540 = 0.39 in water, increasing to Φem = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

(Invited) Split G-quadruplex-based Strategy for Sensitive Detection of Pathogenic Bacteria

In this talk, I will introduce a simple, sequence-specific DNA detection method, utilizing a fluorescent 2-aminopurine (2-AP) nucleobase analogue-containing split G-quadruplex as the key detection component. In the sensor, the 2-AP-containing G-quadruplex is split into two segments and linked to a target-specific overhang sequence. The separate G-quadruplex sequences form an active G-quadruplex structure only in the presence of a complementary target DNA, which leads to a significant increase in the intensity of fluorescence from the 2-AP fluorophore. This simple, one-step, homogenous assay was successfully employed to detect target DNA with a high selectivity. In addition, the practical applicability of the detection method was demonstrated by its use in analyzing target DNAs in human serum. To the best of our knowledge, this is the first time that an investigation was carried out in which a fluorescent nucleobase analogue was incorporated into a split G-quadruplex structure and this structure was utilized as the foundation for a specific DNA sensor.

Fluorescence nucleobase analogue-based strategy with high signal-to-noise ratio for ultrasensitive detection of food poisoning bacteria

We developed a simple and ultrasensitive strategy for the identification of foodborne pathogens utilizing a fluorescent nucleobase analogue [2-aminopurine (2-AP)]-containing split G-quadruplex that binds blocker DNA. Compared to a previous strategy that did not use blocker DNA, this strategy showed a significant increase in the signal-to-noise ratio-by approximately 300%-owing to the displacement of the blocker DNA by the target DNA that induces the formation of an active G-quadruplex structure, thereby leading to a substantial increase in the 2-AP fluorescence signal. The proposed strategy was rationally combined with polymerase chain reaction, which resulted in the successful determination of genomic DNA (within the range of 10-106 copies) derived from the food poisoning bacterium Escherichia coli, with a limit of detection of 5.2 copies and high selectivity. In addition, the practical applicability of this method was demonstrated by analyzing E. coli-spiked lettuce samples.

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue.

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (Pe U), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without Pe U modification; thus suggesting that structural distortion is not induced by the modification. If two Pe Us were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three Pe Us, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.

Enzymatic Incorporation of a Coumarin–Guanine Base Pair

AbstractPrevious expansions beyond nature's preferred base‐pairing interactions have utilized either nonpolar shape‐fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N−H with C−H at a Watson–Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3‐glycosyl‐5‐fluoro‐7‐methoxy‐coumarin‐2′‐deoxyribose (dCC). dGTP is selectively incorporated across from a template dCC during enzymatic DNA synthesis. Likewise, dCC is selectively incorporated across from a template guanine when dCC is provided as the triphosphate dCCTP. DNA polymerase I (Klenow fragment) exhibited about a 10‐fold higher affinity for dCCTP than dCTP, allowing selective incorporation of dCC in direct competition experiments. These results demonstrate that a single C−H can replace N−H at a Watson–Crick‐type interface with preservation of functional selectivity and enhanced activity.

Enzymatic Incorporation of a Coumarin-Guanine Base Pair.

Previous expansions beyond nature's preferred base-pairing interactions have utilized either nonpolar shape-fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N-H with C-H at a Watson-Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3-glycosyl-5-fluoro-7-methoxy-coumarin-2'-deoxyribose (dCC ). dGTP is selectively incorporated across from a template dCC during enzymatic DNA synthesis. Likewise, dCC is selectively incorporated across from a template guanine when dCC is provided as the triphosphate dCC TP. DNA polymerase I (Klenow fragment) exhibited about a 10-fold higher affinity for dCC TP than dCTP, allowing selective incorporation of dCC in direct competition experiments. These results demonstrate that a single C-H can replace N-H at a Watson-Crick-type interface with preservation of functional selectivity and enhanced activity.

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAthaU). We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAthaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.

The use of a 2-aminopurine-containing split G-quadruplex for sequence-specific DNA detection

A simple, sequence-specific DNA detection method, utilizing a fluorescent 2-aminopurine (2-AP) nucleobase analogue-containing split G-quadruplex as the key detection component, is described. In the sensor, the 2-AP-containing G-quadruplex is split into two segments and linked to a target-specific overhang sequence. The separate G-quadruplex sequences form an active G-quadruplex structure only in the presence of a complementary target DNA, which leads to a significant increase in the intensity of fluorescence from the 2-AP fluorophore. This simple, one-step, homogenous assay was successfully employed to detect target DNA with a high selectivity. In addition, the practical applicability of the detection method was demonstrated by its use in analyzing target DNAs in human serum. To the best of our knowledge, this is the first time that an investigation was carried out in which a fluorescent nucleobase analogue was incorporated into a split G-quadruplex structure and this structure was utilized as the foundation for a specific DNA sensor.

The origin of fluorescence in DNA thio-analogues

Fluorescent probes are vital molecular tools for DNA biognostics – often aiding in the detection of malignant tumors. Biomimetic nucleobase analogues are amongst the most widely used probes due to their versatility and biocompatibility. Thieno[3,4-d]pyrimidin-4-amine (TPA) and isothiazolo[4,3-d]pyrimidin-7-amine (iTPA) are two recently developed sulphur-containing heteroaromatic fluorescent nucleobase analogues. Their inherent structures give rise to superior biomimetic properties that ensure their optimal interaction with double-helix DNA. Using high-level electronic structure methods, we simulate the absorption and emission spectra in order to better understand the origins of the strong absorption and emission profiles observed experimentally. Radiationless decay pathways are also computed and compared with those of the canonical DNA nucleobases, in order to unravel the geometric and electronic properties that make the thio-analogues fluorescent.