Fluorescent Nucleobase Analogues Research Articles

Two-photon excitation of the fluorescent nucleobase analogues 2-AP and tC

We present two-photon (2P) excitation measurements of two commonly-used and commercially available fluorescent nucleobase analogues in aqueous solution: the environment-sensitive adenine analogue 2-aminopurine (2-AP), which emits in the UV region, and the environment-insensitive cytosine analogue 1,3-diaza-2-oxophenothiazine (tC), which emits in the green region. We studied the 2P excitation of free 2-AP and found that the same emissive states are accessed as via one-photon (1P) and three-photon (3P) excitation. The 2P cross section of 2-AP was 0.2 GM, which is similar to that of related molecules such as NADH. We studied tC incorporated in three single-stranded DNA oligonucleotides by 1P and 2P spectroscopy and microscopy. We found that the same emissive states were populated via either the resonant or non-resonant pathway with a 2P cross section of 1.5 GM. Time-resolved fluorescence studies suggested that tC exists in solution as two species, which we assigned as the amino and imino tautomers. Using confocal microscopy, we found that similar emission rates could be achieved for 2P excitation, as compared with 1P excitation, but without the strong photobleaching observed in the latter case. Although we were unable to detect tC fluorescence at the single-molecule level, these results indicate that multiphoton excitation of nucleobase analogues is a promising approach for imaging applications.

Cover Picture: Highly Fluorescent 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐Deoxyuridine 5′‐Triphosphate as an Efficient Substrate for DNA Polymerases (ChemBioChem 15/2011)

The cover picture shows the synthesis by DNA polymerase (shown in green) of a fluorescent DNA probe containing a fluorescent nucleobase analogue, 5-(5,6-dimethoxybenzothiazol-2-yl)-2′-deoxyuridine (dbtU, shown as light blue spheres), that exhibits a very high quantum yield (0.701) and brightness (14 000) in aqueous 100 mm NaOH at 460 nm emission. Although the chemical synthesis of short oligodeoxyribonucleotides (ODNs) containing fluorescent nucleobase analogues at specific sites has been reported, the enzymatic incorporation of these fluorescent nucleobase analogues into DNA has not been substantially reported. On p. 2341 ff., A. Matsuda et al. describe the incorporation of dbtUTP (5′-triphosphate of dbtU) efficiently at the site opposite adenine in ODNs, and how dbtU was found to act not only as a triphosphate but also as a template in an identical manner to dT in its base-pairing pattern.

A Powerful Approach for the Selection of 2-Aminopurine Substitution Sites to Investigate RNA Folding

A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.

A Unique Fluorescent Base Analogue for the Expansion of the Genetic Alphabet

Fluorescent nucleobase analogues are useful in a wide variety of biology and biotechnology tools as molecular probes and reporters for nucleic acids. Here we present a novel fluorescent purine analogue, 7-(2,2'-bithien-5-yl)-imidazo[4,5-b]pyridine (denoted as Dss). The nucleoside triphosphates of Dss can be site-specifically incorporated into DNA and RNA by polymerases, opposite its pairing partner, pyrrole-2-carbaldehyde (Pa), in DNA templates. Despite its high specificity in replication and transcription, Dss in oligonucleotides functions as a universal base that pairs with all four natural bases with nearly equal thermal stabilities. Thus, Dss would be a powerful tool for fluorescent base replacements at specific positions in functional DNA and RNA molecules.

Fluorescent nucleoside analogue displays enhanced emission upon pairing with guanine.

A fluorescent nucleobase analogue, 7-aminoquinazoline-2,4-(1H,3H)-dione, is incorporated into a DNA oligonucleotide and senses mismatched pairing by displaying G-specific fluorescence enhancement.

Photophysical and structural properties of the fluorescent nucleobase analogues of the tricyclic cytosine (tC) family

Fundamental insight into the unique fluorescence and nucleobase-mimicking properties of the fluorescent nucleobase analogues of the tC family is not only vital in explaining the behaviour of these probes in nucleic acid environments, but will also be profitable in the development of new and improved fluorescent base analogues. Here, temperature-dependent fluorescence quantum yield measurements are used to successfully separate and quantify the temperature-dependent and temperature-independent non-radiative excited-state decay processes of the three nucleobase analogues tC, tC(O) and tC(nitro); all of which are derivatives of a phenothiazine or phenoxazine tricyclic framework. These results strongly suggest that the non-radiative decay process dominating the fast deactivation of tC(nitro) is an internal conversion of a different origin than the decay pathways of tC and tC(O). tC(nitro) is reported to be fluorescent only in less dipolar solvents at room temperature, which is explained by an increase in excited-state dipole moment along the main non-radiative decay pathway, a suggestion that applies in the photophysical discussion of large polycyclic nitroaromatics in general. New insight into the ground and excited-state potential energy surfaces of the isolated tC bases is obtained by means of high level DFT and TDDFT calculations. The S(0) potential energy surfaces of tC and tC(nitro) possess two global minima corresponding to geometries folded along the middle sulfur-nitrogen axis separated by an energy barrier of 0.05 eV as calculated at the B3LYP/6-311+G(2d,p) level. The ground-state potential energy surface of tC(O) is also predicted to be shallow along the bending coordinate but with an equilibrium geometry corresponding to the planar conformation of the tricyclic framework, which may explain some of the dissimilar properties of tC and tC(O) in various confined (biological) environments. The S(1) equilibrium geometries of all three base analogues are predicted to be planar. These results are discussed in the context of the tC bases positioned in double-stranded DNA scenarios.

The First All-Nucleobase Analog FRET Pair

The fluorescent nucleobase analogs of the tricyclic cytosine (tC) family are promising nucleic acid probes capable of being inserted into double-stranded DNA as a replacement for one of the natural bases without perturbing the overall double helical structure. The high fluorescence quantum yields in both single- and double stranded DNA, combined with a rigid and well-defined position inside the DNA double helix, make these molecules particularly well suited as fluorescence resonance energy transfer (FRET) probes in nucleic acid studies. Recently we reported the first all-nucleobase analog FRET-pair, consisting of tCO as the donor and the newly developed tCnitro as acceptor which will be the focus of this presentation.1-3 The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex and, more importantly, the rigid stacking of the two base analogs, and consequently excellent control of the their exact positions, results in a very high control of the orientation factor in the FRET efficiency. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. We believe the development of this new tool opens up a wide range of possibilities in the structural investigation of nucleic acids, e.g. in characterizing DNA-protein complexes and in monitoring the inherent dynamics and the structural changes of nucleic acids in response to all kinds of stimuli.

Characterization of Nucleobase Analogue FRET Acceptor tCnitro

The fluorescent nucleobase analogues of the tricyclic cytosine (tC) family, tC and tC(O), possess high fluorescence quantum yields and single fluorescence lifetimes, even after incorporation into double-stranded DNA, which make these base analogues particularly useful as fluorescence resonance energy transfer (FRET) probes. Recently, we reported the first all-nucleobase FRET pair consisting of tC(O) as the donor and the novel tC(nitro) as the acceptor. The rigid and well-defined position of this FRET pair inside the DNA double helix, and consequently excellent control of the orientation factor in the FRET efficiency, are very promising features for future studies of nucleic acid structures. Here, we provide the necessary spectroscopic and photophysical characterization of tC(nitro) needed in order to utilize this probe as a FRET acceptor in nucleic acids. The lowest energy absorption band from 375 to 525 nm is shown to be the result of a single in-plane polarized electronic transition oriented approximately 27 degrees from the molecular long axis. This band overlaps the emission bands of both tC and tC(O), and the Forster characteristics of these donor-acceptor pairs are calculated for double-stranded DNA scenarios. In addition, the UV-vis absorption of tC(nitro) is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pK(a) of 11.1. The structure and electronic spectrum of tC(nitro) is further characterized by density functional theory calculations.

Exploring RNA-ligand interactions

Abstract RNA molecules play essential roles in biological processes and are evolving as important targets for therapeutic intervention. Small molecules that specifically bind unique RNA sites and prevent the formation of functional RNA folds or RNA-protein complexes can modulate cell functions and can become of therapeutic potential. To explore such recognition events and to fabricate discovery assays, effective biophysical tools need to be advanced. When carefully designed, new fluorescent nucleosides can serve an unparalleled role in such studies. Our criteria for "ideal" fluorescent nucleoside analogs include: (a) high structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties; (b) red-shifted absorption bands; (c) red-shifted emission band (preferably in the visible); (d) a reasonable emission quantum efficiency; and, importantly, (e) sensitivity of their photophysical parameters to changes in the microenvironment. Our program, aimed at the development of new emissive isomorphic nucleoside analogs, has yielded several useful nucleobases. Selected analogs were implemented in fluorescence-based assays. This overview presents the motivation for this work by introducing RNA-ligand interactions and discusses the design and synthesis of fluorescent isosteric nucleobase analogs and their utilization for the fabrication of "real-time" fluorescence-based biophysical assays.

New fluorescent nucleosides for real-time exploration of nucleic acids

Nucleic acids experience a variety of perturbations. These may include strand cleavage and ligation, local conformational changes, base flipping, as well as structural and environmental perturbations that are induced upon protein and low MW ligand binding. Since the native nucleobases are practically non-emissive, synthetic fluorescent nucleoside analogs that are sensitive to their local environment have become powerful tools for investigating nucleic acids structure, dynamics, recognition and damage. Our criteria for ideal fluorescent nucleoside analogs include: (a) high structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties; (b) red shifted absorption (> 290 nm) to minimize overlap with the absorption of the natural bases; (c) red shifted emission (preferably in the visible range); (d) reasonable emission quantum efficiency; and, importantly, (e) sensitivity/responsiveness of one or all photophysical parameters (λem and/or ΦF, τ) to changes in the probe's microenvironment. The design and synthesis of new fluorescent isosteric nucleobase analogs and their utilization for the fabrication of real-time fluorescence-based discovery and detection assays are outlined.

Fluorescent pyrimidine ribonucleotide: synthesis, enzymatic incorporation, and utilization.

Fluorescent nucleobase analogues that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics, and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analogue. Responsive isosteric fluorescent pyrimidine analogues are, however, rare. Appending five-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogues with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial A-site, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.

Influence of Base Stacking and Hydrogen Bonding on the Fluorescence of 2-Aminopurine and Pyrrolocytosine in Nucleic Acids

Fluorescent nucleobase analogues are used extensively to probe the structure and dynamics of nucleic acids. The fluorescence of the adenine analogue 2-aminopurine and the cytosine analogue pyrrolocytosine is significantly quenched when the bases are located in regions of double-stranded nucleic acids. To allow more detailed structural information to be obtained from fluorescence studies using these bases, we have studied the excited-state properties of the bases at the CIS and TDB3LYP level in hydrogen-bonded and base-stacked complexes. The results reveal that the first excited state (the fluorescent state) of a hydrogen-bonded complex containing 2-aminopurine and thymine is just the first excited state of 2-aminopurine alone. However, the same cannot be said for structures in which 2-aminopurine is base stacked with other nucleobases. Stacking causes the molecular orbitals involved in the fluorescence transition to spread over more than one base. The predicted rate for the fluorescence transition is reduced, thus reducing the fluorescence quantum yield. The decrease in radiative rate varies with the stacking arrangement (e.g., A- or B-form DNA) and with the identity of the nucleobase with which 2-aminopurine is stacked. Stacking 2-aminopurine between two guanine moieties is shown to significantly decrease the energy gap between the first and second excited states. We do not find reliable evidence for a low-energy charge-transfer state in any of the systems that were studied. In the case of pyrrolocytosine, base stacking was found to reduce the oscillator strength for the fluorescence transition, but very little spreading of molecular orbitals across more than one base was observed.

Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI-DNA complexes

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.

Pyrene-labeled deoxyuridine and deoxyadenosine: fluorescent discriminating phenomena in their duplex and hairpin oligonucleotides

Pyrene-labeled deoxyuracil and deoxyadenine units are useful unnatural nucleobases. These fluorescent nucleobase analogues allow strong interstrand stacking interactions to compensate for a loss of hydrogen bonding and exhibit a range of different emission intensities when they form duplexes with one another. These findings may provide new insights into the design of new probes and nucleobase analogues for applications in molecular biology. For this purpose, we have prepared an hairpin molecular beacon (MB) that incorporates an excimer unit in its closed state, and have utilized lambda(max) changing to discriminate between match and mismatch. This hairpin configuration is attractive because the synthesis of such an MB is relatively simple and inexpensive because it does not require two distinct processes to prepare the fluorophore and quencher.