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Fluorescent base analogues (FBAs) are versatile nucleic acid labels that can replace a native nucleobase, while maintaining base pairing and secondary structure. Following the recent demonstration that free FBAs can be detected at the single-molecule level, the next goal is to achieve this level of detection sensitivity in oligonucleotides. Due to the short-wavelength absorption of most FBAs, multiphoton microscopy has emerged as a promising approach to single-molecule detection. We report the multiphoton-induced fluorescence of 5-(5-(4-methoxyphenyl)thiophen-2-yl)-6-aza-uridine (MeO th aU), a polarity-sensitive fluorescent thymidine analogue, as a nucleoside, and in two single-stranded deoxyribo-oligonucleotides, with and without their complementary strands. Ensemble steady-state and time-resolved measurements in dioxane, following one-photon and two-photon excitation, reveals both strongly and weakly emissive species, assigned as rotamers, while in Tris buffer there are additional non-emissive states, which are attributed to tautomeric forms populated in aqueous environments. The two-photon (2P) brightness for MeO th aU is highest as the free nucleoside in dioxane (10 GM) and lowest as the free nucleoside in Tris buffer (0.05 GM). The species-averaged 2P brightness values in DNA are higher for the single strands (0.66 and 0.82 GM for sequence context AXA and AXT, respectively, where X is MeO th aU) than in the duplex (0.31 and 0.25 GM for AXA and AXT, respectively). Using 2P microscopy with pulse-shaped broadband excitation, we were able to detect single- and double-stranded oligos with a molecular brightness of 0.8-0.9 kHz per molecule. This allowed the detection of as few as 7 DNA molecules in the focus, making it the brightest responsive FBA in an oligonucleotide reported to date.

2-aminopurine (2AP) is a responsive fluorescent base analogue that is used widely as a probe of the local molecular environment in DNA. The ability of 2AP to report changes in local conformation and base-stacking interactions arises from the efficient quenching of its fluorescence by the natural DNA bases. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of the collisional quenching of 2AP by the natural bases, in different buffer solutions, showed that dynamic quenching efficiency depends on the identity of the natural base, but disagreed on the relative quenching efficiencies of the bases. We report a comprehensive investigation of inter-base quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies. Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a charge-transfer mechanism. Time-resolved measurements also provide insight into static quenching, revealing formation of 2AP-NMP ground-state complexes in which 2AP displays a very short fluorescence lifetime, comparable to that seen in oligonucleotides. In these complexes, the dependence of the rate of quenching on the partner base also supports a charge-transfer mechanism.

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597-610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The structure- and strand-specific phosphodiesterase flap endonuclease-1 (FEN1), the prototypical 5'-nuclease, catalyzes the essential removal of 5'-single-stranded flaps during replication and repair. FEN1 achieves this by selectively catalyzing hydrolysis one nucleotide into the duplex region of substrates, always targeting the 5'-strand. This specificity is proposed to arise by unpairing the 5'-end of duplex to permit the scissile phosphate diester to contact catalytic divalent metal ions. Providing the first direct evidence for this, we detected changes induced by human FEN1 (hFEN1) in the low-energy CD spectra and fluorescence lifetimes of 2-aminopurine in substrates and products that were indicative of unpairing. Divalent metal ions were essential for unpairing. However, although 5'-nuclease superfamily-conserved active-site residues K93 and R100 were required to produce unpaired product, they were not necessary to unpair substrates. Nevertheless, a unique arrangement of protein residues around the unpaired DNA was detected only with wild-type protein, suggesting a cooperative assembly of active-site residues that may be triggered by unpaired DNA. The general principles of FEN1 strand and reaction-site selection, which depend on the ability of juxtaposed divalent metal ions to unpair the end of duplex DNA, may also apply more widely to other structure- and strand-specific nucleases.

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems., (© 2012 American Chemical Society)

Several important aspects of fluorescence decay analysis are addressed and tested against new experimental measurements. A simulated-annealing method is described for deconvoluting the instrument response function from a measured fluorescence decay to yield the true decay, which is more convenient for subsequent fitting. The method is shown to perform well against the conventional approach, which is to fit a convoluted fitting function to the experimentally measured decay. The simulated annealing approach is also successfully applied to the determination of an instrument response function using a known true fluorescence decay (for rhodamine 6G). The analysis of true fluorescence decays is considered critically, focusing specifically on how a distribution of decay constants can be incorporated in to a fit. Various fitting functions are applied to the true fluorescence decays of 2-aminopurine in water-dioxane mixtures, in a dinucleotide, and in DNA duplexes. It is shown how a suitable combination of exponential decays and non-exponential decays (based on a Γ distribution of decay constants) can provide fits of equal quality to the conventional multi-exponential fits used in the majority of previous studies, but with fewer fitting parameters. Crucially, the new approach yields decay-constant distributions that are physically more meaningful than those corresponding to the conventional multi-exponential fit. The methods presented here should find wider application, for example to the analysis of transient-current or optical decays and in Förster resonance energy transfer (FRET).

When 2-aminopurine (2AP) is substituted for adenine in DNA, it is widely accepted that its fluorescence spectrum is essentially unchanged from that of the free fluorophore. We show that 2AP in DNA exhibits long-wavelength emission and excitation bands, in addition to the familiar short-wavelength spectra, as a result of formation of a ground-state heterodimer with an adjacent, pi-stacked, natural base. The observation of dual emission from 2AP in a variety of oligodeoxynucleotide duplexes and single strands demonstrates the generality of this phenomenon. The photophysical and conformational properties of the long-wavelength-emitting 2AP-nucleobase dimer are examined. Analogous long-wavelength fluorescence is seen when 2AP pi-stacks with aromatic amino acid sidechains in the active sites of methyltransferase enzymes during DNA nucleotide flipping.

Time-resolved fluorescence of 2-aminopurine-labeled DNA duplexes at 77 K reveals the relationship between base dynamics and the conformational heterogeneity that results in the well-known multiexponential fluorescence decay at room temperature. The conformation that exhibits rapid interbase charge transfer at room temperature is not populated in the frozen duplex at 77 K; this geometry is accessed by thermal motion of the bases, it is not a minimum energy structure of the duplex. Three photophysically distinct conformational states persist in the frozen duplex; these are minimum energy structures and do not interconvert at room temperature on the time scale of the 2-aminopurine excited-state lifetime.

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 A 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 (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 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.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.Taql, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short (∼50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by π-stacking interactions with Tyrl 08 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.Taql and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.Taql, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.Taql, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.Taql complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.Taql and suggest that the partner thymine assists in delivering the target base into the active site. [ABSTRACT FROM AUTHOR]