Multifluorophore DNA Origami Beacon as a Biosensing Platform.

Biosensors play increasingly important roles in many fields, from clinical diagnosis to environmental monitoring, and there is a growing need for cheap and simple analytical devices. DNA nanotechnology provides methods for the creation of sophisticated biosensors, however many of the developed DNA-based sensors are limited by cumbersome and time-consuming readouts involving advanced experimental techniques. Here we describe design, construction, and characterization of an optical DNA origami nanobiosensor device exploiting arrays of precisely positioned organic fluorophores. Two arrays of donor and acceptor fluorophores make up a multifluorophore Förster resonance energy-transfer pair that results in a high-output signal for microscopic detection of single devices. Arrangement of fluorophores into arrays increases the signal-to-noise ratio, allowing detection of signal output from singular biosensors using a conventional fluorescence microscopy setup. Single device analysis enables detection of target DNA sequences in concentrations down to 100 pM in <45 min. We expect that the presented nanobiosensor can function as a general platform for incorporating sensor modules for a variety of targets and that the strong signal amplification properties may allow detection in portable microscope systems to be used for biosensor applications in the field.

Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations.

The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20-0.25), resulting in an average brightness of 1900 M-1 cm-1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17-0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.

Optimal Background Estimators in Single-Molecule FRET Microscopy.

Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy.

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity. To elucidate the different G-quadruplex conformations and their dynamics, we investigated telomeric G-quadruplex folding using single molecule FRET microscopy in conditions where it was previously believed to yield low structural heterogeneity. We observed four FRET states in Na(+) buffers: an unfolded state and three G-quadruplex related states that can interconvert between each other. Several of these states were almost equally populated at low to medium salt concentrations. These observations appear surprising as previous studies reported primarily one G-quadruplex conformation in Na(+) buffers. Our results permit, through the analysis of the dynamics of the different observed states, the identification of a more stable G-quadruplex conformation and two transient G-quadruplex states. Importantly these results offer a unique view into G-quadruplex topological heterogeneity and conformational dynamics.

Quantitative single molecule FRET efficiencies using TIRF microscopy.

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2-10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design.

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Second-generation fluorescent quadracyclic adenine analogues: environment-responsive probes with enhanced brightness.

Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putative quadracyclic adenine analogues. The compounds were efficiently synthesized from a common intermediate through a two-step pathway with the Suzuki-Miyaura coupling as the key step. Two of the compounds, qAN1 and qAN4, display brightnesses (εΦF) of 1700 and 2300, respectively, in water and behave as wavelength-ratiometric pH probes under acidic conditions. The other two, qAN2 and qAN3, display lower brightnesses but exhibit polarity-sensitive dual-band emissions that could prove useful to investigate DNA structural changes induced by DNA-protein or -drug interactions. The four qANs are very promising microenvironment-sensitive fluorescent adenine analogues that display considerable brightness for such compounds.

Single molecule FRET analysis of the 11 discrete steps of a DNA actuator.

DNA hybridization allows the design and assembly of dynamic DNA-based molecular devices. Such structures usually accomplish their function by the addition of fuel strands that drive the structure from one conformation to a new one or by internal changes in DNA hybridization. We report here on the performance and robustness of one of these devices by the detailed study of a dynamic DNA actuator. The DNA actuator was chosen as a model system, as it is the device with most discrete states to date. It is able to reversibly slide between 11 different states and can in principle function both autonomously and nonautonomously. The 11 states of the actuator were investigated by single molecule Förster Resonance Energy Transfer (smFRET) microscopy to obtain information on the static and dynamic heterogeneities of the device. Our results show that the DNA actuator can be effectively locked in several conformations with the help of well-designed DNA lock strands. However, the device also shows pronounced static and dynamic heterogeneities both in the unlocked and locked modes, and we suggest possible structural models. Our study allows for the direct visualization of the conformational diversity and movement of the dynamic DNA-based device and shows that complex DNA-based devices are inherently heterogeneous. Our results also demonstrate that single molecule techniques are a powerful tool for structural dynamics studies and provide a stringent test for the performance of molecular devices made out of DNA.

The photoinduced transformation of fluorescent DNA base analogue tC triggers DNA melting.

While fluorescent analogues of the canonical nucleobases have proven to be highly valuable in a large number of applications, up until today, fluorescent DNA base analogues remain virtually inapplicable for single-molecule fluorescence experiments which require extremely bright and photostable dyes. Insight into the photodegradation processes of these fluorophores is thus a key step in the continuous development towards dyes with improved performances. Here, we show that the commercially available fluorescent nucleobase analogue tC under intense long-term illumination and in the presence of O2 is degraded to form a single photoreaction product which we suggest to be the sulfoxide form of tC. The photoproduct is characterized by a blue-shifted absorption and a less intense fluorescence compared to that of tC. Interestingly, when tC is positioned inside double-stranded DNA this photodriven conversion of tC to its photoproduct greatly reduces the duplex stability of the overall double helix in which the probe is positioned. Since tC can be excited selectively at 400 nm, well outside the absorption band of the natural DNA bases, this observation points towards the application of tC as a general light-triggered switch of DNA duplex stability.

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids.

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base-base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.

A DNA based five-state switch with programmed reversibility.

A programmable switch based on a DNA hairpin loop is functionalised with a rigid or flexible porphyrin or FAM and TAMRA FRET pair, which provides insight into the restructuring of the hairpin as well as porphyrin-porphyrin coupling. The switch contains five discrete states which can be accessed independently and followed by real-time spectroscopy, opening the way to a quinary computing code.

Advances in quantitative FRET-based methods for studying nucleic acids.

Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for "on/off" experiments. To use FRET to obtain quantitative distances and three-dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single-molecule levels.

Quadracyclic adenine: a non-perturbing fluorescent adenine analogue.

Fluorescent-base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base-pairing properties of a new environment-sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid-phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B-form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2-aminopurine, and the recently developed triazole adenine, qA shows highly specific base-pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300 nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8 % with an emission maximum at 456 nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2-aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid-containing systems.

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.

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.

Nucleic acid base analog FRET-pair facilitating detailed structural measurements in nucleic acid containing systems.

We present the first nucleobase analog fluorescence resonance energy transfer (FRET)-pair. The pair consists of tC(O), 1,3-diaza-2-oxophenoxazine, as an energy donor and the newly developed tC(nitro), 7-nitro-1,3-diaza-2-oxophenothiazine, as an energy acceptor. The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes as the number of bases separating tC(O) and tC(nitro) is varied. 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. In combination with the good nucleobase analog properties, this points toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid experimental support to the dependence of energy transfer efficiency on orientation of involved transition dipoles as predicted by the Forster theory.