Peter Sandin

Self-Assembled DNA Photonic Wire for Long-Range Energy Transfer

DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several-nanometer regime, and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. The wire utilizes DNA as a scaffold for a chromophore with overlapping absorption and emission bands enabling fluorescence resonance energy transfer (FRET) between pairs of chromophores leading to sequential transfer of the excitation energy along the wire. This allows for the creation of a self-assembled photonic wire using straightforward construction and, in addition, allows for a large span in wire lengths without changing the basic components. The intercalating chromophore, YO, is chosen for its homotransfer capability enabling effective diffusive energy migration along the wire without loss in energy. In contrast to heterotransfer, i.e., multistep cascade FRET, where each step renders a photon with less energy than in the previous step, homotransfer preserves the energy in each step. By using injector and detector chromophores at opposite ends of the wire, directionality of the wire is achieved. The efficiency of the wire constructs is examined by steady-state and time-resolved fluorescence measurements and the energy transfer process is simulated using a Markov chain model. We show that it is possible to create two component DNA-based photonic wires capable of long-range energy transfer using a straightforward self-assembly approach.

Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue

This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tCO, is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tCO is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (ε = 9000 M−1 cm−1) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tCO shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tCO, on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tCO a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).

Fluorescent properties of DNA base analogue tC upon incorporation into DNA — negligible influence of neighbouring bases on fluorescence quantum yield

The quantum yield of the fluorescent tricyclic cytosine analogue, 1,3-diaza-2-oxophenothiazine, tC, is high and virtually unaffected by incorporation into both single- and double-stranded DNA irrespective of neighbouring bases (0.17–0.24 and 0.16–0.21, respectively) and the corresponding fluorescence decay curves are all mono-exponential, properties that are unmatched by any base analogue so far. The fluorescence lifetimes increase when going from tC free in solution (3.2 ns) to single- and double-stranded DNA (on average 5.7 and 6.3 ns, respectively). The mono-exponential decays further support previous NMR results where it was found that tC has a well-defined position and geometry within the DNA helix. Furthermore, we find that the oxidation potential of tC is 0.4 V lower than for deoxyguanosine, the natural base with the lowest oxidation potential. This suggests that tC may be of interest in charge transfer studies in DNA as an electron hole acceptor. We also present a novel synthetic route to the phosphoramidite form of tC. The results presented here together with previous work show that tC is a very good C-analogue that induces minimal perturbation to the native structure of DNA. This makes tC unique as a fluorescent base analogue and is thus highly interesting in a range of applications for studying e.g. structure, dynamics and kinetics in nucleic acid systems.

Synthesis and oligonucleotide incorporation of fluorescent cytosine analogue tC: a promising nucleic acid probe

The tricyclic cytosine, tC, is a fluorescent base analogue with excellent properties for investigating intrinsic characteristics of nucleic acid as well as interactions between nucleic acids and other molecules. Its unique fluorescence properties and insignificant influence on overall structure and dynamics of nucleic acid after incorporation makes tC particularly interesting in fluorescence resonance energy transfer and anisotropy measurements. We here describe a straightforward synthesis of the standard monomer form of tC for DNA solid-phase synthesis, the tC phosphoramidite, and its subsequent incorporation into oligonucleotides. The total synthesis of the tC phosphoramidite takes approximately 8 days and its incorporation and the subsequent oligonucleotide purification an additional day.

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.

Nucleic Acid Structure and Sequence Probing Using Fluorescent Base Analogue tCO

The fluorescent cytosine analog tC(O) is on average the brightest probe of its kind and, moreover, it introduces minimal perturbations to the normal secondary structure of DNA. Here several ways of how tC(O), with an advantage, can be used as a local fluorescent probe in nucleic acid systems are presented. Most importantly, we show that tC(O) is an excellent probe for the detection of individual melting processes of complex nucleic acid structures containing a large number of separate secondary structure motifs. Since conventional UV-melting investigations merely monitor the global melting process of the whole nucleic acid structure, e.g. multi-hairpin systems in RNA/DNA, and thus is incapable of estimating individual melting transitions of such systems, tC(O) represents a new method of characterization. Furthermore, we find that tC(O) may be used to detect bulges and loops in nucleic acids as well as to distinguish a matched base-pair from several of the mismatched.

Exploring a cascade Heck–Suzuki reaction based route to kinase inhibitors using design of experiments

Design of Experiments (DoE) has been used to optimize a diversity oriented palladium catalyzed cascade Heck-Suzuki reaction for the construction of 3-alkenyl substituted cyclopenta[b]indole compounds. The obtained DoE model revealed a reaction highly dependent on the ligand. Guided by the model, an optimal ligand was chosen that selectively delivered the desired products in high yields. The conditions were applicable with a variety of boronic acids and were used to synthesize a library of 3-alkenyl derivatized compounds. Focusing on inhibition of kinases relevant for combating melanoma, the library was used in an initial structure-activity survey. In line with the observed kinase inhibition, cellular studies revealed one of the more promising derivatives to inhibit cell proliferation via an apoptotic mechanism.

On the photostability of scytonemin, analogues thereof and their monomeric counterparts

As a part of their sun-protective strategy, cyanobacteria produce the natural UV-screener scytonemin. Its accumulation in the extracellular sheaths allows the bacteria to thrive in inhospitable locations highly exposed to solar radiation. Scytonemin is often referred to as photostable and has been reported to be non-fluorescent. Taken together, these properties indicate inherently fast non-radiative relaxation processes. Despite these interesting traits, the photophysics of scytonemin is as yet almost completely unexplored. In this study, we have compared the steady-state photophysics of scytonemin itself and four derivatives thereof. Furthermore, the in vitro photostability of scytonemin was studied in different solvents using a solar simulation system. Scytonemin and the investigated derivatives demonstrated a more rapid photoinduced decay in comparison with two commercial UV-screening agents. The photostability could be modulated by varying the solvent, with the protic solvent ethanol providing the most stabilizing environment.

Self-Assembled DNA Photonic Wire

DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several nanometer regime and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. Our results show that it is possible to create two component DNA-based photonic wires capable of long range energy transfer using a straightforward self-assembly approach.

Addressable Molecular Node Assembly – High Information Density DNA Nanostructures

The inherent self-assembly properties of DNA make it ideal in nanotechnology. We present a fully addressable DNA nanostructure with the smallest possible unit cell, a hexagon with a side-length of only 3.4 nm.(2,3) Using novel three-way oligonucleotides, where each side has a unique double-stranded DNA sequence that can be assigned a specific address, we will build a non-repetitive two-dimensional grid.