Fluorescent nucleobase analogues for base-base FRET in nucleic acids: Synthesis, photophysics and applications

Forster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base-base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base-base FRET pair, tC O -tC nitro , has recently been complemented with among others the adenine analogue FRET pair, qAN1-qA nitro , increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ 2 , have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base.base FRET and that are described here. Base-base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications

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 (epsilon Phi(F) = 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

Lighting Up DNA with the Environment-Sensitive Bright Adenine Analogue qAN4

The fluorescent adenine analogue qAN4 was recently shown to possess promising photophysical properties, including a high brightness as a monomer. Here we report the synthesis of the phosphoramidite of qAN4 and its successful incorporation into DNA oligonucleotides using standard solid-phase synthesis. Circular dichroism and thermal melting studies indicate that the qAN4-modification has a stabilizing effect on the B-form of DNA. Moreover, qAN4 base-pairs selectively with thymine with mismatch penalties similar to those of mismatches of adenine. The low energy absorption band of qAN4 inside DNA has its peak around 358 nm and the emission in duplex DNA is partly quenched and blue-shifted (ca. 410 nm), compared to the monomeric form. The spectral properties of the fluorophore also show sensitivity to pH; a property that may find biological applications. Quantum yields in single-stranded DNA range from 1-29 % and in duplex DNA from 1-7 %. In combination with the absorptive properties, this gives an average brightness inside duplex DNA of 275 M-1  cm-1 , more than five times higher than the most used environment-sensitive fluorescent base analogue, 2-aminopurine. Finally, we show that qAN4 can be used to advantage as a donor for interbase FRET applications in combination with adenine analogue qAnitro as an acceptor

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase F\uf6rster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA

Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M−1cm−1in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives

DNA and RNA base analogue FRET - from fluorophore design to biochemical applications

This thesis focuses on the development and use of fluorescent base analogues (FBAs). They are important tools in research concerning nucleic acids structure, dynamics and interactions. FBAs are fluorescent molecules that are structurally similar to the natural nucleobases and can therefore replace them inside nucleic acids without significantly perturbing the properties of the nucleic acid. The design of new FBAs is often challenging due to the limitations imposed on their structure by the overall structure of nucleic acids. This thesis starts by describing the development and characterization of a large set of new potential adenine analogues, all based on the structure of the FBA qA, and how TDDFT calculations were utilized to aid the design. Among these, three fluorescent (qAN1, qAN4 and pA) and one non-fluorescent (qAnitro) analogue have been incorporated and characterized inside DNA as well. They are all good adenine analogues, i.e. they do not perturb the structure or stability of DNA duplexes significantly. The three fluorescent analogues are all significantly brighter than the parent compound qA, and importantly, pA is the brightest adenine analogue inside DNA reported to date. The thesis also describes the development and characterization of a good thymine analogue, bT, which might serve as the starting point for development of brighter thymine analogues, much like qA did for the adenine analogues mentioned above. The second half of the thesis focuses on interbase FRET (F\uf6rster resonance energy transfer) using the new adenine analogues and the previously reported FRET-pair tCO-tCnitro. FRET is confirmed and characterized inside DNA using the three adenine donors (qAN1, qAN4 and pA) with the acceptor qAnitro. These FRET-pairs can monitor energy transfer up to 1.5 turns of DNA and are hence suitable for monitoring structural changes in short DNA. This is exemplified by a study of the effect on DNA structure by binding of netropsin, showing that the interbase FRET is sensitive to small changes in DNA structure. The previously reported tCO-tCnitro are here both incorporated into RNA and interbase FRET in RNA is measured for the first time. This is an important step since RNA, among other things, has proved to be a key player in cell regulation and hence of high interest and importance. Lastly the change in interbase FRET upon inducing a change from A- to Z-form RNA is shown to be significant, again highlighting the potential of interbase FRET in nucleic acid structure investigations

Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by A beta(1-40) and A beta(1-42)

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the A beta(1-40) and A beta(1-42) variants of the Alzheimer\u27s-related peptide amyloid-beta, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with A beta(1-40) compared to A beta(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with A beta(1-42) fibrils producing a longer ThT fluorescence lifetime compared to A beta(140). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed beta-sheet in the mature A beta fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation

Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by A beta(1-40) and A beta(1-42)

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the A beta(1-40) and A beta(1-42) variants of the Alzheimer\u27s-related peptide amyloid-beta, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with A beta(1-40) compared to A beta(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with A beta(1-42) fibrils producing a longer ThT fluorescence lifetime compared to A beta(140). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed beta-sheet in the mature A beta fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation

Interbase FRET in RNA: from A to Z

Published by Oxford University Press on behalf of Nucleic Acids Research. Interbase FRET can reveal highly detailed information about distance, orientation and dynamics in nucleic acids, complementing the existing structure and dynamics techniques. We here report the first RNA base analogue FRET pair, consisting of the donor tCO and the non-emissive acceptor tCnitro. The acceptor ribonucleoside is here synthesised and incorporated into RNA for the first time. This FRET pair accurately reports the average structure of A-form RNA, and its utility for probing RNA structural changes is demonstrated by monitoring the transition from A- to Z-form RNA. Finally, the measured FRET data were compared with theoretical FRET patterns obtained from two previously reported Z-RNA PDB structures, to shed new light on this elusive RNA conformation