Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues.

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e. it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.

Synthesis and Photophysical Characterization of a pH-Sensitive Quadracyclic Uridine (qU) Analogue.

Fluorescent base analogues (FBAs) have become useful tools for applications in biophysical chemistry, chemical biology, live-cell imaging, and RNA therapeutics. Herein, two synthetic routes towards a novel FBA of uracil named qU (quadracyclic uracil/uridine) are described. The qU nucleobase bears a tetracyclic fused ring system and is designed to allow for specific Watson-Crick base pairing with adenine. We find that qU absorbs light in the visible region of the spectrum and emits brightly with a quantum yield of 27 % and a dual-band character in a wide pH range. With evidence, among other things, from fluorescence lifetime measurements we suggest that this dual emission feature results from an excited-state proton transfer (ESPT) process. Furthermore, we find that both absorption and emission of qU are highly sensitive to pH. The high brightness in combination with excitation in the visible and pH responsiveness makes qU an interesting native-like nucleic acid label in spectroscopy and microscopy applications in, for example, the field of mRNA and antisense oligonucleotide (ASO) therapeutics.

Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA.

Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.

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 Förster 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.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Mechanical characterization of base analogue modified nucleic acids by force spectroscopy.

We use mechanical unfolding of single DNA hairpins with modified bases to accurately assess intra- and intermolecular forces in nucleic acids. As expected, the modification stabilizes the hybridized hairpin, but we also observe intriguing stacking interactions in the unfolded hairpin. Our study highlights the benefit of using base-modified nucleic acids in force-spectroscopy.

Interbase FRET in RNA: from A to Z.

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.

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers.

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.

Flexibility and Preorganization of Fluorescent Nucleobase-Pyrene Conjugates Control DNA and RNA Recognition.

We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase-fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase-fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson-Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity.

Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach.

Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair.

Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins.

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

Förster 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, tCO-tCnitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1-qAnitro, 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, κ2, 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.

Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET.

Förster resonance energy transfer (FRET) using fluorescent base analogues is a powerful means of obtaining high-resolution nucleic acid structure and dynamics information that favorably complements techniques such as NMR and X-ray crystallography. Here, we expand the base-base FRET repertoire with an adenine analogue FRET-pair. Phosphoramidite-protected quadracyclic 2'-deoxyadenosine analogues qAN1 (donor) and qAnitro (acceptor) were synthesized and incorporated into DNA by a generic, reliable, and high-yielding route, and both constitute excellent adenine analogues. The donor, qAN1, has quantum yields reaching 21% and 11% in single- and double-strands, respectively. To the best of our knowledge, this results in the highest average brightness of an adenine analogue inside DNA. Its potent emissive features overlap well with the absorption of qAnitro and thus enable accurate FRET-measurements over more than one turn of B-DNA. As we have shown previously for our cytosine analogue FRET-pair, FRET between qAN1 and qAnitro positioned at different base separations inside DNA results in efficiencies that are highly dependent on both distance and orientation. This facilitates significantly enhanced resolution in FRET structure determinations, demonstrated here in a study of conformational changes of DNA upon binding of the minor groove binder netropsin. Finally, we note that the donor and acceptor of our cytosine FRET-pair, tCO and tCnitro, can be conveniently combined with the acceptor and donor of our current adenine pair, respectively. Consequently, our base analogues can now measure base-base FRET between 3 of the 10 possible base combinations and, through base-complementarity, between all sequence positions in a duplex.

Chromophoric Nucleoside Analogues: Synthesis and Characterization of 6-Aminouracil-Based Nucleodyes.

Nucleodyes, visibly colored chromophoric nucleoside analogues, are reported. Design criteria are outlined and the syntheses of cytidine and uridine azo dye analogues derived from 6-aminouracil are described. Structural analysis shows that the nucleodyes are sound structural analogues of their native nucleoside counterparts, and photophysical studies demonstrate that the nucleodyes are sensitive to microenvironmental changes. Quantum chemical calculations are presented as a valuable complementary tool for the design of strongly absorbing nucleodyes, which overlap with the emission of known fluorophores. Förster critical distance (R0) calculations determine that the nucleodyes make good FRET pairs with both 2-aminopurine (2AP) and pyrrolocytosine (PyC). Additionally, unique tautomerization features exhibited by 5-(4-nitrophenylazo)-6-oxocytidine (8) are visualized by an extraordinary crystal structure.

The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation.

Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.

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.

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.

Mammalian transcription factor A is a core component of the mitochondrial transcription machinery.

Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.

Synthesis and photophysical characterisation of new fluorescent triazole adenine analogues.

Fluorescent nucleic acid base analogues are powerful probes of DNA structure. Here we describe the synthesis and photo-physical characterisation of a series of 2-(4-amino-5-(1H-1,2,3-triazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl) and 2-(4-amino-3-(1H-1,2,3-triazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) analogues via Sonogashira cross-coupling and [3 + 2]-cycloaddition reactions as the key steps in the synthesis. Compounds with a nitrogen atom in position 8 showed an approximately ten-fold increase in quantum yield and decreased Stokes shift compared to analogues with a carbon atom in position 8. Furthermore, the analogues containing nitrogen in the 8-position showed a more red-shifted and structured absorption as opposed to those which have a carbon incorporated in the same position. Compared to the previously characterised C8-triazole modified adenine, the emissive potential was significantly lower (tenfold or more) for this new family of triazoles-adenine compounds. However, three of the compounds have photophysical properties which will make them interesting to monitor inside DNA.

Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues.

The parasitic nature of the SARS-CoV-2 virus demands selective packaging of its RNA genome (gRNA) from the abundance of other nucleic acids present in infected cells. Despite increasing evidence that stem-loop 4 (SL4) of the gRNA 5' UTR is involved in the initiation of this process by binding the nucleocapsid (N) protein, little is known about its conformational dynamics. Here, we unravel the stability, dynamics and (un)folding pathways of SL4 using optical tweezers and a base analogue, tCO, that provides a local and subtle increase in base stacking without perturbing hydrogen bonding. We find that SL4 (un)folds mainly in a single step or through an intermediate, encompassing nucleotides from the central U bulge to the hairpin loop. Due to an upper-stem CU mismatch, SL4 is prone to misfold, the extent of which can be tuned by incorporating tCO at different positions. Our study contributes to a better understanding of SARS-CoV-2 packaging and the design of drugs targeting SL4. We also highlight the generalizability of using base analogues in optical tweezers experiments for probing intramolecular states and conformational transitions of various nucleic acids at the level of single molecules and with base-pair resolution.