Red light-triggered nucleic acid-templated reactions for detection of nucleic acids in live cells

Nucleic acid-dependent(templated) reactions are powerful tools for the detection of various nucleic acids in vitro and in vivo. Achieving the additional control of this reaction with non-toxic and biocompatible triggers will broader their application spectrum, e.g. in molecular biology and medicine. In particular, we are interested in using red light (>630 nm) as a trigger. In contrast to usually used UV-light and short wavelength visible light, such a trigger exhibits practically no toxicity towards the cells. The nucleic acid-dependent reactions developed previously in the group of Mokhir are mediated by singlet oxygen (1O2), which is generated upon the irradiation of red light-sensitive photosensitizers. 1O2 reacts with a corresponding substrate, e.g. 9,10- dialcoxyanthracene, initiating its cleavage, which is designed to induce the generation of a fluorescent signal. The latter is achieved by the covalent attachment of a fluorophore to the substrate. Herein I report about the further improvement of the already known red light-triggered fluorogenic nucleic acid - templated reaction as well as the successful application of this reaction for detection of beta-actin mRNA in live human cells. Initially, the dependence of the efficiency of the reaction between 9,10-dialcoxyanthracene and 1 O2 to the presence of substituents within the anthracene structure, having electron-donating properties, was investigated. For that purpose two new compounds based on 9,10-dialcoxyanthracene containing two different electron-donating moieties were synthesized. Using 1H-NMR spectroscopy, their reactivity towards 1O2 was studied and compared with the reactivity of 9,10-dialcoxyanthracene. Next, DNA molecules were chemically modified with these new anthracenes and the resulting conjugates were studied as substrates in red-light dependent templated reactions, both in the absence and in the presence of glutathione (GSH). In the result of these studies, 1O2-sensitive moieties were developed, which do not consume external electrons and, therefore, do not disturb RedOx balance of live cells. In order to further decrease the sensitivity of the templated reaction to external donors of electrons a new linker based on mono 9-alcoxyanthracene was synthesized. Due to the different cleavage mechanism, this compound demonstrated negligible sensitivity to glutathione. Importantly, being applied in the DNA-templated reaction, this compound showed significantincrease of the reaction rate in the presence of the complementary template nucleic acids. Correspondingly, the new linker allowed improving substantially signal-to-noise ratio of the reaction. Further improvement of the latter parameter was achieved by reducing the residual activity of the photosensitizer in the absence of the template by using Disperse Blue 3- oligonucleotide conjugates complementary to corresponding photosensitizer-containing substrates. In the result of all these improvements the limit of the target template detection could be improved by over 104 fold (with respect to the previously reported reaction) down to 1.9 pmol/L. Finally, to test whether the improved templated reaction is compatible with live cells, probes based on 2’-OMe-RNAs were synthesized. The first probe (MeRN1-ANM-FL-UU) was chemically modified with the new linker and fluorescein, whereas the second probe (MeRN2-PS) contained a photosensitizer. 2’OMe-RNAs were chosen, since these chemical analogues of RNAs are known to be highly resistant to hydrolysis in cells and retain hybridization properties of the natural counterparts. Sequences of the 2’-OMe RNA probes were selected to be complementary to the target template – β-actin messenger RNA. For the control experiment a non-complementary probe (MeRN3-PS) modified with a photosensitizer was synthesized. This control probe in combination with the singlet oxygen-sensitive probe is not able to build a duplex with the target template and thus the whole system was not expected to produce a fluorescent signal. Using the common transfection agent (LypofectamineTM 3000), probes MeRN1-ANM-FL-UU and MeRN2-PS as well as MeRN1-ANM-FL-UU and MeRN3-PS were transferred into the cells, which were then irradiated with the red light. It was demonstrated that in cell cultures the fluorescent signal doubles in case of irradiation of fully complementary probes whereas a control experiment showed a significantly lower fluorescence increase

Suppressing catalyst poisoning in the carbodiimide-fueled reaction cycle

In biology, cells regulate the function of molecules using catalytic reaction cycles that convert reagents with high chemical potential (fuel) to waste molecules. Inspired by biology, synthetic analogs of such chemical reaction cycles have been devised, and a widely used catalytic reaction cycle uses carboxylates as catalysts to accelerate the hydration of carbodiimides. The cycle is versatile and easy to use, so it is widely applied to regulate motors, pumps, self-assembly, and phase separation. However, the cycle suffers from side reactions, especially the formation of N-acylurea. In catalytic reaction cycles, side reactions are disastrous as they decrease the fuel’s efficiency and, more importantly, destroy the molecular machinery or assembling molecules. To put that in perspective, a side reaction that irreversibly converts as little as 1% of the fuel into a side product would mean less than 5% of the molecular machine left after 100 cycles. Therefore, this work tested how to suppress N-acylurea by screening precursor concentration, its structure, carbodiimide structure, additives, temperature, and pH. It turned out that the combination of low temperature, low pH, and 10% pyridine as a fraction of the fuel could significantly suppress the N-acylurea side product and keep the reaction cycle highly effective to regulate successful assembly. We anticipate that our work will provide guidelines for using carbodiimide-fueled reaction cycles to regulate molecular function about how to choose an optimal condition

DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries

DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

<div><p>Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye <i>via</i> fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries.</p></div

Structure of system 5 at 0.51 ns (after equilibration with harmonic restraints, above) and at 220.5 ns (below).

<p>Ions and water were omitted for clarity. Figures were created using VMD. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160229#pone.0160229.ref076" target="_blank">76</a>]</p

Structures of system 4 at 0.51 ns (after equilibration with harmonic restraints, above) and at 260.5 ns (below, left) and 220.5 ns (below, right), respectively. Left: Simulation at 310 K, right: simulation at 363 K.

<p>Ions and water were omitted for clarity. Figures were created using VMD. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160229#pone.0160229.ref076" target="_blank">76</a>]</p

Experimental (black) and calculated (red) steady-state absorption (left) and emission spectra of fluorescein, using a subset of the MD ensemble (424 snapshots) and the QM/MM methodology described in reference [35].

<p><b>Additionally, the fitted ensemble of transition dipoles describing the S₁ → S₀ transition (emission spectrum) is shown (transition dipoles multiplied by a factor of 10)</b>. Note that the experimental spectra were recorded for systems with the chromophores attached to DNA and therefore additionally contain typical DNA absorption peaks in the UV region, while the calculated spectra resemble the situation of the chromophores in solution (due to the QM/MM formalism).</p

Experimental (black) and calculated (red) steady-state absorption (left) and emission spectra of PPa, using a subset of the MD ensemble (424 snapshots) and the QM/MM methodology described in reference [35].

<p><b>Additionally, fitted ensembles of transition dipoles describing the S₀ → S_i transition with maximal oscillator strength (absorption spectrum) and the S₁ → S₀ transitions (emission spectrum) are shown (transition dipoles multiplied by a factor of 10)</b>. Note that the experimental spectra were recorded for systems with the chromophores attached to DNA and therefore additionally contain typical DNA absorption peaks in the UV region, while the calculated spectra resemble the situation of the chromophores in solution (due to the QM/MM formalism).</p

1–3: Structures of DNA-dye conjugates (green: PPa, orange: fluorescein) with linkers (magenta) cleavable by ¹O₂.

<p><b>LA, LB</b>: linkers: 1,9-dialkoxyanthracene (left) and 9-alkoxyanthracene derivatives (right).</p

Detecting specific nucleic acid sequences in a templated reacion.

<p><b>F</b>: 5-carboxyfluorescein, <b>PS</b>: photosensitizer (pyropheophorbide-a, PPa), <b>L</b>: linker cleavable by ¹O₂. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160229#pone.0160229.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160229#pone.0160229.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160229#pone.0160229.ref027" target="_blank">27</a>]</p