Nucleic Acids In Living Cells Research Articles

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

Boosting the turn-on fluorescent signaling ability of thiazole orange dyes: The effectiveness of structural modification site and its unusual interaction behavior with nucleic acids

New fluorescent dyes derivatized on a classical thiazole orange framework are able to show unexpectedly strong interaction signal and unusual binding selectivity with different structures of nucleic acid, particularly when bound with double-stranded DNA or G-quadruplex DNA. The present study reveals that these small binding ligands simply bearing an additional amino side group on its parent molecule of thiazole orange have almost no background fluorescence in solution. Conversely, they are able to produce an extremely strong yellow emission signal upon interaction with targeting nucleic acids in live cells. The induced fluorescence intensity was approximately 10–15 times stronger than that of thiazole orange. The dyes are therefore excellent fluorescent stains for bio-sensing and bio-imaging applications. It is particularly suitable for fluorescence microscopy experiments requiring very low working concentration (0.25 μM or less) targeting nucleic acids. This was demonstrated using pu27 G-quadruplex DNA, which has a low limit of detection (LOD = 3–4 nM) while thiazole orange was much higher (LOD = 48.7 nM) under the same conditions. In addition, it was found that structural modification on the quinolinium scaffold of thiazole orange was less effective than modification on the benzothiazole moiety. The findings of the present study provide important information for structural advancement of small molecules based on the widely used thiazole orange skeleton, resulting in analogues that are able to achieve higher sensitivity and selectivity for targeting at a specific class of nucleic acids. Computational docking studies were also conducted to illustrate the interaction behaviors of the dyes with different DNA structures.

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.

Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches

361-Pos Board B116 Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches Stephen P. Mieruszynski 1 , Michelle A. Digman 2 , Enrico Gratton 2 , Mark Jones 1 . Health and Science, University of Western Sydney, Richmond, Australia, Biomedical Engineering Department, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA. Addressing the mobility and dynamics of DNA within live cells requires the characterization of individual particles in real time. Two such techniques enabling the quantification of the dynamics and means of motion of fluorescently labeled particles include the Raster Image Correlation Spectroscopy (RICS) and the image-based Mean Square Displacement (iMSD) approaches. In this study, the RICS and iMSD approaches were applied in order to elucidate the mobility and means of motion of DNA lipoplexes formed from varying sized DNA (20bp- 5.5kbp). The RICS approach demonstrated that two species of mobility were pre- sent within the cytoplasm. Across the DNA fragment sizes, the slower species was not size dependent and were statistically the same. Whereas, faster moving particles which demonstrated a size dependent mobility. Nuclear localized DNA demonstrated to be significantly dynamic compared to other previously pub- lished work, ranging from 1.22 mm2/s (5.5kbp) to 3.67 mm2/s (21bp), consistent to nuclear proteins and RNA. The iMSD approach applied enabled the discrim- ination between different means of motion including random diffusion (RD), active transport (AT), confined diffusion (CD), anomalous subdiffusion (AS) and transient confinement (TC). Motion through RD and TC had demonstrated a size dependency across the DNA sizes assessed. Whereas, motion through AT, CD and AS did not demonstrate size dependency additionally these other means of motion had a higher mobility rate than that of RD.

Fluorogenic, Catalytic, Photochemical Reaction for Amplified Detection of Nucleic Acids

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

Is there any quantitative relationship between the synthesis and the breakdown of nucleic acids in living cells?

Es wurde untersucht, ob die Biosynthese der Nukleinsauren in wachsenden Systemen mit dem Abbau von ursprunglich vorhandenen Nukleinsauren derselben Art gekoppelt ist. Die Versuche wurden mit Bakterien (E. coli) und mit Rattenleber gemacht. Die in der Zelle vorhandenen Nukleinsauren waren mit P32 markiert, und es wurde der Verlust an Radioaktivitat in jeder der beiden Nukleinsauretypen im Laufe des Wachstums gemessen. Die Resultate wiesen darauf hin, dass das Auftreten von neuen Nukleinsauremolekeln in der lebenden Zelle ohne den begleitenden Abbau der alten Molekeln stattfinden kann.