PMC

Activation of functionality by two R/DNA hybrids. (a) Cartoon showing the general principle of functionality activation upon re-association of two non-functional units. (b) Schematic representation of R/DNA hybrids re-association resulting in asymmetric Dicer substrate siRNA release. The color code is kept the same throughout the figure.

Fluorescent studies of R/DNA hybrid re-association in solution at 37 °C. (a) Schematic representations of control DNA duplexes fluorescently labeled with Alexa488 and Alexa546 unable to recombine (upper part) and fluorescently labeled R/DNA hybrids programmed for re-association (lower part). (b) Emission spectra of control DNA duplexes showing no FRET and recombined R/DNA hybrids with increased Alexa546 emission signal. (c) FRET time traces during re-association of R/DNA hybrids labeled with Alexa488 and Alexa546. (d) Fluorescently labeled R/DNA hybrids individually associated with L2K prior to mixing were followed by fluorescent time tracing. Please note that L2K forms complexes with hybrids thus, preventing their re-association and the emission signal of Alexa488 (in green) stays above Alexa546 (in red) comparing to (c). (e–f) Schematic representations and FRET time traces (in green) during re-association of R/DNA hybrids labeled with Alexa488 and quencher IowaBlack FQ with schematic representation of corresponding hybrids programmed for recombination. Please note that as well as in (d), L2K forms complexes with quenched hybrids (in blue) and prevents their recombination.

HIV-1 expression and production is inhibited by siRNAs and recombined R/DNA hybrids. (a) HeLa cells were transfected with pNL4-3, with and without siRNAs. Virus supernatant was harvested and the Reverse Transcriptase (RT) activity was measured (this assay estimates the amounts of virus produced by the cells); data are shown normalized to virus controls (VC.1 and VC.2) without siRNAs. Mock represents non-treated HeLa cells. Error bars denote SD; N=4. (b) At 48 h posttransfection, HeLa cells were metabolically labeled with [³⁵S]MetCys for 4h. Cell lysates were radioimunoprecipitated. Positions of envelope glycoprotein precursor, gp160, and surface glycoprotein gp120; Pr55Gag (Pr55), CA-SP1 (p25) and CA (p24) are indicated. Quantification of total cell-associated Gag: Pr55+p25+p24. Total Gag in virus control (VC.1) without siRNAs set at 100. Error bars denote SD; N=3.

GFP knockdown assays for human breast cancer cells (MDA-MB-231/GFP) which stably express enhanced GFP (eGFP). Three days after the transfection of cells with R/DNA hybrids programmed to release siRNAs against eGFP (H_s and H_ant), (a) eGFP expression was observed by fluorescence microscopy and (b) eGFP expression was statistically analyzed with flow cytometry experiments. As the control, siRNA duplexes against eGFP were used. Please note that the individual R/DNA hybrids cause no decrease in eGFP production (). (c) Dependency of toehold lengths in R/DNA hybrids co-transfected on two different days show their ability to knockdown eGFP expression. R/DNA hybrids containing antisense (H_ant) were transfected one day prior to hybrids with sense (H_s). Three days after, eGFP expression was analyzed.

Re-association and localization of R/DNA hybrids in human breast cancer cells (MDA-MB-231) visualized by confocal fluorescence microscopy. (a) FRET experiments: cells were co-transfected with cognate hybrids labeled with Alexa488 and Alexa546 and images were taken on the next day. (b) Dequenching experiments: cells were co-transfected with cognate duplexes, having one duplex labeled with Alexa488 and IowaBlack FQ. Images were taken on the next day. (c–d) Studying the localization of R/DNA hybrids with commonly used markers for endosomal compartments (c) EEA1 and (d) Rab7.
Image numbers in a–d correspond to: differential interference contrast (DIC) images (1), Alexa488 emission (2), Alexa546 emission (3), bleed-through corrected FRET image (4), 3D chart representation of zoomed fragment indicated by a white box of bleed-through corrected FRET image with the yellow star indicating the correspondence (5), EAA1 antibody staining (6), and Rab7 antibody staining (7). Images (1+2+3), (1+4), (1+2), (1+3+6), (1+3+7) are superpositions of two or three different images.

In vivo and ex vivo studies of R/DNA hybrids in tumor xenograft mouse model. (a) Time-dependant bio-distribution imaging in vivo. In three hours whole mouse image, fluorescent maximums (in red) correspond to the places of injection (1), tumor (2), and blood withdrawal (3). (b) Relative organ uptakes of fluorescently labeled R/DNA hybrids and siRNAs in tumor-bearing mice three hours post tail-vein injection. A relatively high level of hybrid accumulation occurs in tumor tissue. Error bars denote SD; N=3. (c) The amounts of the fluorescent probe (R/DNA hybrids and Dicer substrate siRNAs labeled with IRDye700) in the mouse blood-stream were measured three hours post-injection. Error bars denote SD; N=4 (d) Ex vivo fluorescent imaging of tumors after 5 and 13 days post-injections in vivo demonstrate comparable levels of eGFP silencing caused by siRNA and R/DNA hybrids. (e) Ex vivo quantification (two animals per each experiment) of eGFP expression after 5 and 13 days post-injection.