Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: Magnesium induced assembly of nanoparticles with improved resistance to degradation and miRNA function. (A) Micelle formation under various concentrations of MgCl 2 detected by hydrophobic-incorporation of fluorescent dye (CMC-535). (B) Plots of fluorescence intensity at 535 nm under various concentrations of the delivery platform. The critical micelle concentration (CMC) was 3 × 10 -7 M. (C) Transmission electron microscopy image of the delivery platform nanoparticles using negative staining. Scale bar, 50 nm. Representative image of a single experiment. (D) The particle size distribution of the delivery platform with MgCl 2 (+Mg 2+ ) or without MgCl 2 (-Mg 2+ ) measured by dynamic light scattering. Representative image of two independent experiments. (E) Stability of each component of the miR-26a delivery platform against serum degradation in vitro . The miR-26a chimera of particle form (+Mg 2+ ) or non-particle monomer form (-Mg 2+ ) in various formats ( Fig. S3A ) was incubated with human serum for various time periods. Stability was measured by qPCR for miR-26a. (F) Functional effect of each moiety in the delivery platform for gene silencing by miR-26a. The c-Kit-targeting miR-26a chimera (1 μM) in various formats ( Fig. S3A ) with or without MgCl 2 were incubated with c-Kit + TUBO cancer cells for 2 days. The expression levels of a miR-26a target gene, Ezh2 , were measured by qPCR. Asterisks denote the significant difference compared to vehicle controls. (A, B, E, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: Fluorescence, Concentration Assay, Transmission Assay, Electron Microscopy, Negative Staining, In Vitro, Incubation, Functional Assay, Expressing

Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: The pH-sensitive delivery platform induced endosomal leakage but not cytotoxicity. (A) Target specificity of the c-Kit-targeting delivery platform linked with anti-c-Kit DNA aptamer. The c-Kit-targeting aptamer-positive platform bound specifically to c-Kit + MEF cells in vitro . (B) Visualizing uptake of c-Kit-targeting miR-26a chimera in c-Kit + MEF cells over 120 min. The AF488-conjugated miR-26a chimera (Green) was incubated with the cells at 37 °C. After washing with PBS, the cells were fixed with 4% formaldehyde and images were acquired on a fluorescent microscope. Scale bar, 5 μM. (C) Destabilization of micelle particles of the miR-26a chimera under acidic pH conditions detected by hydrophobic-incorporation of fluorescent dye (CMC-535). Tween-20 is a used as a control. Asterisks denote significant differences compared to pH 7.0. (D) Free magnesium concentration in solution of the delivery platform nanoparticles incubated under various pH conditions for 1 h. Asterisks denote significant differences compared to pH 7.0. (E) Release of an endosomal trafficking fluorescent probe (10 k Dextran, red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Top panels). Release of another fluorescent probe (Cathepsin enzymatic fluorescent substrate, Magic Red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Bottom panels). Scale bar, 5 μM. (F) Lack of cytotoxicity of miR-26a chimera to cKit + MEF cells treated with various concentration of miR-26a chimera for 24 h as detected by LDH release assay. Asterisks denote the significant difference compared to 0 μM of miR-26a chimera treatment. (A, B, E) Representative images of two independent experiments. (C, D, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: In Vitro, Incubation, Microscopy, Control, Concentration Assay, Lactate Dehydrogenase Assay

Journal: Cancers

Article Title: Curcumin-Dichloroacetate Hybrid Molecule as an Antitumor Oral Drug against Multidrug-Resistant Advanced Bladder Cancers

doi: 10.3390/cancers16173108

Figure Lengend Snippet: List of fine chemicals and assay kits used in this study.

Article Snippet: JC-1 dye , G-Biosciences (St. Louis, MO, USA) #786-1322.

Techniques:

Journal: BMC Developmental Biology

Article Title: BMP-SMAD signalling output is highly regionalized in cardiovascular and lymphatic endothelial networks

doi: 10.1186/s12861-016-0133-x

Figure Lengend Snippet: The GFP reporter protein faithfully recapitulates the transcriptional activation of the BRE :: gfp transgene. In situ hybridisation for gfp mRNA ( red ) and direct GFP fluorescence ( green ) on cryosections through the cardinal vein ( a – d ) and the ventricular trabeculae ( e – g ) at E11.5. DAPI is used to stain nuclei. The boxed area in ( a ) is enlarged in panels ( b – d ). The corresponding low magnification view of the trabeculae in the ventricle is not provided because of insufficient intensity of the ISH signal. White arrowheads indicate ECs double positive for gfp mRNA and direct GFP fluorescence. The red and green arrows depict ECs only positive for gfp mRNA or GFP fluorescence respectively. Scale bars: 100 μm ( a ); 50 μm ( b – d ); 10 μm ( e – g )

Article Snippet: The GFP fluorescent in situ hybridisation (ISH) probe was custom designed with the probe designer tool from Stellaris (LGC biosearch technologies).

Techniques: Activation Assay, In Situ, Hybridization, Fluorescence, Staining

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: a) In the absence of tRNAGly transcription terminates prior to the RNA sequence targeted by the fluorescent probe RTprb. The hairpin form of RTprb has minimal background fluorescence due to Cy5 fluorescence being quenched by Dabcyl. b) Transcription readthrough induced by tRNAGly produces the full-length transcript, including the target sequence of RTprb. RTprb binds the target sequence, unfolding the hairpin structure such that Dabcyl no longer quenches Cy5 and fluorescence increases.

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Sequencing, Fluorescence

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: a) Comparison of Readthroughmax of cognate tRNAGly (wt, acceptor end 5′-UCCA-3′) with tRNAGly(U73A), tRNATyr(A73U) and tRNATyr(wt, acceptor end 5′-ACCA-3′). For all reactions with tRNA, [tRNA] = 100 nM. b) Dose-response curve of tRNAGly readthrough induction.

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Comparison

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: Z′ factor analysis of fluorescence monitoring methods

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Fluorescence

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: a) Structure of spermidine. b) Spermidine dose-response effect on glyQS transcription readthrough. In the absence of tRNAGly (open circle), spermidine EC50 = 0.49 mM (95% CI = 0.41-0.59 mM, R2 = 0.96). In the presence of tRNAGly(filled circle), spermidine EC50 = 0.58 mM (95% CI = 0.49-0.68 mM, R2 = 0.98). The Transcription Control data (triangle) reflect the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. For the Transcription control, the spermidine EC50 = 0.82 mM (95% CI = 0.74-0.91 mM, R2 = 0.95). The data points at [spermidine] = 10 mM were excluded from EC50 calculations as discussed in Results.

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Control

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: a) Transcription Control (striped bars), glyQS readthrough in the presence of 100 nM tRNAGly (shaded bars), and glyQS readthrough in the absence of tRNAGly (basal level transcription, unfilled bars) were performed in the presence or absence of 5% (wt/v) PEG-8000. The Transcription Control data reflect the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. b) Effect of molecular crowding on the affinity (EC50) of tRNAGly to induce glyQS readthrough. For tRNAGly EC50 = 30 nM in the absence of PEG (open circle, 95% CI = 22-39 nM, R2 = 0.97) and EC50 = 26 nM in the presence of 5% PEG (filled circle, 95% CI = 13-50 nM, R2 = 0.84).

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Control

Journal: Bioorganic & medicinal chemistry

Article Title: Factors that influence T box riboswitch efficacy and tRNA affinity

doi: 10.1016/j.bmc.2015.07.018

Figure Lengend Snippet: a) Dose-response of compound 1 on glyQS transcription readthrough in the presence of 15 nM tRNAGly. Apparent IC50 = 8 μM (95% CI = 4-15 μM, R2 = 0.86). Inset: Structure of triazole 1. b) Readthroughmax without (open bars) and with 1 (50 μM, shaded bars) for glyQS basal level transcription, glyQS with tRNAGly (15 nM), and, the Transcription Control reaction. For each reaction condition, the percent of ligand-induced inhibition is indicated. The Transcription Control data reflect the effect of 1 on the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. Also shown is the effect of 1 on RTprb fluorescence in the presence of Target Sequence RNA (20 nM).

Article Snippet: 2.1 Nucleic Acids The fluorescently labeled readthrough probe (RT prb ) was obtained from Trilink as 5′ Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl 3′ where the upper case letters represent deoxyribonucleotides and lower case letters 2′- O -methyl ribonucleotides.

Techniques: Control, Inhibition, Fluorescence, Sequencing