oligonucleotide ratio  (New England Biolabs)

Journal: Nature materials

Article Title: Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

doi: 10.1038/nmat4497

Figure Lengend Snippet: a, Schematic showing the self-assembly process of three RNA strands to form a dual-colour RNA triple helix. The RNA triplex nanoparticles consist of stable two-pair FRET donor/quencher RNA oligonucleotides used for in vivo miRNA inhibition (miR-221 antagomiR) strategy and miRNA replacement therapy (miR-205 mimic). b, Secondary structure of an RNA triple helix bearing both miR-205 mimic and miR-221 antagomiR (structure produced/adapted using M-fold software). c, Formation of the RNA-triple-helix hydrogel scaffolds by conjugation of the RNA-triple-helix structure to PAMAM G5 (Cryo-EM image showing the branched sponge-like nanoscopic structure) followed by reaction with oxidized dextran to form the adhesive hydrogel. d,e, High-resolution SEM image of RNA microstructures (3.4 ± 1.1 μm) (d) composed of small RNA-triple-helix-dendrimer nanoparticles (56.6 ± 3.9 nm) (e). f-h, High-resolution SEM images of RNA macrostructures embedded in the dextran-dendrimer hydrogel scaffolds.

Article Snippet: Characterization and competition assays Presence of urea The concentration of single RNA and the RNA-triple-helix oligonucleotides (equal molar ratio 1:1:1, final concentration 1 μM each) was fixed and the samples were loaded on 20% TBE PAGE gel without and with urea (0.007 and 7M) (Invitrogen).

Techniques: In Vivo, Inhibition, Produced, Software, Conjugation Assay, Cryo-EM Sample Prep

Journal: Nature materials

Article Title: Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

doi: 10.1038/nmat4497

Figure Lengend Snippet: a, Evaluation of the fluorescence intensity of the different RNA oligonucleotides at several molar ratios at room temperature using a live imaging system, to compare quenching efficiencies induced by the proximity of the chromophores with the quencher BHQ2. b, RNA combinations are depicted in the table by ‘+’ signs, indicating the presence of a specific RNA strand. c, Gel electrophoresis showing self-assembly of the RNA triple helix (lane 7) and all other oligo combinations depicted in b. d, First derivative of the melting temperature (Tm) curves for the assembly of the different oligo combinations described in b. e, Flow cytometry revealed the specific uptake of fluorescent RNA-dendrimer nanoparticles into MDA-MB-231 cells. Negative and positive controls were cells only and cells treated with the RNA double helices (Q705:BHQ2 and Q570:BHQ2), respectively. Q1, the cell population containing the dye Quasar 705; Q4, containing the dye Quasar 570; Q2 containing both dyes and Q3 with no fluorescence signal. f, Confocal images showing strong uptake of the RNA-dendrimer complexes, as demonstrated by the outstanding co-localization of the dual-colour RNA triple helix (Q705:Q570:BHQ2). Nuclei (in blue) were stained with DAPI. Scale bars, 10 μm. All experiments were done in triplicate and errors reported as standard deviation (s.d.).

Article Snippet: Characterization and competition assays Presence of urea The concentration of single RNA and the RNA-triple-helix oligonucleotides (equal molar ratio 1:1:1, final concentration 1 μM each) was fixed and the samples were loaded on 20% TBE PAGE gel without and with urea (0.007 and 7M) (Invitrogen).

Techniques: Fluorescence, Imaging, Nucleic Acid Electrophoresis, Flow Cytometry, Staining, Standard Deviation

Journal: Nature materials

Article Title: Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

doi: 10.1038/nmat4497

Figure Lengend Snippet: a, Dual-colour hydrogel scaffolds made of RNA-triple-helix nanoconjugates pre-lncubated with complementary mIR targets. b, Cryosectlon of dendrlmer-dextran adhesive hydrogel (12 μm thickness) depicting adhesive morphology (dextran aldehyde was tagged with Alexa Fluor 405). Red spots represent the triple-helix nanoparticles containing Q705 (red) and Q570 (green) oligonucleotides. c, Live imaging of female SCID hairless congenic mice with triple-negative breast tumour xenograft implanted with hydrogels embedded with RNA-triple-helix nanoparticles and with a control triplex (scrambled miRs), (n = 5 per group). Ex vivo images of breast tumours and whole body organs (T, tumour; Lv, liver; K, kidneys; S, spleen; H, heart; Lu, lung; Int, intestines) are also presented. d, Haematoxylin and eosin (H&E) stains of tumours from treated groups with hydrogels embedded with RNA-triple-helix nanoparticles and with a control triplex (scrambled miRs). e, Immunohistochemical evaluation of tumours treated with hydrogels embedded with RNA-triple-helix nanoparticles and with a control triplex for Ki67 to evaluate tumour cell proliferation. f, Tumour size following treatment (n = 5, statistical analysis performed with a two-way ANOVA, **, P< 0.01; *, P< 0.05). Individual tumours were measured using a Vernier calliper and tumourvolume was calculated by: tumourvolume (mm3) = width × (length2)/2. g, Kaplan-Meier curves for mice treated with hydrogel scaffolds loaded with triple-helix and control triple-helix nanoparticles, as well as for the drugs (DOX, PTX and Avastin). Statistical analysis (n = 5) was performed with a Log-Rank Mantel-Cox test (P = 0.006). Survival cutoff criteria included tumour ulceration or compassionate euthanasia, aggregate tumour burden >1 cm in diameter, or if tumour impedes eating, urination, defecation or ambulation. Arrow represents the day of hydrogel implantation (day 22 post tumour induction). h, Heat-map summary of gene expression profiling of the miRs and their related genes that play key roles in cancer progression and migration.

Article Snippet: Characterization and competition assays Presence of urea The concentration of single RNA and the RNA-triple-helix oligonucleotides (equal molar ratio 1:1:1, final concentration 1 μM each) was fixed and the samples were loaded on 20% TBE PAGE gel without and with urea (0.007 and 7M) (Invitrogen).

Techniques: Imaging, Ex Vivo, Immunohistochemical staining, Expressing, Migration