Polyvalent RNA-nanoparticle composition
A technology of nanoparticles and compositions, applied in the field of nanoparticles, can solve problems such as inability to establish experimental reactions
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Embodiment 1
[0139] Example 1. Preparation of RNase-free nanoparticles
[0140] Citrate-stabilized gold nanoparticles (13 nm) were prepared using a published method [Frens, Nature Physical Science 241:20-22 (1973)]. After synthesis, the particles were treated with 0.1% diethylpyrocarbonate (DEPC) for 12 hours with stirring and then autoclaved at 121° C. for 60 minutes. Importantly and rather surprisingly, the optical and physical properties of the nanoparticles were not affected by this relatively extreme treatment, as can be analyzed by UV spectroscopy and TEM ( figure 1 )Measurement. Subsequent ligand functionalization was also unaffected by this treatment. Detection of RNase activity in these solutions using the RNaseAlert kit (Ambion) showed no detectable RNase activity compared to control or untreated particles ( figure 2 ).
Embodiment 2
[0141] Example 2. Modification of RNase-free nanoparticles
[0142] The resulting RNase-free nanoparticles were further appropriately modified by thiolated oligonucleotides using published procedures [Demers et al., Anal. Chem. 72:5535 (2000)]. Subsequent functionalization with RNA could not be achieved without this pretreatment, presumably due to rapid degradation of the RNA-based surface capping ligand. A duplex consisting of a 27-base RNA strand and a 25-base complement terminated with an ethylene glycol spacer and an alkylthiol was hybridized and added to RNase-free Au NPs , which allow them to be absorbed chemically via thiol-gold bonds. For this work, the sequence was designed to target the firefly luciferase gene.
[0143] RNA oligonucleotides were synthesized using TOM-RNA reagents (Glen Research) and MerMade 6 (Bioautomation), or were prepared commercially (Integrated DNA Technologies). Oligonucleotides synthesized from non-commercial sources were purified using th...
Embodiment 3
[0148] Example 3. Cellular uptake of RNA-nanoparticle compositions
[0149] Using the fluorescent (cyanine 5, Cy5) compositions prepared above, the ability of the compositions to enter cells was studied by confocal microscopy. RNA-Au NPs were added to cultures of HeLa cells. Cells were grown on glass coverslips and treated with nanoparticles functionalized with fluorophore-labeled RNA duplexes. After 6 hours of treatment, coverslips were removed, washed with PBS, and mounted in slide-mounted chambers filled with PBS. All images were acquired by a scanning confocal microscope (Zeiss 510LSM) at 63x magnification and a 633 nm HeNe laser excitation source. Imaging studies showed fluorescence in all HeLa cells after 6 hours ( Figure 4 a). It is interesting to note that, like DNA Au-NPs, RNA Au-NPs do not require transfection agents to enter cells [Giljohann et al., Nano Lett. 7:3818 (2007)]. Indeed, analytical flow cytometry confirmed RNA-Au NP uptake in >99% of the cell popu...
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