Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods

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Methods

Gel electrophoresis         SAXS         FRET         Dicer         Dual Luciferace assay

Gel electrophoresis

Gel electrophoresis uses an electric field to drive charged molecules through a porous gel, thereby separating them according to charge and size. Gels can be made of e.g. agarose or polyacrylamide. Phosphate in the sugar-phosphate backbone of DNA and RNA provide the molecules with an overall evenly distributed negative charge. DNA and RNA will therefore move toward the positive pole, the anode. Because the charge to mass ratio of nucleic acids is rather uniform, the separation is essentially determined by the mass. If the nucleic acids are considered to be homogeneous in shape, the mass becomes proportional to the size, which then becomes the determining factor. Small molecules are allowed easy access through the porous matrix and travel fast, while larger molecules are subject to more friction and travel slower. Because molecules of different size travel with different speeds in a gel, it can be used to determine the difference between a folded structure and a non-folded structure. The technique is called electrophoretic mobility shift assay (EMSA) or band shift assay. Visualization of the nucleotides can be done with intercalating dyes such as SYBR Safe DNA Gel Stain.

SAXS

Small Angle X-ray Scattering (SAXS) is a method based on detection of elastic scattering of X-rays on macromolecules in solution recorded at very low angles. The technique provides information of size and three-dimensional structure in the nanometer range. The SAXS intensity is recorded as a function of q = 4πsin(θ)/λ, where 2θ is the angle between the incident beam and the scattered beam and λ is the wave length of the X-rays. From these data we are able to determine the real-space distance distribution, p(r) using indirect Fourier transformation. The p(r) distribution corresponds to a histogram over all distances between pairs of points inside the particle and leads thereby to direct insight into shape and size of the particles. Furthermore the direct scattering (i.e. the scattering at θ=0) is proportional to the weight-average molecular weight, and we can thereby get information of the molecular weight of our particles by extrapolating our data to 0.

FRET

Förster Resonance Energy Transfer (FRET) has been used as a workhorse to measure distances at the nanoscale, by doing calculations of the transfer energy, which correlates to the distance. Single-molecule (SM) measurements can provide more detailed conformational and time dependent information than is achievable by ensemble measurements. Techniques for SM FRET measurements continuously improve, but are still time consuming, which is why only ensemble measurements have been performed in this study. Resonance energy transfer (RET) is a non-radiative energy transfer that happens at distances shorter than the wavelength of a photon. The transfer of energy from the donor molecule to the acceptor molecule is only possible if there is a partial overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. The donor and acceptor fluorophores used for FRET measurements are often aromatic with delocalized electrons. By measuring the fluorescence intensity with a spectrofluorometer it is possible to calculate the transfer efficiency, which in combination with the Förster radius can be used to calculate the distance between the fluorophores.

Dicer

Dicer holds a central role in both RNAi pathways. Dicer consists of several domains, the three most noticeable being two RNaseIII domains and a PAZ domain. Structural analysis reveals that the Dicer from Giardia intestinalis has a long “hatchet like” structure, with the two RNaseIII domains at one end and the PAZ domain at the other. The structure of Dicer suggests its function to be that of a molecular ruler. The PAZ domain recognizes a 2 nt 3' overhang thus fixing the end of the dsRNA. The long flat side of Dicer contains many basic amino acids and thus has a net positive charge, attracting the negatively charged RNA. The connector helix puts the catalytic site at exactly the right distance from the end to create siRNAs. The affinity of the PAZ domain for 3' overhangs may be why circular viral dsRNA shows resistance to RNAi.

Dual Luciferace assay

The Luciferase construct containing Firefly luciferase (FLuc), Renilla luciferase (RLuc) and an EGFP site in 3’ UTR.
The Luciferase construct containing Firefly luciferase (FLuc), Renilla luciferase (RLuc) and an EGFP site in 3’ UTR.

The dual luciferase assay is a common tool for measuring gene knockdown from RNAi in cells. Cells can be transfected with siRNAs with the help of either electroporation or the use of transfection agents like Lipofectamine. This leads to a knockdown which can be measured 2-3 days later. The usage of luciferase genes is standard in RNAi work, as their elumination is easy detectable. In our experiment we use the H1299 cell line that has a gene construct producing both Renilla luciferase and Firefly luciferase, designed with an EGFP site in 3’-UTR of the Renilla Luciferase gene (Figure 1). An siRNA complementary to the EGFP site will therefore forsake knockdown of Renilla luciferase but not of Firefly Luciferase. By determining the relation between the expression levels of these two genes, the knockdown effect of an siRNA can be determined. Our structure is designed to have a knockdown effect on Renilla luciferase as the sequence of the structure is similar to the luciferase contruct in H1299 cells around the EGFP site. When making a dual luciferase assay it is always recommended to run a non-transfected control, so as to measure the toxicity of the transfection agent, and a control with a siRNA that does not target a luciferase gene.


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