Purification of poly-dA oligonucleotides and mRNA-protein fusions with dT25-OAS resin
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Section snippets
Materials
Oligo A25 was purchased from Integrated DNA Technologies. OAS resin (Oligo-Affinity Support PS, catalog number 26-4101-41) can be purchased from Glen Research (Sterling, VA). dT14-SeraMag™ magnetic beads were purchased from Sigma Aldrich. dT25-OAS and pF30P ssDNA (A21-Spacer 93-ACC-Puromycin) were purchased from Keck Biotechnology Resource Lab at Yale University. Retic Lysate IVTTM Kit was purchased from Thermo Fisher Scientific. Easy Tag Express Protein Labeling Mix [35S]-Methionine was
Polyadenosine binding assay
Resins were washed 5x with dT binding buffer (20 mM Tris, pH 8.0; 1 M NaCl; 1 mM EDTA; 0.2% [v/v] Triton-X 100) and resuspended at 10 mg/mL concentration. These stock solutions were serially diluted 2-fold with dT binding buffer from 50 µg to 0.78 µg in 10 µL total volume. To these were added 83 pmol of A21 ssDNA oligo to a total volume of 20 µL in dT binding buffer. After shaking for 1 h at room temperature, resins were pelleted and the A260 measured on a NanoDrop1000. The pmol of bound oligo
Resin binding specificity assay
Her2 affibody DNA was purchased as a gBlock® from Integrated DNA Technologies and PCR amplified with primers containing a T7 promoter (forward primer) and a DNA ligation spacer sequence (reverse primer). Following T7 RNA polymerase transcription, the mRNA was splint ligated to pF30P ssDNA and purified by Urea-PAGE. Fusion templates were translated in rabbit reticulocyte lysate using 20× high salt mixture without methionine according to manufacturer’s instructions. For each translation reaction,
Autoradiography of fusions
Affibody fusions (20 picomoles input) purified via dT25-OAS were ethanol precipitated, resuspended in 40 µL of water, split into thirds and incubated either alone, with RNAse A (10 µg), or RNAse plus micrococcal nuclease (12,500 gel units) with supplied buffer overnight at room temperature. Reaction solutions were separated by reducing tris-glycine SDS-PAGE, gel washed in water, dried onto filter paper, exposed on autoradiography film and imaged on a FLA 5100 Scanner (FujiFilm).
Funding
This work was supported by MD Anderson Startup Funds (SWM), a GE In-kind Multi-investigator Imaging (MI2) Research Award (SWM), the MD Anderson Cancer Center Ovarian Cancer SPORE (P50 CA2167685), The National Institutes of Health (2R44CA206771 SWM, 1T32CA196561 BJG, F32EB024379 BJE), and the MD Anderson Cancer Center Support Grant (5P30CA016672).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
We would like to thank the Keck Oligo Synthesis Facility at Yale for their helpful suggestions.
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