Purification of poly-dA oligonucleotides and mRNA-protein fusions with dT25-OAS resin

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Abstract

Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT25-conjugated Oligo Affinity Support resin (dT25-OAS) alongside poly-dT14 magnetic beads and dT25-cellulose. dT25-OAS was found to have the highest dA21 oligo binding capacity at 4 pmol/µg, followed by dT14-magnetic beads (1.1 pmol/µg) and dT25-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT25-cellulose showed the highest mRNA-affibody purification specificity, followed by dT25-OAS and dT14-magnetic beads. Overall, dT25-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment.

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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