Purification of poly-dA oligonucleotides and mRNA-protein fusions with dT 25 -OAS resin.

Autor: Engel BJ; Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, United States., Grindel BJ; Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, United States., Gray JP; Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, United States., Millward SW; Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, United States. Electronic address: smillward@mdanderson.org.
Jazyk: angličtina
Zdroj: Bioorganic & medicinal chemistry letters [Bioorg Med Chem Lett] 2020 Feb 15; Vol. 30 (4), pp. 126934. Date of Electronic Publication: 2019 Dec 30.
DOI: 10.1016/j.bmcl.2019.126934
Abstrakt: 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 dT 25 -conjugated Oligo Affinity Support resin (dT 25 -OAS) alongside poly-dT 14 magnetic beads and dT 25 -cellulose. dT 25 -OAS was found to have the highest dA 21 oligo binding capacity at 4 pmol/µg, followed by dT 14 -magnetic beads (1.1 pmol/µg) and dT 25 -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 dT 25 -cellulose showed the highest mRNA-affibody purification specificity, followed by dT 25 -OAS and dT 14 -magnetic beads. Overall, dT 25 -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.
(Copyright © 2019 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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