ArticleJournal of separation science2026
Affinity Chromatography With Selective Nucleic Acid Hybridization: Direct Coupling With Size Exclusion Chromatography.
Article in Journal of separation science, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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4 authors.
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Abstract
Affinity liquid chromatography (aLC) with immobilized oligonucleotides provides high selectivity for nucleic acid targets through on-column hybridization. We describe the preparation and evaluation of prototype 2.1 mm × 20 mm affinity columns packed with a 3.5 µm nonporous polystyrene/divinylbenzene sorbent covalently modified with streptavidin. Two different affinity columns were prepared by immobilizing biotinylated oligonucleotides on-column via biotin-streptavidin bond: (i) biotinylated 25-nt oligodeoxythymidine, dT25 or (ii) biotinylated 21 nt TP53 oligonucleotide. dT25 column was used for poly(A) denylated messenger RNA (mRNA) and adenosine oligonucleotides variants analysis. The TP53 column was applied to single-nucleotide polymorphism analysis. Nucleic acid binding was performed in 0.1 M sodium phosphate buffer (pH 7.4); retained species were eluted with water or 10% acetonitrile solution in water. We demonstrate that dT25 column selectively retains poly(A)-tailed EPO, EGFP, and Cas9 mRNAs, while tailless mRNA is unretained. dT25 column resolved dA14-dA25 oligoadenosine length variants and dA25 variants bearing cytidine substitutions. The TP53 column baseline-separated a fully complementary 21-nt sequence from single-nucleotide mismatches. Direct coupling of the dT25 column with a 250 Å, 1.7 µm size exclusion chromatography (SEC) column enabled improved poly(A) tail sizing after RNase T1 digestion of intact mRNA; mRNA recovery exceeded 98%, and carryover remained below 0.1% after cleanup.
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