Affinity Chromatography With Selective Nucleic Acid Hybridization: Direct Coupling With Size Exclusion Chromatography

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.