Alkaline-tolerant RNA aptamers useful to purify acid-sensitive antibodies in neutral conditions.

Recently, several oligonucleotides have been launched for clinical use and a number of therapeutic oligonucleotides are under clinical trials. Aptamer is one of the oligonucleotide therapeutics and has received a lot of attention as a new technology and an efficacious pharmaceutical compound comparable to antibody. Aptamer could be used for various purposes, not only therapeutics but also diagnostics, and applicable to affinity chromatography as a carrier molecule to purify proteins of interest. Here we demonstrate the usage and advantages of RNA aptamer to Fc region of human IgG (i.e., IgG aptamer) for purification of human antibodies. IgG aptamer requires divalent cations for binding to IgG and bound IgG dissociates easily upon treatment with chelating reagent, such as EDTA, under neutral conditions. This elution step is very mild and advantageous for maintaining active conformations of therapeutic antibodies compared to the widely used affinity purification with Protein A/G, which requires acidic elution that often damages the active conformation of antibodies. In fact, of several monoclonal antibodies tested, three antibodies were prone to aggregate on acidic elution from the Protein A/G resin, while remained fully active upon neutral elution from the IgG aptamer resin. The IgG aptamer was fully manipulated to alkaline resistant by ribose 2'-modifications, and thereby reusable numerous times with 1 N NaOH washing. The capacity of the aptamer resin to bind IgG was equivalent to that of the Protein A/G resin. Therefore, the IgG aptamer will provide us with a unique tool to uncover and purify human monoclonal antibodies, which hold therapeutic potential but lose the activity upon acidic elution from Protein A/G-based affinity resin.

Docking simulation of polyamines on a kissing-loop RNA dimer.

Polyamines, especially branched polyamines such as tetrakis(3-aminopropyl)ammonium (Taa), stabilize the tertiary structure of RNA molecules. In this study, we examined the polyamine binding site of the HIV-1 dimerization initiation site (DIS) in the kissing-loop dimer by the docking simulation. It was found that Taa binds predominantly to the kissing loop interaction site of DIS.

Selective structural change of bulged-out region of double-stranded RNA containing bulged nucleotides by spermidine.

Polyamines are essential for cell growth due to effects mainly at the level of translation. These effects likely involve a structural change, induced by polyamines, of the bulged-out region of double-stranded RNA that is different from changes induced by Mg(2+). Structural changes were studied using U6-34, a model RNA of U6 small nuclear RNA containing bulged nucleotides. Binding of NS1-2 peptide derived from the RNA binding site of NS1 protein, to U6-34 was inhibited by spermidine but not by Mg(2+). A selective conformational change of the bases in the bulged-out region of U6-34 induced by spermidine was observed by NMR. The selective effect of spermidine was lost when the bulged-out region of U6-34 was removed in U6-34(Delta5). The binding of NS1-2 peptide to U6-34(Delta5) was inhibited both by spermidine and Mg(2+). The selective structural change of U6-34 by spermidine was confirmed by circular dichroism.