Retention Of Oligonucleotides Research Articles

Control of oligonucleotide retention on a pH-stabilized strong anion exchange column

Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alkaline conditions. In this article, we report the use of an anion exchange column prepared from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and composition. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5′ or 3′ terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column’s hydrophobic interactions when CH 3CN is used with NaClO 4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base composition-dependent) separations.

Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography

Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50×4.6-mm column packed with porous, 2.5 μm C 18 sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC–MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy™3) dyes, as well as dually-labeled TaqMan™ probes. Purification of a 0.1-μmole oligonucleotide synthesis in a single injection was demonstrated.

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C 18 stationary phases. A small sorbent particle size (2.5 μm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA–HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Reverse-phase ion-pair chromatography of polyvalent ions using oligonucleotides as model substances

The retention of oligonucleotides of uridylic and adenylic acids as model substances was investigated employing the reverese-phase ion-pair chromatography technique and using LiChrosorb RP-18 and tetra- n-alkylammonium compounds. The retention sequence is controlled by the charge of the oligonucleotides, thus determining the number of ion pairs formed. The chain length of the n-alkyl group of the ion pair reagent and the content of organic solvent in the hydroorganic eluant also contribute to retention. A semiquantitative model was developed to describe the dependence of k′ on the ion pair concentration in a certain range. A comparison of retention in the reverse-phase ion-pair and ion-exchange chromatographic techniques established distinct differences in the dependence of log k′ on the charge of the oligonucleotide. Hydrophobic interactions also appear to make a larger contribution to retention in reverse-phase ion-pair chromatography than in ion-exchange chromatography.

Application of a weakly basic dimethylamino-modified silica ion exchanger to the separation of oligonucleotides

LiChrosorb RP-8, RP-18 and Diol as well as a newly synthesized basic dimethylamino-modified silica ion-exchanger (DMA-silica) were applied for the separation of adenylic acid, cytidylic acid and uridylic acid oligoribonucleotides. On LiChrosorb RP-8 and RP-18, respectively, in aqueous buffered eluents (K 2HPO 4- H 3PO 4), the retention of oligonucleotides was increased with decreasing number of nucleotide units in the solute, i.e., with increasing hydrophobic character. The elution behaviour of ologonucleotides on LiChrosorb Diol followed the same order but took place according to a size-exclusion mechanism. The retention of oligonucleotides on DMA-silica is assumed to be based on electrostatic interactions between the charged. solute and the ionic surface sites of DMA. This is evidenced by (i) the exponential decrease of k′ of the solute with increasing salt concentration of buffer, and (ii) the increase of log k′ of the solute with the number of nucleotide units, i.e., negative charges in the oligoribonucleotide solute. The relative contribution of ionic and molecular interactions to total retention of the solutes is descussed.

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.