Study On The Method Of Aptamers Selection Based On Monolithic Capillary

Systematic evolution of ligands by exponential enrichment (SELEX) is the workhorse method for selecting aptamers that are capable of binding target molecules from a random oligonucleic acid library. Aptamer works as antibody’s substitute which not only has the specificity of antibody, but also it is more stable, easier to be modified and could be synthesis in vitro. A key step in SELEX is to isolate target-binding single stranded DNA (ssDNA) from the random pool. Widely used SELEX methods mainly rely on affinity chromatography, membrane filtration, magnetic beads, capillary electrophoresis (CE), and surface plasmon resonance (SPR). The frequently used methods are all associated with apparent drawbacks.Affinity chromatography, magnetic beads, and SPR based methods require tedious procedures to immobilize targets and to recover bound species, magnetic beads and filtration based methods suffer strong nonspecific binding toward targets and oligonucleic acids, while filtration based method is limited to large target molecules. Therefore, novel methods that can effectively overcome these drawbacks are of great importance.In this work, we first chose boronate affinity monolithic column as the platform to select aptamers. The boronate affinity monolithic column allow for reversible covalent capture/release of cis-diol-containing compounds in a pH-switchable fashion (high pH on, low pH off).What’s more, the monolithic column has some advantages such as easy fabrication, fast convective mass transfer and low volume. Herein, we used 3-carboxybenzoboroxole-functionalized monolithic capillary as the selection platform to select aptamers for glycoprotein (HRP).By using boronate affinity monolithic capillary as a platform for target immobilization and aptamer isolation, the proposed SELEX method allowed for efficient selection of glycoprotein-binding aptamers by 6 rounds and the dissociation constants were at 10-8 M level. Because of the employment of boronate affinity monolithic capillary, the new SELEX approach overcame the above-mentioned drawbacks and provided several significant advantages, including rapid selection speed (only 2 days were needed), high specificity toward the target molecules, and minute reagent consumption (10-20μL per cycle).Next, as an extension of boronate affinity monolithic column SELEX, we developed other monolithic column to select aptamers for nonglycoproteins. N-(β-hydroxy propyl) ethylene diamine modified monolithic column was chosen as a platform to select BSA aptamers. This column could capture and separate proteins by making the pore size nearly the same as protein sizes (～3-30 nm in this study). Modified with an appropriate ligand (N-(β-hydroxy propyl)ethylene diamine), the monolithic column exhibited boronate affinity-like pH-controlled binding properties toward proteins. By using this monolithic column as a platform to do SELEX, the ssDNA that could recognize BSA had been acquired after 3 rounds. The dissociation constants were also at 10-8 M level. This method inherited the merits of boronate affinity monolithic column SELEX, including fast screening, high specificity toward the target molecules, and minute reagent consumption.