Aptamer Selection for Human Serum Albumin By

ii Human serum albumin (HSA) is a highly abundant protein in the human plasma, causing the discovery of new biomarkers challenging. Here we introduced a new method: aptamers as new affinity ligands for the depletion of HSA from serum. Aptamers are short RNA or DNA oligonucleotides (15-120 nucleotides) that bind to different targets, including proteins, peptides, and cells. Aptamers are like chemical antibodies, able to form 3-dimensional structures with targets and bind with high affinity and specificity. In this research, we selected aptamers against recombinant HSA (rHSA) using two different combinatorial DNA libraries: N40 and structured DNA. The method we used is called Nonequilibrium Capillary Electrophoresis of Equilibrium Mixture (NECEEM) with Systemic Evolution of Ligands in Exponential Enrichment (SELEX). This method uses capillary electrophoresis to separate target-bound DNA from non-target bound DNA in an equilibrium mixture containing both the DNA and the target. The collected target-bound nucleic acids are amplified with Polymerase Chain Reaction (PCR) and used for the next round of selection. By repeating this procedure, SELEX generates a pool of aptamers with the highest binding affinity to the target. The final aptamer pool is later cloned and sequenced. Prior to selection, we optimized both mastermix and PCR conditions for the amplification of the structured DNA library. Unfortunately, the selection of aptamers against rHSA with the two DNA libraries was unsuccessful. The structured DNA library did not generate high enough aptamer concentration in the first round for further selection rounds. The N40 DNA library showed no enrichment of aptamers with 5 rounds of selection