Detection of single enzyme molecules and catalytic nucleic acids using microfluidics and selection of aptamers using capillary electrophoresis-SELEX

by Jing Yang

Institution: University of Minnesota
Year: 2014
Keywords: Chemistry
Record ID: 2025334
Full text PDF: http://hdl.handle.net/11299/172104


Aptamers are single stranded DNA or RNA molecules that can bind targets with high affinity and specificity. They are selected in vitro from a large library of synthetic oligos via Systematic Evolution of Ligand by Exponential enrichment (SELEX) process. Conventional SELEX techniques using affinity chromatography and nitrocellulose filtration have identified aptamers for a wide range of targets. Although proven successful, the conventional SELEX is also time-consuming and labor-intensive. Capillary electrophoresis-SELEX (CE-SELEX) has later been introduced for aptamer selection, which greatly reduces the selection time and the number of cycles needed and minimizes the non-specific interactions and steric hindrance.CE-SELEX has been previously performed mainly against large protein targets. A small molecule target N-methyl mesoporphyrin (NMM) is now proved to be suitable for CE-SELEX. Aptamers with high nM to low µM affinity are selected by CE-SELEX and two out eight randomly picked sequences are also capable of catalyzing the corresponding metal insertion reaction. It greatly expands the capability of CE-SELEX and indicates that any target that can generate enough change in hydrodynamic size of the oligos upon binding can be used in CE-SELEX. Not only have we explored the low size limitation of targets for CE-SELEX, but also we have applied CE-SELEX against a fully intact membrane target - transferrin receptor (TfR) which is difficult for conventional SELEX. Aptamers with a few tens of nM Kd have been selected after four rounds of CE-SELEX. These aptamers binding receptors involved in receptor-mediated endocytosis process could be potentially engineered for transporting therapeutic cargos for treating intracellular targets.Besides aptamer selection using CE-SELEX, binding interactions between DNA/RNA analogs and important cardiac muscle proteins have also been studied. Using affinity CE and fluorescence polarization measurements, various DNA/RNA analogs, such as oligos of different lengths, L-DNA, O-methyl RNA, and Ca2+ pump Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and regulatory protein phospholamban (PLN), and contractile proteins such as troponin and myosin have been employed in the study. This sequence-independent binding affinity indicates possible direct nucleic acid regulation effect on protein functions and shows their potential as drug candidates for PLN super-inhibition caused heart failure.Finally, other than binding affinities, catalytic activities of nucleic acid oligos have also been assessed. A novel platform allowing multiple turnover reactions of catalytic sequences has been designed and fabricated in PDMS. Single enzyme molecules have been detected successfully and their kinetics have been studied using the microwell array device as a proof-of-concept. Unfortunately, when applying DNA/RNA libraries, we can't detect any catalytic sequences, indicating either the abundance or catalytic activity is too low to be detected.