Microfluidic aptamer selection
We are developing integrated microfluidic systems for the rapid and high-efficiency selection of nucleic acid aptamers.
Aptamers are single strands of RNA of DNA that adopt three-dimensional folds and bind to a variety of target molecules with high affinity. Aptamers are isolated from large libraries of random sequences, most often using Systematic Evolution of Ligands through Exponential Enrichment (SELEX) (Tuerk and Gold, 1990). SELEX has been used to generate high-affinity, high-specificity aptamers against a variety of whole-cell, protein, and carbohydrate targets. Aptamers have advantages over other affinity reagents including large starting libraries that are easy to generate using standard chemistries, simple reagent manipulation, and amplification to enrich high-affinity binding sequences. Unfortunately, currently available aptamers suffer several drawbacks that limit their utility in analysis of complex samples. First, the specificity of aptamers is largely uncontrolled and can vary widely. In addition, aptamers are not easily selected against subtly differing targets, such as different post-translational modifications of the same protein. Finally, some aptamer selections present idiosyncrasies that can confound the selection process, such as a decrease in affinity in later selection rounds. For these reasons, it is important to improve the quality of aptamer selection methods in order to develop high-affinity reagents for the analysis of complex samples.
SELEX as currently practiced can be challenging to perform due to poor selection efficiencies, requiring up to 17 rounds of selection and months of time. Robotic methods for aptamer selection in parallel have been presented, resulting in faster completion of selections, although no improvement in the efficiency of such selections was observed. Recent SELEX approaches using capillary electrophoresis and surface plasmon resonance have demonstrated more efficient selection than previous approaches, resulting in fewer necessary rounds of selection. To date, these methods are still manually intensive, and therefore not amenable to large-scale application.
We will improve aptamer selection methods by developing microsystems capable of selecting or evolving such affinity reagents more rapidly and with better efficiency than has thus far been possible. By integrating high-efficiency selection with amplification and sample preparation steps, we hope to dramatically decrease the time and effort needed to select nucleic acid aptamers against a wide variety of targets of clincal utility. Specificlaly, we are designing systems that can select for undreds of aptamers in parallel in less than a day.