Investigating Protein Capture at Aptamer‑Coated Surfaces

The ever increasing importance of protein analysis in biological, medical, energy, environmental, industrial and defense research has led to growing demand for reagents that bind to target proteins with high affinity and specificity.  Antibodies have long been unrivaled as affinity reagents for proteins due to their strong and selective binding; however, drawbacks associated with their production, stability and manipulation have prompted researchers to seek alternatives. Foremost among these are aptamers, which are short, generally single-stranded oligonucleotides comprising DNA, RNA or related nucleotides.  Aptamers are most commonly selected from random, combinatorial oligonucleotide libraries although alternative pathways to their identification have been developed. Regardless of the discovery route, aptamers offer important advantages over antibodies in terms of stability, size, ease of production and manipulation and reusability.

In the analytical arena, aptamers have been employed in various devices and processes for applications involving protein detection, quantification, separation, isolation, purification and imaging. Binding is generally indicated by a label that produces a non-specific signal (e.g., direct or enzymatically generated fluorescence, chemiluminescence, electrochemical, etc.) since it is the aptamer, not the label, that determines the specificity. Therefore, in developing an aptamer-based analytical method or process, it is essential to know what is being captured and how capture is affected by experimental conditions in order to avoid inaccuracies due to cross-reactivity, non-specific binding and other sources of chemical and biological interference.

This talk will describe studies of the processes that govern affinity protein capture at aptamer-modified surfaces, as indicated by mass spectrometry and fluorescence imaging. The technique of Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS) provides a profile of protein capture as a function of experimental conditions, including changes in the protein capture profile and therefore specificity over time. Fluorescence microscopy allows us to visualize the spatial distribution of immobilized aptamer at the surface as well as protein capture at the surface. These factors are fundamentally important for design of aptamer-based sensors and devices, and for accurate interpretation of resulting data.