A Reverse Transcription Polymerase Chain Reaction-Based Method for Determining Aptamer–Protein Interactions at Nanoparticle Surfaces

Nucleic acid–protein interactions influence the activity and stability of DNA- and RNA-functionalized nanomaterials. Herein, a restriction enzyme-mediated approach coupled with reverse transcription-polymerase chain reaction (RT-PCR) for probing aptamer–protein interactions on a nanoparticle (NP) surface as a general method for identifying interactions between proteins and an RNA on a NP is described. This process is enabled by utilizing a restriction enzyme (RE)-specific sequence at the NP surface that can be masked and subsequently revealed depending on the bound state of a protein at the particle's surface. The method is suitable for investigating a specific protein interaction versus a nonspecific interaction, using an RT-PCR assay designed to detect exposed nucleic acid ligands at the NP surface. This approach is highly modular and can be applied to a variety of RNA–protein interactions for bioanalytical purposes, including as a selection pressure for in vitro selections and Systematic Evolution of Ligands by EXponential enrichment protocols.