Magnetically Detected Protein Binding Using Spin-Labeled Slow Off-Rate Modified Aptamers.
Shutian LuCatherine R FowlerBrian ReamSheela M WaughTheresa M RussellJohn C RohloffLarry GoldJason P ClevelandStefan StollPublished in: ACS sensors (2023)
Recent developments in aptamer chemistry open up opportunities for new tools for protein biosensing. In this work, we present an approach to use immobilized slow off-rate modified aptamers (SOMAmers) site-specifically labeled with a nitroxide radical via azide-alkyne click chemistry as a means for detecting protein binding. Protein binding induces a change in rotational mobility of the spin label, which is detected via solution-state electron paramagnetic resonance (EPR) spectroscopy. We demonstrate the workflow and test the protocol using the SOMAmer SL5 and its protein target, platelet-derived growth factor B (PDGF-BB). In a complete site scan of the nitroxide over the SOMAmer, we determine the rotational mobility of the spin label in the absence and presence of target protein. Several sites with sufficiently tight affinity and large rotational mobility change upon protein binding are identified. We then model a system where the spin-labeled SOMAmer assay is combined with fluorescence detection via diamond nitrogen-vacancy (NV) center relaxometry. The NV center spin-lattice relaxation time is modulated by the rotational mobility of a proximal spin label and thus responsive to SOMAmer-protein binding. The spin label-mediated assay provides a general approach for transducing protein binding events into magnetically detectable signals.
Keyphrases
- binding protein
- single molecule
- growth factor
- protein protein
- room temperature
- density functional theory
- amino acid
- minimally invasive
- randomized controlled trial
- computed tomography
- cancer therapy
- mass spectrometry
- gold nanoparticles
- pet imaging
- drug delivery
- small molecule
- magnetic resonance imaging
- energy transfer
- transcription factor
- smooth muscle
- molecular dynamics
- blood brain barrier
- electron microscopy
- magnetic nanoparticles
- label free
- solid state