Crossing the Halfway Point: Aptamer-Based, Highly Multiplexed Assay for the Assessment of the Proteome.

Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.

Magnetically Detected Protein Binding Using Spin-Labeled Slow Off-Rate Modified Aptamers.

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.

Spectral properties of microcantilevers in viscous fluid.

We explore analytically, numerically, and experimentally the spectral properties of the flexural vibrations of micron scale cantilevers in a viscous fluid that are driven externally or by Brownian motion. Although the physical origins of driven and thermal cantilever dynamics are quite different, we show that in each case the dynamics can be calculated deterministically using an impulse or step force, respectively. The stochastic dynamics of the cantilever are related to the removal of a step force by the fluctuation-dissipation theorem to yield the autocorrelation and noise spectral density of equilibrium fluctuations. The dynamics of a cantilever driven externally is related to an impulse in force by transfer function theory. Using this approach, we explore the differences between the driven and thermal spectra of microcantilevers. We find that higher order cantilever modes and the spatial distribution of the applied load for the external drive can be critical to the relationship between the thermal and driven spectra.

Molecular nanosprings in spider capture-silk threads.

Spider capture silk is a natural material that outperforms almost any synthetic material in its combination of strength and elasticity. The structure of this remarkable material is still largely unknown, because spider-silk proteins have not been crystallized. Capture silk is the sticky spiral in the webs of orb-weaving spiders. Here we are investigating specifically the capture spiral threads from Araneus, an ecribellate orb-weaving spider. The major protein of these threads is flagelliform protein, a variety of silk fibroin. We present models for molecular and supramolecular structures of flagelliform protein, derived from amino acid sequences, force spectroscopy (molecular pulling) and stretching of bulk capture web. Pulling on molecules in capture-silk fibres from Araneus has revealed rupture peaks due to sacrificial bonds, characteristic of other self-healing biomaterials. The overall force changes are exponential for both capture-silk molecules and intact strands of capture silk.