Linus Hein

All Publications

• Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using An Aptamer Switch-Conjugated Hydrogel Matrix. Advanced materials (Deerfield Beach, Fla.) Park, C. H., Thompson, I. A., Newman, S. S., Hein, L. A., Lian, X., Fu, K., Pan, J., Eisenstein, M., Soh, H. T. 2023: e2306704

  Abstract

  Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adma.202306704

  View details for PubMedID 37947789

• A massively parallel screening platform for converting aptamers into molecular switches. Nature communications Yoshikawa, A. M., Rangel, A. E., Zheng, L., Wan, L., Hein, L. A., Hariri, A. A., Eisenstein, M., Soh, H. T. 2023; 14 (1): 2336

  Abstract

  Aptamer-based molecular switches that undergo a binding-induced conformational change have proven valuable for a wide range of applications, such as imaging metabolites in cells, targeted drug delivery, and real-time detection of biomolecules. Since conventional aptamer selection methods do not typically produce aptamers with inherent structure-switching functionality, the aptamers must be converted to molecular switches in a post-selection process. Efforts to engineer such aptamer switches often use rational design approaches based on in silico secondary structure predictions. Unfortunately, existing software cannot accurately model three-dimensional oligonucleotide structures or non-canonical base-pairing, limiting the ability to identify appropriate sequence elements for targeted modification. Here, we describe a massively parallel screening-based strategy that enables the conversion of virtually any aptamer into a molecular switch without requiring any prior knowledge of aptamer structure. Using this approach, we generate multiple switches from a previously published ATP aptamer as well as a newly-selected boronic acid base-modified aptamer for glucose, which respectively undergo signal-on and signal-off switching upon binding their molecular targets with second-scale kinetics. Notably, our glucose-responsive switch achieves ~30-fold greater sensitivity than a previously-reported natural DNA-based switch. We believe our approach could offer a generalizable strategy for producing target-specific switches from a wide range of aptamers.

  View details for DOI 10.1038/s41467-023-38105-4

  View details for PubMedID 37095144

  View details for PubMedCentralID 4215550