Linus Hein

All Publications

• A Rationally Designed Split Lettuce Aptamer Based on Large-Scale Mutational Analysis. ACS sensors Adams, A. M., Pimentel, E. B., Loh, N. D., Gidi, Y., Hein, L. A., Eisenstein, M., Soh, H. T. 2026; 11 (8): 6867-6877

  Abstract

  Split-aptamer biosensors offer exceptionally low background by assembling only in the presence of a target analyte; however, their performance is frequently limited by the lack of robust design rules for selecting effective split sites. Existing approaches largely rely on heuristic, structure-based assumptions that are poorly validated and often yield suboptimal signal. Herein, we introduce a systematic, data-driven strategy for identifying high-performance split sites within fluorogenic DNA aptamers. Using our massively parallel aptamer performance analyzer platform, we performed comprehensive single- and double-mutant analysis of the DFAME-binding region of the fluorogenic DNA aptamer Lettuce, informed by its three-dimensional structure. Dimensionality reduction and clustering of the resulting sequence-function landscape revealed mutation-tolerant elements within the binding domain that are suitable for splitting while preserving fluorophore activation. Sensors constructed using these nonintuitive split sites, which are unconventional by standard design principles, exhibited a nearly four-fold improvement in the fluorescence signal-to-background ratio for SARS-CoV-2 RNA detection compared to a prior split-Lettuce design. The same split architecture also enabled robust detection of high-pathogenicity H5Nx avian influenza RNA. These results demonstrate that large-scale, data-driven interrogation of aptamer sequence-function relationships can identify nonintuitive split sites and provide a proof-of-concept framework for developing measurement-based design principles for split-aptamer biosensors.

  View details for DOI 10.1021/acssensors.6c00688

  View details for PubMedID 42687284

• A Rationally Designed Split Lettuce Aptamer Based on Large-Scale Mutational Analysis ACS SENSORS Adams, A. M., Pimentel, E. B., Loh, N., Gidi, Y., Hein, L. A., Eisenstein, M., Soh, H. 2026

  View details for DOI 10.1021/acssensors.6c00688

  View details for Web of Science ID 001836183100001

• Single-molecule peptide sequencing through reverse translation of peptides into DNA. Nature biotechnology Zheng, L., Sun, Y., Hein, L. A., Eisenstein, M., Soh, H. T. 2026

  Abstract

  Despite advances in mass spectrometry and emerging single-molecule approaches, sequencing peptides at the single-molecule level remains a central challenge in proteomics. Here we present a 'reverse translation' strategy that enables single-molecule peptide sequencing with single-amino-acid resolution. In this approach, peptides undergo a modified Edman degradation that iteratively releases N-terminal amino acids tagged with peptide-specific DNA barcodes. Antibody-mediated proximity extension assays identify these barcoded amino acids and generate PCR-amplifiable DNA reporters that record the identity, position and originating peptide of each amino acid. The resulting DNA library is directly read by high-throughput sequencing, converting peptide sequences into digital DNA outputs. Using this approach, we demonstrate true single-molecule peptide sequencing, achieving full sequence coverage in millions of reads and accurate differentiation of both native and post-translationally modified peptides. These results establish a framework that redefines protein sequencing as a DNA sequencing problem and lays the foundation for high-throughput, de novo single-molecule protein sequencing.

  View details for DOI 10.1038/s41587-026-03061-z

  View details for PubMedID 41851307

  View details for PubMedCentralID 4191988

• Theoretical Framework and Experimental Validation of Multiplexed Analyte Quantification Using Cross-Reactive Affinity Reagents. Analytical chemistry Newman, S. S., Hein, L. A., Adams, A. M., Soh, H. T. 2025

  Abstract

  Gold standard immunoassays depend on specific affinity reagents for accurate molecular quantification. Any cross-reactivity of affinity reagents, wherein the reagent nonspecifically binds to unintended molecules, can create false positive binding signals and result in inaccurate quantification of analytes. Mitigating cross-reactivity represents one of the greatest challenges in molecular diagnostics, and remains an unsolved problem. To mitigate the burden of cross-reactive reagents, we present a mathematical framework that uses generalized binding equations and noise estimation to enable the accurate use of multiple cross-reactive reagents for molecular quantification. As a proof of concept, we experimentally demonstrate accurate 2-plex quantification of a small molecule for which no specific affinity reagents are available, even in the presence of high concentrations of a cross-reactive molecule in 10% human serum. This robust schema yields well-defined bounds of quantification that make it easier to assess the quality of assay results, and predicts conditions under which assay performance is likely to break down. This work turns cross-reactive affinity reagents, which are conventionally unused, into a tool for achieving accurate quantification of analytes.

  View details for DOI 10.1021/acs.analchem.5c00763

  View details for PubMedID 40844373

• Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix (Adv. Mater. 4/2024) ADVANCED MATERIALS Park, C., Thompson, I. A. P., Newman, S. S., Hein, L. A., Lian, X., Fu, K. X., Pan, J., Eisenstein, M., Soh, H. 2024; 36 (4)

  View details for DOI 10.1002/adma.202470026

  View details for Web of Science ID 001149659100061

• 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