Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels
Year: 2024
Type: article
Abstract: Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.
Source: Nature Nanotechnology
Institution: University of Chicago
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $12,290
Funders: National Science Foundation, U.S. Department of Health and Human Services, University of Chicago, Neubauer Family Foundation, National Institutes of Health, Directorate for Mathematical and Physical Sciences, Materials Research Science and Engineering Center, Harvard University, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences, Division of Materials Research, Office of Multidisciplinary Activities
Awards: G1023082496, G1828835790, G1965089940, G2289426772, G2732840215, Medical Scientist National Research Service Award, G302334235, Materials Research Science and Engineering Center, QLCI-CI: NSF Quantum Leap Challenge Institute for Quantum Sensing in Biophysics and Bioengineering, Function and Regulation of Stress-Induced Adaptive Condensates, G4262658549, G4657484576, G4843848388, G5471082878, G6256675488, G7542389844, G8048112891, G8411689924, Investigating Biomolecular Condensates and Heat Shock Proteins in Cellular Responses to Sublethal Heat Shock and Fever, G9000080134, G9000084438, G9000202268, G9000314923, G935552465
OpenAlex ID: W4396929329