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Abstract Allostery is a hallmark of cellular function and important in every biological system. Still, we are only starting to mimic it in the laboratory. Here, we introduce an approach to study aspects of allostery in artificial systems. We use a DNA origami domino array structure which–upon binding of trigger DNA strands–undergoes a stepwise allosteric conformational change. Using two FRET probes placed at specific positions in the DNA origami, we zoom in into single steps of this reaction cascade. Most of the steps are strongly coupled temporally and occur simultaneously. Introduction of activation energy barriers between different intermediate states alters this coupling and induces a time delay. We then apply these approaches to release a cargo DNA strand at a predefined step in the reaction cascade to demonstrate the applicability of this concept in tunable cascades of mechanochemical coupling with both spatial and temporal control. 

ABSTRACT Single‐walled carbon nanotubes (SWCNTs) are promising optical biosensing platforms due to their intrinsic near‐infrared fluorescence and environmental sensitivity. While DNA‐SWCNT hybrids have been widely studied, the structural arrangement of double‐stranded DNA (dsDNA) on SWCNTs and its impact on exciton–fluorophore interactions remain insufficiently characterized. Here, we introduce carbon nanotube energy transfer with vertical nucleic acids (CNETvNA), in which fluorophores are positioned at defined distances from SWCNTs using guanine‐defect anchored capture sequences hybridized with complementary oligonucleotides. By systematically varying the duplex length from 12 to 24 base pairs, we probe the distance dependence of dye–SWCNT interactions at the single‐molecule level. Fluorescence lifetime imaging microscopy reveals efficient quenching of ATTO542 and ATTO643 dyes, with lifetime distributions reflecting heterogeneous duplex conformations. Molecular dynamics simulations demonstrate that dsDNA duplexes adopt a predominantly perpendicular orientation relative to the SWCNT axis, with increasing tilt and conformational variability at longer lengths. Combining experimental and computational results, we establish a distance dependence of d⁻⁵with 7.4 ± 0.7 nm for 50% quenching efficiency, consistent with theoretical predictions for point dipole donors and 1D acceptors. These findings provide structural insights into DNA‐SWCNT conjugates and establish CNETvNA as a rational design principle for SWCNT‐based biosensors. 

Abstract Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.