Accurate drift-invariant single-molecule force calibration using the Hadamard variance.

Single-molecule force spectroscopy (SMFS) techniques play a pivotal role in unraveling the mechanics and conformational transitions of biological macromolecules under external forces. Among these techniques, multiplexed magnetic tweezers (MT) are particularly well suited to probe very small forces, ≤1 pN, critical for studying noncovalent interactions and regulatory conformational changes at the single-molecule level. However, to apply and measure such small forces, a reliable and accurate force-calibration procedure is crucial. Here, we introduce a new approach to calibrate MT based on thermal motion using the Hadamard variance (HV). To test our method, we perform bead-tether Brownian dynamics simulations that mimic our experimental system and compare the performance of the HV method against two established techniques: power spectral density (PSD) and Allan variance (AV) analyses. Our analysis includes an assessment of each method's ability to mitigate common sources of additive noise, such as white and pink noise, as well as drift, which often complicate experimental data analysis. We find that the HV method exhibits overall similar or higher precision and accuracy, yielding lower force estimation errors across a wide range of signal-to-noise ratios (SNRs) and drift speeds compared with the PSD and AV methods. Notably, the HV method remains robust against drift, maintaining consistent uncertainty levels across the entire studied SNR and drift speed spectrum. We also explore the HV method using experimental MT data, where we find overall smaller force estimation errors compared with PSD and AV approaches. Overall, the HV method offers a robust method for achieving sub-pN resolution and precision in multiplexed MT measurements. Its potential extends to other SMFS techniques, presenting exciting opportunities for advancing our understanding of mechanosensitivity and force generation in biological systems. To make our methods widely accessible to the research community, we provide a well-documented Python implementation of the HV method as an extension to the Tweezepy package.