Biology subjects

Ulugol, A.

Publications and source records attributed to Ulugol, A..

3 recordsLinked to original sources

bioRxiv2026

CALISTO: A Standardized and Open-Source Software for Magnetic Tweezer Force Calibration

Magnetic tweezers (MT) enable multiplexed single-molecule force spectroscopy while applying controlled forces and torques to biomolecules. They resolve forces below 1 pN and maintain constant forces over extended periods, but accurate calibration remains challenging due to drift, tracking artifacts, camera noise, and bead variability. Here, we present CALISTO (CALIbration Suite for magnetic Tweezer Operation), an open-source software for MT force calibration. CALISTO analyzes position traces of DNA-tethered magnetic beads and surface-bound reference beads for drift correction. It implements three thermal-motion-based calibration methods: power spectral density, Allan variance, and Hadamard variance, with the latter providing increased robustness against drift and correlated measurement noise. The graphical user interface combines automated analysis with user-guided quality control, including reference bead selection, z-offset determination, magnet-position classification, bead-wise force estimation, and tether extension analysis. Together, these steps generate a master force calibration curve. We systematically evaluated their effects on calibration performance. The largest improvement resulted from automated filtering of problematic force plateaus using time-series characterization to identify traces affected by drift, tracking artifacts, or irregular fluctuations. Filtering reduced scatter and improved force estimation while preserving the underlying force-magnet position relationship. CALISTO thus provides a standardized, reproducible framework for reliable magnetic-tweezers force calibration.

biophysics↗

bioRxiv2026

Molecular Structure, DNA Binding, and Photophysical Properties of SYTOX Orange and SYTOX Green

Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1){degrees} and 20.5(1){degrees} for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 {micro}M), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

biophysics↗

bioRxiv2024

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 (MTs) are particularly well suited to probe very small forces, [≤]1 pN, critical for studying non-covalent 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 MTs based on thermal motion using the Hadamard variance (HV). To test our method, we develop a bead-tether Brownian dynamics simulation that mimics 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 methods ability to mitigate common sources of additive noise, such as white and pink noise, as well as drift, which often complicate experimental data analysis. Our findings demonstrate that the HV method exhibits overall similar or even higher precision and accuracy, yielding lower force estimation errors across a wide range of signal-to-noise ratios (SNR) and drift speeds compared to 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 to 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 mechano-sensitivity and force generation in biological systems. Therefore, we provide a well-documented Python implementation of the HV method as an extension to the Tweezepy package. Statement of Signi[fi]canceSingle-molecule force spectroscopy techniques are vital for studying the mechanics and conformations of bio-macromolecules under external forces. Multiplexed magnetic tweezers (MTs) excel in applying forces [≤] 1 pN, which are critical for examining non-covalent interactions and regulatory changes at the single-molecule level. Precise and reliable force calibration is essential for these measurements. In this study, we present a new force calibration method for multiplexed MTs using Hadamard variance (HV) based on thermal motion. The HV method shows similar or even higher precision and accuracy to established techniques like power spectral density and Allan variance. Most significantly, it is drift-invariant, maintaining consistent performance across varying experimental conditions. This robustness against drift ensures reliable force application and measurements at sub-pN resolution.

biophysics↗