LOOP-TAG: massively-parallel measurement of length-dependent protein-mediated DNA looping probabilities by tagmentation in vitro
- 1Mayo Clinic College of Medicine and Science
Abstract
We are interested in DNA stiffness in vitro and in vivo. Classical approaches for measuring this parameter in vitro involve tedious T4 DNA ligase-mediated cyclization kinetics experiments performed for one DNA fragment length at a time to establish effective local end-end concentrations (J-factors). Protein-mediated DNA looping is a more relevant biological reaction. An efficient approach to determine length-dependent protein-mediated DNA J-loop values in vitro has been lacking. We describe LOOP-TAG, a massively-parallel method. We leverage intramolecular DNA looping and tagmentation catalyzed by Tn5 transposase (Tnp) tethered to the terminus of a ~1,000-bp bead-bound duplex DNA. We detected loops terminating with Tnp. Counts of the resulting DNA looping-dependent bead-bound tagmentation products from deep sequencing determine DNA loop length probabilities at single-base pair resolution for all bead-bound fragments. When normalized to DNA fragment length probabilities from tagmentation using known concentrations of free Tnp, length-dependent J-loop values are directly determined. We demonstrate LOOP-TAG for Tnp tethered in two ways and in the presence of DNA-kinking architectural DNA binding protein Nhp6A. Intermediate length J-loop values are consistent with expectations of wormlike chain theory after accounting for strong sequence-dependence of Tnp. DNA loops are shifted to smaller sizes in the presence of Nhp6A, as predicted.
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BibTeX
@misc{kennel2026looptag,
title = {{LOOP-TAG: massively-parallel measurement of length-dependent protein-mediated DNA looping probabilities by tagmentation in vitro}},
author = {N. R. Kennel and N. A. Becker and J. P. Peters and L. J. Maher},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.753159},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.753159v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - LOOP-TAG: massively-parallel measurement of length-dependent protein-mediated DNA looping probabilities by tagmentation in vitro
AU - N. R. Kennel
AU - N. A. Becker
AU - J. P. Peters
AU - L. J. Maher
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.753159
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.753159v1
AB - We are interested in DNA stiffness in vitro and in vivo. Classical approaches for measuring this parameter in vitro involve tedious T4 DNA ligase-mediated cyclization kinetics experiments performed for one DNA fragment length at a time to establish effective local end-end concentrations (J-factors). Protein-mediated DNA looping is a more relevant biological reaction. An efficient approach to determine length-dependent protein-mediated DNA J-loop values in vitro has been lacking. We describe LOOP-TAG, a massively-parallel method. We leverage intramolecular DNA looping and tagmentation catalyzed by Tn5 transposase (Tnp) tethered to the terminus of a ~1,000-bp bead-bound duplex DNA. We detected loops terminating with Tnp. Counts of the resulting DNA looping-dependent bead-bound tagmentation products from deep sequencing determine DNA loop length probabilities at single-base pair resolution for all bead-bound fragments. When normalized to DNA fragment length probabilities from tagmentation using known concentrations of free Tnp, length-dependent J-loop values are directly determined. We demonstrate LOOP-TAG for Tnp tethered in two ways and in the presence of DNA-kinking architectural DNA binding protein Nhp6A. Intermediate length J-loop values are consistent with expectations of wormlike chain theory after accounting for strong sequence-dependence of Tnp. DNA loops are shifted to smaller sizes in the presence of Nhp6A, as predicted.
ER -