Estimating RNA Polymerase Protein Binding Sites on lambda DNA Using Solid-State Nanopores
by Kaur, H.; Nandivada, S.; Acharjee, M. C.; McNabb, D. S.; Li, J. L.
In this work, using a silicon nitride nanopore based device, we measure the binding locations of RNA Polymerase (RNAP) on 48.5 kbp (16.5 mu m) long lambda DNA. To prevent the separation of bound RNAPs from a lambda DNA molecule in the high electric field inside a nanopore, we cross-linked RNAP proteins to lambda DNA by formaldehyde. We compare the current blockage event data measured with a mixture of lambda DNA and RNAP under cross-link conditions with our control samples: RNAP, lambda DNA, RNAP, and lambda DNA incubated in formaldehyde separately and in a mixture. By analyzing the time durations and amplitudes of current blockage signals of events and their subevents, as well as subevent starting times, we can estimate the binding efficiency and locations of RNAPs on a lambda DNA. Our data analysis shows that under the conditions of our experiment with the ratio of 6 to 1 for RNAP to lambda DNA molecules, the probability of an RNAP molecule to bind a lambda DNA is similar to 42%, and that RNAP binding has a main peak at 3.51 mu m +/- 0.53 mu m, most likely corresponding to the two strong promoter regions at 3.48 and 4.43 mu m of lambda DNA. However, individual RNAP binding sites were not distinguished with this nanopore setup. This work brings out new perspectives and complications to study transcription factor RNAP binding at various positions on very long DNA molecules.
- Journal
- ACS Sensors
- Volume
- 4
- Issue
- 1
- Year
- 2019
- Start Page
- 100-109
- URL
- https://dx.doi.org/10.1021/acssensors.8b00976
- ISBN/ISSN
- 2379-3694
- DOI
- 10.1021/acssensors.8b00976