Estimating RNA Polymerase Protein Binding Sites on λ DNA Using Solid-State Nanopores.

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 μm) long λ DNA. To prevent the separation of bound RNAPs from a λ DNA molecule in the high electric field inside a nanopore, we cross-linked RNAP proteins to λ DNA by formaldehyde. We compare the current blockage event data measured with a mixture of λ DNA and RNAP under cross-link conditions with our control samples: RNAP, λ DNA, RNAP, and λ 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 λ DNA. Our data analysis shows that under the conditions of our experiment with the ratio of 6 to 1 for RNAP to λ DNA molecules, the probability of an RNAP molecule to bind a λ DNA is ∼42%, and that RNAP binding has a main peak at 3.51 μm ± 0.53 μm, most likely corresponding to the two strong promoter regions at 3.48 and 4.43 μm of λ 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.