Probing the Effect of Ubiquitinated Histone on Mononucleosomes by Translocation Dynamics Study through Solid-State Nanopores

Abstract

Post-translational modifications (PTMs), such as ubiquitination, are critically important in regulating genetic expressions by adjusting the nucleosome stability. A fast and label-free technology inspecting dynamic nucleosome structures can facilitate the interrogation of PTMs effects. Here we leverage the advantages of mechanically stable solid-state nanopores and detect the effect of a ubiquitinated histone on mononucleosomes at the single-molecule level. By comparing the translocation dynamics of natural and crosslinked mononucleosomes, we verified that the nucleosomal DNA unravelled from histones in natural mononucleosomes. Furthermore, we found that a turning point of voltage corresponds to the onset of nucleosome rupture. More importantly, we reveal that ubH2A stabilizes the nucleosome by shifting the turning point to a larger value and investigated the effect of ubiquitination on different histones (ubH2A and ubH2B). These findings open promising possibilities for developing a miniaturized and portable device for the fast screening of PTMs on nucleosomes.

Nanopore Sensing of DNA–Histone Complexes on Nucleosome Arrays

“…40 Although there is growing interest on applying this technique to study the stability of chromatin systems, studies are currently limited to simpler systems such as distinguishing unmodified from methylated 41,42 and ubiquitinated nucleosomes. 43 In this paper, we demonstrate the first report on detection of nucleosomal arrays using the nanopore sensing platform. We observe well-resolved multilevel signatures in nanopore conductance where the individual levels correspond to distinct local structures present on the arrays that are distinctly detected during translocation through the pore.…”

“…A quantitative analysis of mononucleosomes and their subcomponents using nanopores was reported earlier. , Previous studies have also demonstrated the measurement of interactions involved in stabilizing the streptavidin–biotin complex and the Eco RI–DNA complex . Although there is growing interest on applying this technique to study the stability of chromatin systems, studies are currently limited to simpler systems such as distinguishing unmodified from methylated , and ubiquitinated nucleosomes …”

Experimental study on single biomolecule sensing using MoS2–graphene heterostructure nanopores

“…In recent decades, solid-state nanopores have emerged as a new tool with great potential in the detection of single biomolecules such as DNA, 1–9 RNA 10–12 and proteins. 13–15 In order to achieve single biomolecule sequencing and reveal more detailed information from the corresponding ionic blockage signals, the low spatial resolution and the ultra-fast translocation speed are two critical problems that remain to be solved.…”

Oligonucleotide Discrimination Enabled by Tannic Acid-Coordinated Film-Coated Solid-State Nanopores

“…Biological nanopores (helicase-modified MspA or alpha-hemolysin) have made great progress in next-generation sequencing technology that is capable of reading ultralong strands and have been used in the production of a commercial DNA sequencing portable device, MinION . Solid-state nanopores, which are fabricated in a suspending thin film (commonly an insulated inorganic silicon-based material) have a tunable geometry and superior robustness compared to their biological counterpart and have expanded the diversity of analytes to include nucleic acids, − proteins, − and biomolecular complexes. − However, discriminating each nucleotide for DNA sequencing using solid-state nanopores has been a big challenge due to the inferior spatial and temporal resolution compared to biological nanopores …”