Sizing up DNA nanostructure assembly with native mass spectrometry and ion mobility

Abstract

Recent interest in biological and synthetic DNA nanostructures has highlighted the need for methods to comprehensively characterize intermediates and end products of multimeric DNA assembly. Here we use native mass spectrometry in combination with ion mobility to determine the mass, charge state and collision cross section of noncovalent DNA assemblies, and thereby elucidate their structural composition, oligomeric state, overall size and shape. We showcase the approach with a prototypical six-subunit DNA nanostructure to reveal how its assembly is governed by the ionic strength of the buffer, as well as how the mass and mobility of heterogeneous species can be well resolved by careful tuning of instrumental parameters. We find that the assembly of the hexameric, barrel-shaped complex is guided by positive cooperativity, while previously undetected higher-order 12- and 18-mer assemblies are assigned to defined larger-diameter geometric structures. Guided by our insight, ion mobility-mass spectrometry is poised to make significant contributions to understanding the formation and structural diversity of natural and synthetic oligonucleotide assemblies relevant in science and technology.

Functional Nanopores Enabled with DNA

“…[151] Six-helical bundle DNA pores were also shown to have clinical application, puncturing live cell membranes and transporting anti-tumour drugs. [152] The assembly pathway of the six-helix bundle pore was recently monitored by ion mobility mass spectrometry, [153] while their structural conformations have been investigated using cryo-electron microscopy and molecular dynamics simulations. [154][155][156] The dimensions of DNA pore structures were further reduced by Göpfrich et al, with the design of a DNA channel comprising solely four duplexes that enclosed a subnanometer lumen (Figure 5f).…”

“…Six‐helical bundle DNA pores were also shown to have clinical application, puncturing live cell membranes and transporting anti‐tumour drugs [152] . The assembly pathway of the six‐helix bundle pore was recently monitored by ion mobility mass spectrometry, [153] while their structural conformations have been investigated using cryo‐electron microscopy and molecular dynamics simulations [154–156] …”

Dominant Analytical Techniques in DNA Nanotechnology for Various Applications

“…A few techniques have been explored in a limited range for analyzing DNA nanostructures, namely, mass spectrometry-based tools and capillary electrophoresis . The former, in the MALDI-TOF format, currently falls short in the molecular weights of biomolecules that can be characterized (<300 kDa), and advancements in the technology are needed.…”

Proton Transfers to DNA in Native Electrospray Ionization Mass Spectrometry: A Quantum Mechanics/Molecular Mechanics Study

“…Native electrospray ionization-ion mobility mass spectrometry (N-ESI/IM-MS) is a very powerful tool for low-resolution structural studies of DNA oligomers and their complexes with ligands. − It requires quantities much smaller than those required for high-resolution techniques, such as nuclear magnetic resonance, X-ray crystallography, or cryo-electron microscopy. , The biomolecular analytes are sprayed from solution under quasi-physiological conditions (aqueous ammonium solutions at neutral pH, without organic cosolvents) , into the gas phase through a capillary applied to an electric field. , The initially formed droplets are shrunk by fast solvent evaporation and jet fission events to yield ESI nanodroplets. The analyte ions are released into the gas phase from the latter.…”