Article pubs.acs.org/ac
Reversible and Irreversible Mechanical Damaging of Large DoubleStranded DNA upon Electrospraying Yuri M. Shlyapnikov,*,† Elena A. Shlyapnikova,† and Victor N. Morozov†,‡ †
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia 20110, United States
‡
S Supporting Information *
ABSTRACT: Electrohydrodynamic spraying (or electrospaying, ES) of DNA solutions is an attractive technique for applications in mass spectrometry, in microarray fabrication, and in generation of DNA nanoaerosols. Here we report how ES affects DNA structure and evaluate possible ways to reduce DNA damage upon ES. It is shown that under any ES conditions, linear λ-phage DNA is subjected to intensive rupture producing a mixture of fragments. In addition to such fragmentation, notable reversible changes in the DNA structure were revealed by a slight increase in DNA electrophoretic mobility. The degree of fragmentation was shown to decrease with decreased DNA length and with increased flow rate through the ES capillary. Fragments shorter than 5 kbp did not show any notable damage upon ES. Both experimental data and theoretical estimations of the forces acting on DNA during ES indicate that DNA is damaged by mechanical forces, and the damage takes place in the vicinity of the Taylor cone tip, presumably due to the high shear stress or/and viscous drag forces operating there. Condensation of λ-DNA with hexamminecobalt(III) ions completely protected it from any damage upon ES. lectrohydrodynamic spraying (ES) finds numerous applications in the ionization of different substances.1 Although ES of nucleic acids has not yet become a widespread method, it is employed in mass spectrometry,2 gene transfection,3 and fabrication of microarrays.4 ES-generated DNA nanoaerosols may become efficient agents for gene therapy with precise and selective drug delivery.5−8 The ES process involves high-energy events such as (i) intensive flows and vortexes in the Taylor cone,9,10 (ii) high-speed flow in the liquid jet,11,12 and (iii) high-energy molecular species formed in the corona discharge which often accompanies ES.13 Therefore, when biological molecules are subjected to electrospraying, the potential for them to be chemically modified, fragmented, or somehow otherwise damaged upon ES must be considered. For proteins, measurements of enzymatic activity have enabled us to identify the ES conditions that preserve the proteins’ functional properties.13−15 However, because large nucleic acids are much more sensitive than globular proteins to the shear flow and other mechanical forces operating in ES, these conditions cannot be automatically transferred to the ES of DNA solutions and require special study. Thundat et al.16 were the first to apply ES to prepare DNA samples for scanning tunneling microscopy. While depositing plasmid DNA on the gold surface, the authors noted fragmentation of the plasmids. Later, Cheng et al.2 confirmed this result by showing that supercoiled plasmids formed nicked forms even when the plasmids were electrosprayed into a buffer solution. Limited data on the effects of ES on large linear DNAs were found in the literature. Thus, a broad dispersion of ion masses was observed when λ-phage DNA was subjected to ES. 17 The authors explained this fact by the DNA
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© 2016 American Chemical Society
fragmentation, but neither estimate of the degree of fragmentation nor its dependence on the ESI conditions, were made. Also, the possible changes in DNA secondary structure could not be observed by the methods used in ref 17. At the same time, short linear DNA sequences (
