nn9b04752_si_002.pdf (4.92 MB)
Enzymatic Degradation of DNA Probed by In Situ X‑ray Scattering
journal contribution
posted on 2019-09-18, 19:33 authored by Kurinji Krishnamoorthy, Sumit Kewalramani, Ali Ehlen, Liane M. Moreau, Chad A. Mirkin, Monica Olvera de la Cruz, Michael J. BedzykLabel-free in situ X-ray scattering from protein
spherical nucleic acids (Pro-SNAs, consisting of protein cores densely
functionalized with covalently bound DNA) was used to elucidate the
enzymatic reaction pathway for the DNase I-induced degradation of
DNA. Time-course small-angle X-ray scattering (SAXS) and gel electrophoresis
reveal a two-state system with time-dependent populations of intact
and fully degraded DNA in the Pro-SNAs. SAXS shows that in the fully
degraded state, the DNA strands forming the outer shell of the Pro-SNA
were completely digested. SAXS analysis of reactions with different
Pro-SNA concentrations reveals a reaction pathway characterized by
a slow, rate determining DNase I-Pro-SNA association, followed by
rapid DNA hydrolysis. Molecular dynamics (MD) simulations provide
the distributions of monovalent and divalent ions around the Pro-SNA,
relevant to the activity of DNase I. Taken together, in situ SAXS in conjunction with MD simulations yield key mechanistic and
structural insights into the interaction of DNA with DNase I. The
approach presented here should prove invaluable in probing other enzyme-catalyzed
reactions on the nanoscale.