10.1021/acsami.8b18157.s009
Samuel
J. W. Krerowicz
Samuel
J. W.
Krerowicz
Juan P. Hernandez-Ortiz
Juan P.
Hernandez-Ortiz
David C. Schwartz
David C.
Schwartz
Microscale
Objects via Restructuring of Large, Double-Stranded
DNA Molecules
American Chemical Society
2018
nonrepetitive microscale structures
RNA
Double-Stranded DNA Molecules
DNA nanotechnology increases
fluorescence intensity measurements
model system
single-stranded DNA flaps
40 nm diameter neutravidin bead
single-molecule fluorescence microscopy
2018-11-07 00:00:00
Media
https://acs.figshare.com/articles/media/Microscale_Objects_via_Restructuring_of_Large_Double-Stranded_DNA_Molecules/7361060
As
the interest in DNA nanotechnology increases, so does the need
for larger and more complex DNA structures. In this work, we describe
two methods of using large, double-stranded (ds) DNA to self-assemble
sequence-specific, nonrepetitive microscale structures. A model system
restructures T7 DNA (40 kb) through sequence-specific biotinylation
followed by intramolecular binding to a 40 nm diameter neutravidin
bead to create T7 “rosettes”. This model system informed
the creation of “nodal DNA” where “nodes”
with single-stranded DNA flaps are attached to a large dsDNA insert
so that a complementary oligonucleotide “strap” bridges
the two nodes for restructuring to form a DNA “bolo”.
To do this in high yield, several methodologies were developed, including
a protection/deprotection scheme using RNA/RNase H and dialysis chambers,
which remove excess straps while retaining large DNA molecules. To
assess these restructuring processes, the DNA was adsorbed onto supported
lipid bilayers, allowing for a visual assay of their structure using
single-molecule fluorescence microscopy. Good agreement between the
expected and observed fluorescence intensity measurements of the individual
features of restructured DNA for both the DNA rosettes and bolos gives
us a high degree of confidence that both processes give sequence-specific
restructuring of large, dsDNA molecules to create microscale objects.