Biomedical Engineering Reference
In-Depth Information
a
c
b
100nm
5.8 Hz
1
345
6
2
2
H frequency (Hz)
200nm
Fig. 16.3
Production and characterization of the DNA six-helix bundle. (
a
) Gel analysis of the
six-helix bundles after folding and purification. The lanes are 1 kb ladder.
1
, p7308 “scaffold.”
2
and
3
, front and rear monomers folded before purification.
4
and
5
, front and rear monomers
after purification.
6
, heterodimer. 1
L drop solution exhibited birefringence between crossed
polarizers by DNA-nanotube heterodimers at 25 mg/mL. (
b
) Negative-stain transmission electron
micrograph of a purified sample of six-helix bundle heterodimer. (
c
) 1D NMR spectrum of
2
H
2
Oat
2
H frequency of the six-helix bundle sample 25 mg/mL in 2 mM MgCl
2
,20mMTris-HClpH7.5,
100 mM DPC, 90%/10% H
2
O/D
2
O. The 1D spectrum was recorded at
2
H frequency of 600 MHz
at 25
ı
C on a Bruker 600 MHz spectrometer
of the concentration and quality of the DNA-nanotube liquid crystal, and liquid
crystals on the lower end of this spectrum (
4 Hz) have been demonstrated to be
sufficient to induce a weak alignment of membrane proteins in solution. The lower
limit of nanotube concentration required for such alignment can vary depending
on the charge of the protein being analyzed; specifically, the negative charge of
DNA's phosphate backbone results in a global negative surface charge of the DNA
nanotubes, thus producing stronger than typical interactions with positively charged
proteins. The six-helix DNA-nanotube technique represents a simple, versatile, and
stable method to align macromolecules for the measurement of dipolar-coupling
interactions, thus representing the first detergent-compatible liquid crystal that
is generally suitable for high-resolution NMR study of membrane proteins. The
ability to tune the alignment of molecules over a large range of detergents, buffers,
and temperatures makes the DNA nanotubes especially attractive for studies of
membrane proteins as compared with other alignment media, which are subject to a
much more limited range of conditions.
16.5
Overview of the Procedure
16.5.1
Nanomole-Scale Production of DNA-Nanotube Dimers
As mentioned before, the assembly of
0.8
m DNA nanotubes involves thermal
annealing a 7,308 nt M-13 phage-derived “scaffold” strand with a pool of 168
42 nt linear, single-stranded “staple” strands. The 42 nt linear staple strands
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