DNA-Nanotube-Enabled NMR Structure Determination of Membrane Proteins - DNA Nanotechnology: From Structure to Function - page 328

be a dilute liquid crystal that exhibits weak and highly transient interactions ( ns

lifetime) with the protein of interest. To this end, a number of liquid crystal
alignment media have been developed to accurately measure RDCs, including
filamentous phage [ 29 ]; DMPC/DHPC bicelles [ 26 ]; C12E5 polyethylene glycol
[ 30 ]; ternary mixtures of cetylpyridinium Cl/Br, hexanol, and sodium Cl/Br [ 30 ,
31 ]; cellulose crystallites [ 32 ]; and a highly hydrated anisotropically compressed
polyacrylamide gel [ 33 ]. Unfortunately, with the exception of a few cases involving
the polyacrylamide gel [ 34 - 37 ] and the fd bacteriophage [ 38 ], most of these media
have been demonstrated to be incompatible with the detergents and lipids needed to
solubilized membrane proteins. More recently, two new detergent-compatible liquid
crystals have been reported, one based on collagen [ 39 ] and the other nucleic-acid
G-tetrad structures [ 40 ]. However, measuring RDCs for membrane proteins remains
as a challenging topic. In 2007, Douglas et al. introduced DNA-nanotube liquid
crystals as the first detergent-compatible liquid crystal generally suitable for high-
resolution NMR study of membrane proteins (Fig. 16.1 a,b) [ 11 ].

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16.4 Six-Helix Bundle, DNA-Nanotube Liquid Crystals

Scaffolded DNA origami offers a means to build well-defined particles with
arbitrary shapes of dimensions approaching the micrometer scale. Inspired by the
architecture of the established phage-based alignment method [ 26 , 41 ], Douglas
et al. successfully designed DNA-nanotube liquid crystals [ 11 ] as detergent-resistant
shape mimetics of the Pf1 filamentous phage. The core design of the DNA
nanotube involves a parallel array of six DNA double helices held together by
Holliday-junction crossovers occurring every 42 base pairs (bps) between adjacent
helices; in addition, any set of three adjacent helices forms a dihedral angle of
120 ı (Fig. 16.2 a,b). The basic unit of the DNA nanotubes was constructed by
using a 7,308-nucleotide (nt), M13-phage-derived, single-stranded circular DNA
as “scaffold” and 168 linear single strands of 42 nt long DNA as “staples.” Each
of the staple strands is designed to complimentarily pair with three separate and
unique regions of the scaffold, thus inducing the scaffold strand to self-assemble
when the strands are mixed together and annealed at the appropriate temperature,
in this case forming a 0.4

m DNA nanotube (Fig. 16.2 a,b).To achieve a target

uniform length of 0.8 m DNA nanotube was designed to assemble

in two separate pools of unique monomers with three distinct sections: core, head,
and tail (Fig. 16.2 a,b). This design paradigm then allows the nanotube monomers
to form heterodimer linkages in a head-to-tail fashion when the two separate pools
are mixed. To ensure that heterodimerization occurs in a specific and controlled
manner, three special capping strands were designed to pair with the unpaired head
region of the front monomer, and four were designed to cap the unpaired region
tail of the rear monomer. Heterodimerization is facilitated by having three hanging
staple strands with unpaired bases from the tail end of the front monomer pairing
with three complementary strands hanging from the head of the rear monomer

m, each 0.4