Single Molecule Detection with an Aptamer-Integrated Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 57

95 , 101 , 125 ], such as the function block of telomerase active in cancer cells [ 89 ],
and serve as targets for drugs in cancer treatment [ 85 ]. G-quadruplexes can also
be created in vitro through molecular design. Synthetic G-quadruplexes with
controllable folding/unfolding properties act as building blocks for assembling
nano-structures [ 19 ] and nanomachines [ 1 , 72 ]. Due to the high affinity for target
proteins, G-quadruplex aptamers are ideal candidates for biosensor construction
[ 10 ] and show usefulness as potent pharmaceuticals [ 107 ]. Understanding the
cation-selective folding/unfolding of the G-quadruplex is very useful because a
properly folded quadruplex is necessary for the molecular recognition involved in
many quadruplex functions and is beneficial for designing quadruplex applications.
The 15 bases thrombin-binding aptamer (TBA) [ 10 ] is a famous one that folds the
simplest quadruplex structure, with two parallel intra-molecular G-tetrads coordi-
nated by a metal ion (Fig. 3.3a )[ 60 ]. The top tetrad is assembled with guanine 1, 6,
10 and 15, and the bottom one with guanine 2, 5, 11 and 14 [ 77 , 88 , 116 ] (Fig. 3.1b ).
The TBA's capability in forming G-quadruplex is cation-selective. Upon folding,
the TBA quadruplex functions as an ultra-sensitive thrombin detector and an
efficient inhibitor of thrombin clotting activity [ 41 , 43 , 48 ].
We demonstrated that the protein pore can be used to learn various issues on
the folding of G-quadruplex [ 99 , 100 ]: how to discriminate a single TBA molecule
in the G-quartet form or in linear form? What is the interaction of G-quadruplex
with nanopore? What is the approach to the understanding of G-single quadruplex
folding/unfolding kinetics? And how do cations regulate the folding process of the
G-quadruplex.

3.2.1 Encapsulation of a G-Quadruplex
in the Nanopore Nanocavity

-hemolysin pore from the cis side in the
presence of K + or Na + , by generating the signature blocks that are characterized by
a long duration (~15 s in K + and 3 s in Na + ) and partial reduction of the pore
conductance (Fig. 3.4b traces) [ 99 ]. These signature blocks are distinguishable from
the ~10 2

a

TBA was first found to interact with the

s short blocks caused by translocation of a linear form DNA through the
pore (Fig. 3.4b right model), and should be attributed to a single TBA G-quadruplex
entering the pore from the cis opening and accommodating inside the nanocavity
domain (left model). The 2.1-nm wide G-quadruplex is slightly narrower than the
2.6-nm cis opening of

m

-barrel in middle of
the pore (Fig. 3.3 ), making it possible for the G-quadruplex to enter and be trapped
in the nanocavity (Fig. 3.4a ). Moreover, the widest part of nanocavity is 4.6 nm
(Fig. 3.3 ), therefore upon binding of TBA, the unoccupied space in the nanocavity
forms an ion pathway. This is evidenced by the large residual current in the long
blocks (Fig. 3.4b traces). The TBA's ability to bind thrombin was further
investigated. Because thrombin chelates free TBA G-quadruplex in the solution,
and the formed G-quadruplex/thrombin complex is too large to enter the pore,

a -hemolysin but larger than the 1.4 nm b