Solid-State Nanopore Sensors for Nucleic Acid Analysis - Nanopores: Sensing and Fundamental Biological Interactions - page 7

chemistry, charge and size of these chemical tags, suggesting the possibility of base
discrimination based on peptide labeling with application to DNA sequencing.
Another novel nanopore-based sequencing approach was proposed by Cockcroft
et al. that exploited the selective, base-by-base activity of DNA Polymerase [ 19 ].
By anchoring a DNA/DNA-Polymerase complex in the nanopore, the authors were
able to electrically monitor single nucleotide primer extension events. Primer
extensions were controlled by providing each nucleotide set sequentially and tempo-
rally extracting sequence information [ 19 ]. The Bayley group recently demonstrated
the ability to continuously resolve indigenous single nucleotides (dAMP, dCMP,
dGMP, dTMP) through nanopore based resistive current measurements [ 18 ]. Remark-
ably, individual bases could be discriminated based on current blockade levels
without any prior base labeling or chemical modification as shown in Fig. 1.2b, c .
Base selectivity was achieved by modifying the mutant

a - hemolysin pore with a

cyclodextrin adapter (am 6 amPDP 1 b barrel of the

transmembrane domain, thereby constricting the nanopore channel while enhancing
the chemical specificity of the sensor. Raw bases were read with over 99% confidence
under optimal operating conditions. By integrating this base identification platform
with a highly processive exonuclease (chemical attachment or genetic fusion of
exonuclease), a single molecule sequencing by digestion approach may be feasible.

CD) covalently bound within the b

1.2.2 Bacteriophage phi29 Connector

Another biological nanopore that is receiving much interest of late is the connector
protein from the bacteriophage phi29 DNA packaging motor. In bacteriophage
phi29 , linear dsDNA is packaged into a viral capsid, an entropically unfavorable
process that requires the hydrolysis of ATP. During packaging, the linear DNA
passes through a narrow channel of inner diameter ~3.6 nm, termed the connector.
The connector is comprised of 12 GP10 protein subunits that readily self assemble in
solution to form a stable, repeatable do-decameric structure [ 95 ]. As the crystal
structure of this biological nanopore channel has been resolved [ 100 ], explicit site
engineering is possible. Wendell et al. modified the phi29 connector protein to
include hydrophilic sites on the crown and base, thereby allowing its integration
into liposomes [ 95 ]. The insertion of the phi29 connector into preformed lipid
bilayers was achieved through vesicle fusion resulting in steady, repeatable pore
conductance. Pore conductance was comparatively approximately five times higher
than that observed in - hemolysin under similar conditions and did not show

any voltage gating effects. Phi29 has one distinct advantage over

a

- hemolysin .The
larger barrel allows the translocation of dsDNA and a variety of proteins that are
simply too large to pass through

a

- hemolysin .Thusthe phi29 connector system allows
the experimentalist to examine a broader spectrum of biomolecule interactions at the
single molecule level. Wendell et al. demonstrated the successful detection of 5.5 kbp
dsDNA and 35 bp dsDNA using this novel biological nanopore platform.

a