1.

Arrange a four arm junction in correct pattern using four individual

DNA strands each of 10bases in length.

2. Identify the two important transmembrane proteins which function as

nanopores and examine the process of engineering nanopores with suitable
examples.
Nanopores are pores with around 1nm diameter. They can be naturally occurring or
engineered nanopores. Most important of biological nanopores are transmembrane proteins.
The transmembrane proteins consist of Helix bundles and beta barrels.
Helix bundles:
It contains various receptors and channels. The functional groups are more strongly bonded to
the helix structure. Therefore, on modification its structure changes. These are difficult to
engineer.
Beta barrels:
It consists of an open structure and permits alteration. Side chains does not interact with each
other and can be altered to manipulate the properties of pores.

EXAMPLES: The natural channels that have been engineered as nanopores includes Porins
and the α-Haemolysin. Porins are proteins that control the permeability of the bacterial outer
membrane. α-Haemolysin is a pore forming toxin secreted by staphylococcus aureus. It is a
heptameric structure. The transmembrane beta barrel is tapped by a cap domain that contains a
large internal cavity. Both the cap and barrel can be engineered in several ways.
ENGINEERED NANOPORES:
Protein pores are engineered by using mutagenesis and targeted chemical modification ie.,
engineering the interior of the pores.
MUTAGENESIS:
i. Natural: It includes the introduction of naturally occurring 20 amino acids as side
chains.
ii. Artificial: It includes the engineering of side chains that do not occur in nature. Eg:
Ketones, alkenes and azides.
TARGETED CHEMICAL MODIFICATION:
An amino acid chain is selectively modified with a reagent – Diversity of functionalities can
be incorporated into a protein.
NON-COVALENT MODIFICATION:
cyclodextrins and other host molecules (adapters) were allowed to bind to positions within the
lumen of the transmembrane barrel of the αHL pore that could be defined by mutagenesis. The
adapters remain capable of binding guests and therefore noncovalent modification can
introduce new binding sites within the pore.
α-Hemolysin pore modified with noncovalent
adapters. The illustration is based on the
superposition of structures of α-HL and
cyclodextrins. The positions of the
cyclodextrins were surmised from mutagenesis
experiments. In the structure shown, sites for
two different cyclodextrins were engineered.
An organic molecule can be trapped in the
cavity between the two sites.

Potential Applications of Nanopores:

i. Separations can be effected with modified nanopores using principles of molecular
mass/ charge/ hydrophobicity/ stereochemistry.
ii. Cell permeabilization
iii. Therapeutics – Targeted drug delivery
iv. Nanopore sensing
Latest Technology in Nanopores:
Supported Bilayers: To accommodate protein nanopores, it is clear that additional work is
needed on supported bilayers and related areas. The formation of defect-free bilayers appears
to require ultrasmooth surfaces. “Edge effects” – that is, leaks at the perimeter of the bilayers
– must also be tackled. In addition, improved electrical properties demand a substantial
reservoir of electrolyte between the bilayer and the support surface. Finally, a reasonable
fraction of the incorporated nanopores should be active.
Membrane Arrays: Arrays of lipid bilayers would be useful for screening the properties of
membrane proteins and for monitoring their interactions with other molecules.
Alternative Protein Pores: Advances in protein folding and structural biology will drive the
search for alternative protein pores. While research on αHL has been extremely fruitful, the
pore is a heptamer and therefore intricate to handle. A single-chain, monomeric pore would be
ideal, and we have begun to explore the monomeric porin OmpG. It is likely that yet more
sophisticated engineering techniques will be applied to protein nanopores than have been in
the past. For example, multistep syntheses on the lumen wall might be used to produce complex
host molecules within a pore. Because highly stable bilayers with desirable properties have
been difficult to obtain, the possibility of directly housing a protein nanopore inside a
mechanically stable pore of slightly greater diameter should be considered. The surfaces of
both the protein nanopore and the recipient pore (e.g., an Au nanotubule) could be tailored for
compatibility.

3.Indicate the significance of DNA Templated Electronics in

Microelectronics with suitable diagram.
DNA TEMPLATED ELECTRONICS:
The heart of the microelectronics is the integrated circuit and photolithography. The technology
developed to embed the vast complexity of microelectronic circuits in a virgin silicon wafer.
At present, there is no equivalent concept for the assembly of complex structures from
molecular building blocks. It is clear that the invention of such strategy is critical for turning
molecular electronics and more generally nanotechnology into reality.
Advantages:
i. It allows the precise localization of a large number of devices at molecularly accurate
addresses on the substrate.
ii. It provides construction by inter-device wiring.
iii. It wires the molecular network to the macroscopic world.

Difficulties:
i. Biological processes need to be adapted and modified to enable the in-vitro
construction.
ii. Hybridization of electronic materials with biological molecules need to be advanced
for precise localization of electronic devices.
iii. These devices should be compatible with the assembly and functionalization chemistry.
iv. DNA molecules need to be converted into conducting for interconnecting.
The first two difficulties can be addressed by Homologous recombination. There are
various candidates for molecular-scale switching devices. One of the promising possibilities is
the single-electron transistor, which is based on charging effects.
HEURISTIC APPROACH FOR DNA TEMPLATED ELECTRONICS:

(a) Gold pads are defined on an inert substrate. (b–d) correspond to the circle of (a) at different
stages of circuit construction. (b) Oligonucleotides of different sequences are attached to the
different pads. (c) DNA network is constructed and bound to the oligonucleotides on the gold
electrodes. (d) Metal clusters or molecular electronic devices are localized on the DNA
network. The DNA molecules are finally converted into metallic wires, rendering the construct
into a functional electronic circuit.
EXPERIMENTAL PROCEDURE FOR DNA TEMPLATED ELECTRONICS:

Gold pattern of specific size was defined on a passivated glass using microelectronics
techniques. The pattern comprised four bonding pads connected to two long parallel gold
electrodes separated apart. (a) The electrodes were each wetted with a droplet of disulfide-
derivatized oligonucleotide solution of a given sequence (Oligos A and B). (b) After rinsing,
the structure was covered with 100 mL of a solution of l-DNA having two sticky ends that are
complementary to Oligos A and B. A flow was applied to stretch the l-DNA molecule between
the two electrodes, allowing its hybridization. (c) The DNA bridge was loaded with silver ions
by Na+/Ag+ ion exchange. (d) The silver ion– DNA complex was reduced using a basic
hydroquinone solution to form metallic silver aggregates bound to the DNA skeleton. (e) The
DNA-templated wire was “developed” using an acidic solution of hydroquinone and silver
ions.
Under the experimental conditions, metal deposition therefore occurred only along the DNA
skeleton, leaving the passivated substrate practically clean of silver. The coating of DNA
molecules with metal destroys their molecular-recognition capabilities and hence, further
utilization of molecular biology after metalization.

4.Indicate the significance of Sequence Specific Molecular Lithography in

Microelectronics with suitable diagram.
SEQUENCE SPECIFIC MOLECULAR LITHOGRAPHY:
DNA-templated electronics requires elaborate manipulations on scales ranging from
nanometers to many micrometers. In conventional microelectronics, the circuit structure is
dictated by lithography, but the use of an elaborate molecular assembly requires an alternative
approach which is applicable at considerably smaller scales.
The process of sequence-specific molecular lithography that utilizes homologous
recombination processes by RecA protein, operating on the double-stranded DNA (ds-DNA)
substrate molecules. Homologous recombination is a protein-mediated reaction by which two
DNA molecules, possessing some sequence homology, crossover at equivalent sites. RecA is
the major protein responsible for this process in Escherichia coli.

PROCESS:

It is a four step process:

(i) POLYMERIZATION: RecA monomers polymerize on a ss-DNA probe molecule
to form a nucleoprotein filament.
(ii) HOMOLOGOUS RECOMBINATION: The nucleoprotein filament binds to an
aldehydederivatized ds-DNA substrate molecule at an homologous sequence.
(iii) MOLECULAR LITHOGRAPHY: Incubation in AgNO3 solution results in the
formation of silver aggregates along the substrate molecule at regions unprotected
by RecA.
(iv) GOLD METALIZATION: The silver aggregates catalyze specific gold deposition
on the unprotected regions. A highly conductive gold wire is formed, with a gap in
the protected segment.