Biology Reference
In-Depth Information
Recent developments have seen orders of magnitude increases in complexity (i.e. more
nucleic acid components or components with multiple functionalities) for more
predictable and complicated artificial biochemical circuits. In one approach, Erik Winfree
s
'
team designed entropy-driven catalytic gates to provide an amplifying circuit element,
increasing the speed and performance of cell-free circuits.
37
Later, the same group made
additional advances in creating scalable DNA architecture by generating gates that could
amplify downstream signal with minimal noise and errors. Specifically, they designed a
DNA computer capable of calculating the square root of numbers up to 15 with up to
130 DNA strands, albeit in a much slower timeframe than digital computers. This
technology could eventually enable the production of large-scale circuits involving
thousands of gates.
38,39
However, with the scaling-up of circuits comes the added burden
of designing the DNA. To address this challenge, a programming language to facilitate
the design of DNA circuits based on DNA strand displacement has now been created.
40
This tool will help generate sequences for DNA circuit construction after simulation of
nucleic acid strand displacement behavior. In other advances, researchers in the
Tuberfield group demonstrated the creation of a reversible DNA circuit that continuously
changed output depending on changing input (unlike in an irreversible circuit).
41
Further,
modular DNA circuits have been created to detect different analytes and give off various
output signals (electrochemical, colorimetric, and fluorescent outputs).
42
While DNA computation enables us to model and build sophisticated biochemical
circuits, RNA transcriptional logic is used more frequently for cellular regulation.
Additionally, RNA has unique properties (relative to DNA) that add a new dimension
when engineering circuits. RNA can act as a sensor, be catalytic, and exhibit different
conformations to elicit a response.
43
Further, the ability of RNA to base pair enables the
creation of synthetic circuits when placed in
cis
and
trans
or in layers.
44
The development
of hybrid nucleic acid systems, comprising both DNA and RNA, is enabling us to
understand new principles of biological circuitry from a constructive biology approach.
282
Erik Winfree
s group at Caltech has contributed pioneering efforts to the development of
hybrid systems. Their first foray into programmable biochemical circuitry was made using
simplified circuit construction that utilized nucleic acids as regulatory molecules. As a
stripped-down version of a genetic circuit, they created an artificial transcriptional network
with modular and scalable molecular switches. They also attempted to control nucleic acid
production and degradation by including T7 polymerase and RNAse H.
12
'
Highlighting the utility of cell-free systems for accelerating our understanding of the
principles of biological modules that could be used to obtain the same functional output,
several studies in 2011 have reported synthetic in vitro oscillators with different molecular
implementations. Montagne et al.
45
demonstrated a remarkably elegant (and simple)
oscillator that uses just three nucleic acid strands and can be quantitatively modeled. At the
exact same time, Kim et al. reported in vitro oscillators using transcriptional circuits from
seven nucleic acid strands that can be modularly wired into artificial networks by
changing the regulatory and coding domains of DNA.
46
In this system, RNA (not DNA)
was the only molecule synthesized and degraded, thereby preventing accumulation of
mutations in the DNA. An interesting observation is that even simple systems such as
this seven-component oscillator challenge our engineering and analysis abilities. Franco
et al. later expanded on this oscillator work of Kim et al. by combining the synthetic
in vitro oscillator with DNA nanomachines (discussed below), as well as the synthesis
of a functional RNA structure.
47
The key aspect of this research was the ability to use
an insulator circuit to isolate the DNA nanomachine operation from the oscillator
dynamics, allowing for much larger loads in the circuit. In the future the oscillator
can be coupled to more complex downstream processes, and could even be
incorporated into artificial cells (see below). However, waste product accumulation,
