9 research outputs found
DNA Computation in Mammalian Cells: MicroRNA Logic Operations
DNA
computation can utilize logic gates as modules to create molecular
computers with biological inputs. Modular circuits that recognize
nucleic acid inputs through strand hybridization activate computation
cascades to produce controlled outputs. This allows for the construction
of synthetic circuits that can be interfaced with cellular environments.
We have engineered oligonucleotide AND gates to respond to specific
microRNA (miRNA) inputs in live mammalian cells. Both single and dual-sensing
miRNA-based computation devices were synthesized for the cell-specific
identification of endogenous miR-21 and miR-122. A logic gate response
was observed with miRNA expression regulators, exhibiting molecular
recognition of miRNA profile changes. Nucleic acid logic gates that
are functional in a cellular environment and recognize endogenous
inputs significantly expand the potential of DNA computation to monitor,
image, and respond to cell-specific markers
DNA Computation: A Photochemically Controlled AND Gate
DNA computation is an emerging field that enables the
assembly
of complex circuits based on defined DNA logic gates. DNA-based logic
gates have previously been operated through purely chemical means,
controlling logic operations through DNA strands or other biomolecules.
Although gates can operate through this manner, it limits temporal
and spatial control of DNA-based logic operations. A photochemically
controlled AND gate was developed through the incorporation of caged
thymidine nucleotides into a DNA-based logic gate. By using light
as the logic inputs, both spatial control and temporal control were
achieved. In addition, design rules for light-regulated DNA logic
gates were derived. A step-response, which can be found in a controller,
was demonstrated. Photochemical inputs close the gap between DNA computation
and silicon-based electrical circuitry, since light waves can be directly
converted into electrical output signals and vice versa. This connection
is important for the further development of an interface between DNA logic gates and electronic devices, enabling the connection of biological
systems with electrical circuits
Optically Controlled Signal Amplification for DNA Computation
The
hybridization chain reaction (HCR) and fuel–catalyst
cycles have been applied to address the problem of signal amplification
in DNA-based computation circuits. While they function efficiently,
these signal amplifiers cannot be switched ON or OFF quickly and noninvasively.
To overcome these limitations, a light-activated initiator strand
for the HCR, which enabled fast optical OFF → ON switching,
was developed. Similarly, when a light-activated version of the catalyst
strand or the inhibitor strand of a fuel–catalyst cycle was
applied, the cycle could be optically switched from OFF → ON
or ON → OFF, respectively. To move the capabilities of these
devices beyond solution-based operations, the components were embedded
in agarose gels. Irradiation with customizable light patterns and
at different time points demonstrated both spatial and temporal control.
The addition of a translator gate enabled a spatially activated signal
to travel along a predefined path, akin to a chemical wire. Overall,
the addition of small light-cleavable photocaging groups to DNA signal
amplification circuits enabled conditional control as well as fast
photocontrol of signal amplification
Optically Controlled Signal Amplification for DNA Computation
The
hybridization chain reaction (HCR) and fuel–catalyst
cycles have been applied to address the problem of signal amplification
in DNA-based computation circuits. While they function efficiently,
these signal amplifiers cannot be switched ON or OFF quickly and noninvasively.
To overcome these limitations, a light-activated initiator strand
for the HCR, which enabled fast optical OFF → ON switching,
was developed. Similarly, when a light-activated version of the catalyst
strand or the inhibitor strand of a fuel–catalyst cycle was
applied, the cycle could be optically switched from OFF → ON
or ON → OFF, respectively. To move the capabilities of these
devices beyond solution-based operations, the components were embedded
in agarose gels. Irradiation with customizable light patterns and
at different time points demonstrated both spatial and temporal control.
The addition of a translator gate enabled a spatially activated signal
to travel along a predefined path, akin to a chemical wire. Overall,
the addition of small light-cleavable photocaging groups to DNA signal
amplification circuits enabled conditional control as well as fast
photocontrol of signal amplification
Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells
Photocaging provides a method to
spatially and temporally control
biological function and gene expression with high resolution. Proteins
can be photochemically controlled through the site-specific installation
of caging groups on amino acid side chains that are essential for
protein function. The photocaging of a synthetic gene network using
unnatural amino acid mutagenesis in mammalian cells was demonstrated
with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase
was expressed in cells with an expanded genetic code and used in the
photochemical activation of genes under control of an orthogonal T7
promoter, demonstrating tight spatial and temporal control. The synthetic
gene expression system was validated with two reporter genes (luciferase
and EGFP) and applied to the light-triggered transcription of short
hairpin RNA constructs for the induction of RNA interference
Site-Specific Promoter Caging Enables Optochemical Gene Activation in Cells and Animals
In
cell and molecular biology, double-stranded circular DNA constructs,
known as plasmids, are extensively used to express a gene of interest.
These gene expression systems rely on a specific promoter region to
drive the transcription of genes either constitutively (i.e., in a
continually “ON” state) or conditionally (i.e., in response
to a specific transcription initiator). However, controlling plasmid-based
expression with high spatial and temporal resolution in cellular environments
and in multicellular organisms remains challenging. To overcome this
limitation, we have site-specifically installed nucleobase-caging
groups within a plasmid promoter region to enable optochemical control
of transcription and, thus, gene expression, via photolysis of the
caging groups. Through the light-responsive modification of plasmid-based
gene expression systems, we have demonstrated optochemical activation
of an exogenous fluorescent reporter gene in both tissue culture and
a live animal model, as well as light-induced overexpression of an
endogenous signaling protein
Optical Control of CRISPR/Cas9 Gene Editing
The
CRISPR/Cas9 system has emerged as an important tool in biomedical
research for a wide range of applications, with significant potential
for genome engineering and gene therapy. In order to achieve conditional
control of the CRISPR/Cas9 system, a genetically encoded light-activated
Cas9 was engineered through the site-specific installation of a caged
lysine amino acid. Several potential lysine residues were identified
as viable caging sites that can be modified to optically control Cas9
function, as demonstrated through optical activation and deactivation
of both exogenous and endogenous gene function
Regulation of Transcription through Light-Activation and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian Cells
Triplex-forming oligonucleotides (TFOs) are efficient
tools to
regulate gene expression through the inhibition of transcription.
Here, nucleobase-caging technology was applied to the temporal regulation
of transcription through light-activated TFOs. Through site-specific
incorporation of caged thymidine nucleotides, the TFO:DNA triplex
formation is blocked, rendering the TFO inactive. However, after a
brief UV irradiation, the caging groups are removed, activating the
TFO and leading to the inhibition of transcription. Furthermore, the
synthesis and site-specific incorporation of caged deoxycytidine nucleotides
within TFO inhibitor sequences was developed, allowing for the light-deactivation
of TFO function and thus photochemical activation of gene expression.
After UV-induced removal of the caging groups, the TFO forms a DNA
dumbbell structure, rendering it inactive, releasing it from the DNA,
and activating transcription. These are the first examples of light-regulated
TFOs and their application in the photochemical activation and deactivation
of gene expression. In addition, hairpin loop structures were found
to significantly increase the efficacy of phosphodiester DNA-based
TFOs in tissue culture
Conditional Control of Alternative Splicing through Light-Triggered Splice-Switching Oligonucleotides
The
spliceosome machinery is composed of several proteins and multiple
small RNA molecules that are involved in gene regulation through the
removal of introns from pre-mRNAs in order to assemble exon-based
mRNA containing protein-coding sequences. Splice-switching oligonucleotides
(SSOs) are genetic control elements that can be used to specifically
control the expression of genes through correction of aberrant splicing
pathways. A current limitation with SSO methodologies is the inability
to achieve conditional control of their function paired with high
spatial and temporal resolution. We addressed this limitation through
site-specific installation of light-removable nucleobase-caging groups
as well as photocleavable backbone linkers into synthetic SSOs. This
enables optochemical OFF → ON and ON → OFF switching
of their activity and thus precise control of alternative splicing.
The use of light as a regulatory element allows for tight spatial
and temporal control of splice switching in mammalian cells and animals