|
We are developing
light-activated
oligonucleotides to answer fundamental questions regarding the role of
many genes, particularly transcription factors, during embryogenesis.
Initially, we succeeded in photomodulating 25-fold the activity of DNA
polymerase by incorporating a fluorescein donor and a photocleavable
DABSYL acceptor moiety at adjacent cytidines within a 25-mer
oligonucleotide. This created a method for controlling enzyme-DNA
interactions with light, and was one of the first examples of a “caged”
molecule whose activity was linked to its fluorescent state.
|
 Figure 1.
Controlling gene expression using negatively charged peptide nucleic
acid (ncPNA) attached to complementary 2'-OMe RNA via a photocleavable
linker (PL). Photolysis promoted ncPNA binding to mRNA, thereby
blocking protein synthesis in zebrafish embryos.
|
| In order
to photomodulate
gene expression for in vivo studies, we created “caged” DNA
hairpin-like molecules (Figure 1) with high melting temperatures (Tm
> 80 °C) comprised of an antisense oligonucleotide joined via a
photocleavable linker to a blocking sense strand. UV activation breaks
the photocleavable linker, and generates a much less stable oligo-oligo
duplex. This is a uniquely sensitive strategy, as it only
requires a single photocleavable linker to modulate the activity of the
antisense molecule. In one example using caged DNA, formation of
a DNA-mRNA duplex upon photoactivation recruits RNase H, which
hydrolyzes the target mRNA. Using nuclease-resistant phosphorothioated
photoactive oligos, the lab was able to conditionally degrade mRNA from
the c-myb oncogene in human leukemia cells. This is the first example
of directing photoactive oligos at potentially therapeutic targets in
human cells. |
 Figure 2.
Phenotypic response to UV-irradiating zebrafish embryos injected with
PNA-boz clearly observed at 24 hpf.
|
| By a related antisense strategy, negatively charged peptide nucleic acid (ncPNA)-photocleavable linker (PL)-2′-OMe RNA conjugates that block ribosomal translation in zebrafish embryos upon photoactivation were designed (Figure 2). One such construct, PNA-boz, was targeted against bozozok, a gene with important roles in organizer formation during early zebrafish development. A remarkable difference in stability, ΔTm ≈ -41 oC, was observed between the intact conjugate and the photoactivated product duplex. Upon photo-uncaging, PNA-boz was observed to be very biologically active (Figure 2), with no apparent background activity from caged PNA-boz. This new class of oligonucleotides should have tremendous utility in a wide range of model organisms, and provide unprecedented spatiotemporal control over gene expression. The James Chen lab at Stanford and a start-up company Syntrix BioSystems are now also making use of these strategies. We have recently developed related photochemical methods for restoring gene expression in cells, neurons, and embryos. |
 Figure 3. Three
basic steps required for in vivo photo-uncaging.
|
| These
tools enable both up-
and down-regulation of proteins using our state-of-the-art Olympus
FV1000 UV confocal laser scanning microscope (CLSM). We have begun
focal UV uncaging experiments in zebrafish embryos (Figure 3). One
important goal is to modulate the concentration of transcription
factors on a cell-by-cell basis and control the course of development.
These photochemical tools will bring much higher spatial and temporal
resolution to the study of biological processes such as brain formation
in the developing zebrafish embryo. |