3 research outputs found
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
Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy
Antisense
oligonucleotides are powerful tools to regulate gene
expression in cells and model organisms. However, a transfection or
microinjection is typically needed for efficient delivery of the antisense
agent. We report the conjugation of multiple HIV TAT peptides to a
hairpin-protected antisense agent through a light-cleavable nucleobase
caging group. This conjugation allows for the facile delivery of the
antisense agent without a transfection reagent, and photochemical
activation offers precise control over gene expression. The developed
approach is highly modular, as demonstrated by the conjugation of
folic acid to the caged antisense agent. This enabled targeted cell
delivery through cell-surface folate receptors followed by photochemical
triggering of antisense activity. Importantly, the presented strategy
delivers native oligonucleotides after light-activation, devoid of
any delivery functionalities or modifications that could otherwise
impair their antisense activity
Site-Specific DNA–Doxorubicin Conjugates Display Enhanced Cytotoxicity to Breast Cancer Cells
Doxorubicin (Dox) is widely used
for breast cancer treatment but
causes serious side effects including cardiotoxicity that may adversely
impact patient lifespan even if treatment is successful. Herein, we
describe selective conjugation of Dox to a single site in a DNA hairpin
resulting in a highly stable complex that enables Dox to be used more
effectively. Selective conjugation of Dox to G15 in the hairpin loop
was verified using site-specific labeling with [2-<sup>15</sup>N]-2′-deoxyguanosine
in conjunction with [<sup>1</sup>H–<sup>15</sup>N] 2D NMR,
while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF
mass spectrometry and UV spectroscopy. Molecular modeling indicated
covalently bound Dox also intercalated into the stem of the hairpin
and stability studies demonstrated the resulting Dox-conjugated hairpin
(DCH) complex had a half-life >30 h, considerably longer than alternative
covalent and noncovalent complexes. Secondary conjugation of DCH with
folic acid (FA) resulted in increased internalization into breast
cancer cells. The dual conjugate, DCH-FA, can be used for safer and
more effective chemotherapy with Dox and this conjugation strategy
can be expanded to include additional anticancer drugs