| Резюме: | <p>Nucleic acids encode the information of all biological functions, which are regulated through the expression of genes. Today, various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) structures can be reliably synthesized and manipulated to recreate naturallyoccurring phenomena or introduce new functions in cell-free and living systems. Controlling the expression of a gene to protein has become a fundamental tool for therapeutic and synthetic biology research. One way of controlling gene expression is by using specific wavelengths of light. Light is an ideal stimulus as it can be externally applied to a system with high spatiotemporal precision, and it is bioorthogonal to most biological processes.</p>
<p>The synthesis of a protein can be inhibited by degrading or deactivating the corresponding gene, a process called gene knockdown. The two nucleic acids mainly used for gene knockdown are antisense oligonucleotides (ASOs), short single stranded DNA sequences, and small interfering RNAs (siRNAs), short double stranded RNA sequences. ASOs and siRNAs can be synthesized and modified to be activated in response to light irradiation.</p>
<p>This work presents the development of an orthogonal two-wavelength control of cellfree gene expression. Light-activated (LA)-ASOs and LA-siRNAs were generated from commercially available amino modified sequences. The amine groups were functionalized with UV or blue light-activatable photocages, which inactivated the nucleic acids by steric hinderance. Upon irradiation with a specific wavelength of light, the knockdown activity was restored, as the photocages were cleaved off, uncaging to the initial amino-modified nucleic acid structure. Cell-free protein synthesis (CFPS) was controlled with UV lightactivated ASOs, by triggering RNase H-mediated mRNA degradation on demand. Additionally, by combining UV LA-ASOs to a previously developed blue LA-DNA template, transcription was activated with UV and translation halted orthogonally with blue light. Finally, ASOs targeting two different genes were modified with orthogonal photocages for precise and selective two-wavelength gene knockdown in CFPS.</p>
<p>Progress towards light-activated gene knockdown in mammalian cells using siRNA will also be presented. Two different strategies for generating LA-siRNA will be discussed. The siRNA photocages were decorated with a cell-penetrating peptide and a fluorophore, for self-delivery to cells and fluorescent tracing of the nucleic acid. Images demonstrating cellular uptake without the use of transfection reagents will be finally shown.</p>
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