| Popis: |
Ribonucleic acids (RNAs), and messenger RNAs (mRNAs) in particular, have the potential to play a leading role in future therapeutic research. During the SARS-CoV-2 pandemic, mRNA vaccines have proven useful and highly effective. Therefore, it is of great interest to further investigate RNA and to broaden the current knowledge about RNA function, structure as well as modifications and their effects. Expansion of the genetic alphabet by use of unnatural bases (UB) can contribute to this, both by modifying RNA and extracting new information from the modified RNA. In this thesis, unnatural base modifications were utilized for site-specific introduction of various functionalities into different RNA sequences. A valuable contribution was made towards structure elucidation of the non-coding and complex folded regulatory Xist A repeat region. Here, incorporation of UB-attached nitroxide spin labels enabled inter-spin distance measurements and thus support for a previously proposed structure by targeting different labeling positions. Multifaceted analysis of cellular applications was presented for protein coding mRNA sequences carrying cyclopropene (CP)-functionalized UB modifications in their 3′-untranslated regions. Employing inverse electron demand Diels-Alder click chemistry, live-cell labeling of CP-modified mRNAs with tetrazine-conjugated fluorophores allowed excellent spatiotemporal mRNA visualization in cells. In addition, highly modified mRNA sequences with a combination of site-specific unnatural and random positioned natural base modifications were investigated regarding their influence on mRNA stability and functionality. A combined temporal quantification was performed for cellular mRNA levels and cellular expression of the mRNA encoded reporter protein. The combination of unnatural and natural base modifications was shown to synergistically improve both mRNA stability in cells and cellular protein expression through outstanding mRNA translation efficiency. Briefly, UB modifications proved advantageous for research on both coding and non-coding RNA. Moreover, site-specific UB modifications facilitated non-disruptive investigations on different parameters such as structure, function and visualization of RNA. The applications and methods developed in this thesis will support future RNA research and therapeutic development. |
