Developing new methods for specific RNA modification

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

Stronger together for in-cell translation: natural and unnatural base modified mRNA

The preparation of highly modified mRNAs and visualization of their cellular distribution are challenging. We report in-cell application of in vitro transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities. While non-specific introduction of reporter groups often leads to loss in mRNA functionality, we combined the benefits from site-specificity in the 3 '-UTR incorporated unnatural nucleotides with the improved translation efficiency of the natural base modifications psi and 5mC. A series of experiments is described to observe, quantify and verify mRNA functionality. This approach represents a new way to visualize mRNA delivery into cells and monitor its spread on a cellular level and translation efficiency. We observed increased protein expression from this twofold chemically modified, artificial mRNA counterbalancing a reduced transfection rate. This synergetic effect can be exploited as a powerful tool for future research on mRNA therapeutics

Strategies for Covalent Labeling of Long RNAs

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field