5 research outputs found
Expanding the toolbox of modified nucleobases and click chemistry for investigations on non-coding RNA
Non-coding ribonucleic acid (RNA) consists of highly structured molecules whose arrangement and function can be investigated using different chemical labeling approaches. Contemporary labeling techniques show restrictions either in labeling specificity or oligonucleotide length. Literature describes highly efficient and fast labeling strategies as well as different approaches of unnatural base pairs (ubp) specifically recognized by enzymes, but previous work failed to address a combined technique for labeling. In this thesis, an enzymatic template-directed labeling approach is shown based on unnatural ribonucleoside triphosphates either suitable for structural investigations using electron paramagnetic resonance (EPR) spectroscopy or modified for subsequent inverse electron demand Diels-Alder (IEDDA) click chemistry on non-coding RNA. Chemical synthesis of ubp and IEDDA click building blocks, successful template-directed T7 in vitro transcriptions with subsequent fluorescent labeling or EPR-based distance measurement will be described. This novel approach reveals a site-specific labeling technique of long RNA strands to get a better insight in catalysis and regulation on RNA level
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
The Novel Chemical Mechanism of the Twister Ribozyme
We describe the multifactorial
origins of catalysis by the twister
ribozyme. We provide evidence that the adenine immediately 3′
to the scissile phosphate (A1) acts as a general acid. Substitution
of ring nitrogen atoms indicates that very unusually the N3 of A1
is the proton donor to the oxyanion leaving group. A1 is accommodated
in a specific binding pocket that raises its p<i>K</i><sub>a</sub> toward neutrality, juxtaposes its N3 with the O5′
to be protonated, and helps create the in-line trajectory required
for nucleophilic attack. A1 performs general acid catalysis while
G33 acts as a general base. A 100-fold stereospecific phosphorothioate
effect at the scissile phosphate is consistent with a significant
stabilization of the transition state by the ribozyme, and functional
group substitution at G33 indicates that its exocyclic N2 interacts
directly with the scissile phosphate. A model of the ribozyme active
site is proposed that accommodates these catalytic strategies
Diels–Alder Cycloadditions on Synthetic RNA in Mammalian Cells
Inverse
electron demand Diels–Alder cycloadditions are extremely
useful tools for orthogonal labeling of biomolecules such as proteins
or small molecules in a cellular context. In-cell labeling of dienophile-modified
RNA oligonucleotides using Diels–Alder cycloaddition reactions
has not been demonstrated before. In this study we report site-specific
labeling of RNA oligonucleotides modified with norbornene derivatives
at a predefined sequence position within an RNA sequence <i>in
vitro</i> and in mammalian cells using various tetrazine–fluorophore
conjugates. The approach could in future be used as a chemical tool
for the detection and investigation of RNA functions in cells minimizing
the presumed distortion of RNA functions by a large chemical reporter
group such as a fluorophore