Reverse transcription as key step in RNA in vitro evolution with unnatural base pairs.

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can thus enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. However, owing to a lack of methodologies, the reverse transcription of UBPs, a key step in RNA aptamer selection, has not been sufficiently characterized. Here, we present a series of versatile assays to investigate the reverse transcription of the TPT3:NaM base pair as a representative for hydrophobic unnatural base pairs. We determine the fidelity and retention of the UBP for four different reverse transcriptases (RT) in the context of RNA in vitro evolution. The retention of the TPT3:NaM pair during the RNA in vitro selection process was investigated using a novel click-chemistry based electromobility shift assay. Real-time monitoring of reverse transcription kinetics revealed considerable differences in polymerase activity processing the TPT3:NaM base pair. Our findings identified SuperScript IV RT as the most efficient RT for processing the TPT3:NaM pair. Our approach can be applied universally to study newly developed UBPs, not only at the reverse transcription level, but also during PCR and in vitro transcription.

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 Ψ 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.

EPR Distance Measurements on Long Non-coding RNAs Empowered by Genetic Alphabet Expansion Transcription.

We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3NO and its application for site-specific spin-labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin-labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter-spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self-complementary RNA sequences and in a well-studied 185 nucleotide non-coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A-region of the long non-coding RNA Xist, by PELDOR.

Posttranscriptional spin labeling of RNA by tetrazine-based cycloaddition.

The site-specific introduction of spin labels into RNA for distance measurements by EPR gives insight into its solution structure. We here present a method for spin labeling of in vitro transcribed RNA. Distance distributions between two nitroxide spin labels are determined by PELDOR in a self-complementary RNA duplex.

Iluminated by foreign letters - Strategies for site-specific cyclopropene modification of large functional RNAs via in vitro transcription.

The synthesis of sequence-specifically modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods is experimentally challenging. We are using a new approach based on an expanded genetic alphabet preparing site-specifically modified RNA molecules via standard in vitro transcription. In this report, the site-specific labeling of functional RNAs, in particular ribozymes and a long non-coding RNA with cyclopropene moieties, is presented. We provide detailed instructions for RNA labeling via in vitro transcription and include required analytical methods to verify production and identity of the transcript. We further present post-transcriptional inverse electron demand Diels-Alder cycloaddition reactions on the cyclopropene-modified sequences and discuss applications of the genetic alphabet expansion transcription for in vitro preparation of labeled functional RNAs with complex foldings. In detail, the glmS and CPEB3 ribozymes were site-specifically decorated with methyl cyclopropene moieties using the unnatural TPT3CP triphosphate and were proven to be still functional. In addition, the structurally complex A region of the Xist lncRNA (401nt) was site-specifically modified with methyl cyclopropene and detected by fluorescence after cycloaddition reaction with a tetrazine-BODIPY conjugate.

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 pKa 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.

Site-specific enzymatic introduction of a norbornene modified unnatural base into RNA and application in post-transcriptional labeling.

Inverse electron demand Diels-Alder cycloadditions have proven to be extremely useful for mild and additive-free orthogonal labeling of biomolecules, amongst others, for RNA labeling in vitro and in a cellular context. Here we present a method for site-specific introduction of an alkene modification into RNA via T7 in vitro transcription. For this, an unnatural, hydrophobic base pairing system developed by Romesberg and coworkers was modified introducing one or two norbornene moieties at predefined positions into RNA oligonucleotides in an in vitro transcription reaction. This allows post-transcriptional functionalization of these RNA molecules with tetrazine derivatives containing for instance fluorophores or biotin.

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 in vitro 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.