Reverse transcription as key step in RNA 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 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.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) :, novel modified nucleoside triphosphates with a dual modification pattern were synthesized.

Two are not enough: synthetic strategies and applications of unnatural base pairs.

Nucleic acid chemistry is a rapidly evolving field, and the need for novel nucleotide modifications and artificial nucleotide building blocks for diagnostic and therapeutic use, material science or for studying cellular processes continues unabated. This review focusses on the development and application of unnatural base pairs as part of an expanded genetic alphabet. Not only recent developments in "nature-like" artificial base pairs are presented, but also current synthetic methods to get access to C-glycosidic nucleotides.

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 transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities.

Preparation of Site-Specifically Spin-Labeled RNA by in Vitro Transcription Using an Expanded Genetic Alphabet.

Recent advances in pulsed electron paramagnetic resonance (EPR) spectroscopy enable studying structure and folding of nucleic acids. An efficient introduction of spin labels at specific positions within the oligonucleotide sequence is a prerequisite. We here present a step-by-step guide to synthesize long RNA oligonucleotides bearing spin labels at specific positions within the sequence.

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.

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 TPT3 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.

Site-Directed Spin Labeling of RNA with a -Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability.

Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information.

Towards Reverse Transcription with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX).

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.

Dinosaur origin of egg color: oviraptors laid blue-green eggs.

Protoporphyrin (PP) and biliverdin (BV) give rise to the enormous diversity in avian egg coloration. Egg color serves several ecological purposes, including post-mating signaling and camouflage. Egg camouflage represents a major character of open-nesting birds which accomplish protection of their unhatched offspring against visually oriented predators by cryptic egg coloration.

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.

The 5'-AG CC-3' Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G-Quadruplex.

An unusually thermostable G-quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G-quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.

Cycloadditions for Studying Nucleic Acids.

Cycloaddition reactions for site-specific or global modification of nucleic acids have enabled the preparation of a plethora of previously inaccessible DNA and RNA constructs for structural and functional studies on naturally occurring nucleic acids, the assembly of nucleic acid nanostructures, therapeutic applications, and recently, the development of novel aptamers. In this chapter, recent progress in nucleic acid functionalization via a range of different cycloaddition (click) chemistries is presented. At first, cycloaddition/click chemistries already used for modifying nucleic acids are summarized, ranging from the well-established copper(I)-catalyzed alkyne-azide cycloaddition reaction to copper free methods, such as the strain-promoted azide-alkyne cycloaddition, tetrazole-based photoclick chemistry and the inverse electron demand Diels-Alder cycloaddition reaction between strained alkenes and tetrazine derivatives.

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.

Flexibility of C3h -Symmetrical Linkers in Tris-oligonucleotide-Based Tetrahedral Scaffolds.

Flexibility of tris-oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris-oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C3h -symmetrical linker core at these chains. Four tris-oligonucleotides hybridise into a tetrahedral nanocage by sequence-determined self-assembly.

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.

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.

General acid-base catalysis mediated by nucleobases in the hairpin ribozyme.

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5'-oxygen atom at the scissile phosphate by sulfur (5'-PS), which is a much better leaving group.