Andres Jäschke

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock‐in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα‐DM) by crossing APPsα‐KI mice onto an APLP2‐deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα‐DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα‐DM muscle showed fragmented post‐synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα‐DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABAA receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Allylic amination by a DNA-diene-iridium(I) hybrid catalyst.

DNA hybrid catalysis goes organometallic: A DNA strand functionalized with diene ligands forms iridium(I) complexes that can efficiently catalyze an allylic amination in aqueous medium (see scheme). The DNA-based complexes show high stability and activity, and their secondary structure influences the stereoselectivity of the reaction.

Reversibly Photoswitchable Nucleosides: Synthesis and Photochromic Properties of Diarylethene-Functionalized 7-Deazaadenosine Derivatives

Photochromic nucleosides were designed that combine the structural features and molecular recognition properties of nucleic acids with the light-sensitivity of diarylethenes. Target compounds 1a-c consist of a 7-deazaadenosine unit that is linked to a thiophene as the second aryl functionality via a 1,2-cyclopentenyl linker. These nucleoside analogues undergo a reversible electrocyclic rearrangement, generating strongly colored closed-ring isomers upon irradiation with UV-light, while exposure to light in the visible range triggers the cycloreversion to the colorless opened-ring form. UV-vis spectroscopy, HPLC, and (1)H NMR measurements revealed recognition of complementary thymidine and up to 97% conversion to the thermally stable closed-ring isomers after illumination with UV-light. The required wavelength for ring closure was found to vary depending on the substituents attached to the thiophene moiety. In a first design step, we used this important feature of diarylethenes to shift the switching wavelength from initially 300 nm (1a) to 405 nm (1cH(+)). In a second step, we generated a pair of orthogonal switches, differing enough in their respective switching wavelengths to be controlled independently in the same sample. Finally, a molecular switch was developed that showed both photochromism and acidichromism, thereby illustrating the possibility to gate the spectral properties to multiple stimuli. These new photochromic nucleosides represent useful building blocks for the generation of light-sensitive nucleic acids either by inducing conformational changes upon isomerization or by exploring the different spectral properties of the closed and opened isomers, for example, for use as reversible fluorescence quenchers.

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg2+ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg2+ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg2+ concentration that arises from association with several Mg2+ ions. This transition is followed by a second Mg2+-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ∼100 ms timescale offers insight into the folding dynamics of this ribozyme.

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

Detection of small organic analytes by fluorescing molecular switches.

A sensor system was developed for the determination of theophylline concentrations based on a theophylline-dependent allosteric ribozyme (Soukup, G. A.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3584) in combination with an RNA substrate which is double-labeled with a fluorophore and a quencher dye. In the presence of theophylline, a hammerhead ribozyme domain is switched into an active conformation by the action of a theophylline-binding aptamer domain. Upon substrate cleavage, the quencher is removed from the vicinity of the fluorophore, causing an increased fluorescence signal. Real-time analysis of the cleavage reactions both under single and multiple turnover conditions revealed a dependence on the cleavage rate within a range from 0.01 to 2mM theophylline. The structurally similar molecule caffeine, however, had no detectable influence on the fluorescence signal.

NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs

A distinctive feature of prokaryotic gene expression is the absence of 5′-capped RNA. In eukaryotes, 5′,5′-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5′-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

Nucleoside‐Based Diarylethene Photoswitches and Their Facile Incorporation into Photoswitchable DNA

The artificial control of DNA structure and function is an attractive field in chemical and synthetic biology, and light is a powerful and convenient trigger: it is non-invasive, provides high spatio-temporal resolution, and offers the option of orthogonality. DNA is reactive to light: the UVlight-induced cyclodimerization of pyrimidine nucleosides is an important type of DNA damage and has been studied intensively. Interestingly, this chemical property has never been exploited for the construction of DNA-based reversible photoswitches, and all published work in this field is based on the covalent functionalization of DNA with small autonomous photoactive molecules. Different classes of such photoactive molecules have been studied for DNA photoregulation; azobenzenes have been used most frequently and were, for example, employed for modulating oligonucleotide duplex and triplex formation and DNA transcription. Other substance classes studied in this context include arylvinyl derivatives, spiropyranes, and recently also diarylethenes. 7] While a certain influence of photoisomerization on the properties of the nucleic acid was always observed, these approaches share one common limitation: because an autonomous photoswitch was attached to the DNA, either as an appendage or substituting for nucleosides, the rearrangement of chemical bonds upon encountering a photon (i.e., the photochemical reaction) was strictly confined to this non-nucleosidic moiety. Recently, our lab reported a new type of diarylethene photoswitches in which one of the two aryl moieties was replaced by a nucleoside, namely 7-deaza-8-methyldeoxyadenosine (Scheme 1). These photoswitches were synthesized in a convergent multi-step approach in which a substituted cyclopentenyl boronic ester was reacted with protected 7-iodo-8-methyl-7-deazadeoxyadenosine by Suzuki crosscoupling, followed by deprotection. Upon irradiation with light, these compounds were found to undergo a highly efficient, reversible, electrocyclic rearrangement and the switching wavelength could be tuned by the chemical nature of substituents. Switching was found to be near-quantitative in aprotic solvents, and the compounds retained the key properties of nucleotides, such as their capability to form Watson– Crick base-pairs. Unfortunately, the photoisomerization was found to proceed with low efficiency in aqueous solvents, and the demanding synthesis involved limited the application of these photoswitches to oligonucleotides. To develop straight-forward access to truly photoswitchable DNA, we reconsidered our design approach; in contrast to 7-iodo-8-methyl-7-deazadeoxyadenosine, the 5-iodo-substituted pyrimidine nucleosides 5I-dU and 5I-dC represent oligonucleotide modifications readily available from commercial suppliers, and offer the desired reactivity for different cross-coupling reactions. This could allow for the postsynthetic conversion of an iodo-modified oligonucleotide into a photoswitch, leading to the target compounds shown in Scheme 1c. We note that this design challenges some of the common design principles for diarylethene photoswitches: neither unsubstituted nor substituted pyrimidines have ever been applied as components of diarylethene photoswitches, and our target compounds contain just one (rather than two) alkyl groups attached to the carbon atoms that form the new bond in the cyclization reaction, a feature that is thought to be important for the reversibility of photoswitching. This design was first tested on the nucleoside level employing 5-iodoScheme 1. a) Isomerization of typical diarylethene photoswitches. b) A diarylethene photoswitch involving 7-deazadeoxyadenosine. c) New photoswitchable DNA where a pyrimidine nucleotide is part of the photoswitch.