Florian Schäfer

Dependence of aptamer activity on opposed terminal extensions: improvement of light-regulation efficiency

Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable ‘caged’ intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5′- to 3′-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation.

Wavelength-Selective Uncaging of dA and dC Residues

Nitrodibenzofuran (NDBF) groups are used as photolabile caging groups to temporarily mask the Watson-Crick interaction of dA and dC residues. They show improved masking capabilities and are photodeprotected 12 times more efficiently than 1-(o-nitrophenyl)-ethyl (NPE) caging groups in these positions. Furthermore, NDBF groups can be removed wavelength-selectively in the presence of NPE groups. This will allow more complex (un)caging strategies of oligonucleotides--beyond the usual irreversible triggering.

Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice

MicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally control gene expression. Inhibition of miRs by antisense RNAs (antimiRs) might be a therapeutic option for many diseases, but systemic inhibition can have adverse effects. Here we show that light-activatable antimiRs efficiently and locally restricted target miR activity in vivo. We use an antimiR-92a and establish a therapeutic benefit in diabetic wound healing. AntimiR-92a is modified with photolabile protecting groups, so called ‘cages. Irradiation activates intradermally injected caged antimiR-92a without substantially affecting miR-92a expression in other organs. Light activation of caged antimiR-92a improves healing in diabetic mice to a similar extent as conventional antimiRs and derepresses the miR-92a targets Itga5 and Sirt1, thereby regulating wound cell proliferation and angiogenesis. These data show that light can be used to locally activate therapeutically active antimiRs in vivo.

Regulating angiogenesis with light-inducible AntimiRs.

The inhibition of microRNAs (miRs) in a spatiotemporally defined manner by an exogenous trigger would help to specifically target the biological activity and avoid off-target effects. Novel antimiRs directed against miR-92a can be activated by irradiation (see scheme; 3-UTR=3-untranslated region) In this way miR-92a is inhibited, the miR-92a target integrinu2005α5 is derepressed, and angiogenesis of endothelial cells is enhanced.

Comparison of the duplex-destabilizing effects of nucleobase-caged oligonucleotides

AbstractNucleobase-caged oligonucleotide residues have photolabile “caging groups” that prevent the formation of Watson–Crick base pairs until the unmodified nucleobase is restored in a photolysis event. This principle can be used to put a growing variety of powerful nucleic acid-based applications under the precise spatiotemporal control using light as an addressing mechanism. Examples for applications include light control of transcription, RNAi, nucleic acid folding, primer extension, and restriction endonuclease as well as DNAzyme, aptamer, and antisense activity. However, a comparison of the duplex-destabilization properties of the various caged residues that have been used up to date and rules for achieving a maximal duplex destabilization with a minimum amount of modified residues are still missing. We present both a comparison of the duplex-destabilizing capabilities of various nucleobase-caged residues and address the question of influence on neighboring base pairs.n FigureNucleobase-caged nucleosides act as mismatches until irradiation with UV light which cleaves off the caging group. This technology allows to add spatiotemporal control over nucleic acid-based applications. This study provides a first systematic evaluation of residues that have been presented in individual studies over the recent years and tries to establish rules for their optimal use.

Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

Influence of the absolute configuration of npe-caged cytosine on DNA single base pair stability.

Photolabile protecting groups are a versatile tool to trigger reactions by light irradiation. In this study, we have investigated the influence of the absolute configuration of the 1-(2-nitrophenyl)ethyl (NPE) cage group on a 15-base-pair duplex DNA. Using UV melting, we determined the global stability of the unmodified and the selectively (S)- and (R)-NPE-modified DNA sequences, respectively. We observe a differently destabilizing effect for the two NPE stereoisomers on the global stability. Analysis of the temperature dependence of imino proton exchange rates measured by NMR spectroscopy reveals that this effect can be attributed to decreased base pair stabilities of the caged and the 3-neighbouring base pair, respectively. Furthermore, our NMR based structural models of the modified duplexes provide a structural basis for the distinct effect of the (S)- and the (R)-NPE group.