A kinetic model explains why shorter and less affine enzyme-recruiting oligonucleotides can be more potent

Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs

Managing the sequence-specificity of antisense oligonucleotides in drug discovery

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery

Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3′UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity

Indflydelse i arbejdslivet – status, muligheder, alternativer?

I de formende konflikter på arbejdsmarkedet i slutningen af det 19. århundrede var indfl ydelse i arbejdet et centralt konfliktpunkt, i Danmark og i den industrialiserede verden i øvrigt. Arbejdsgivere og fagforeninger var i konflikt om, hvem der havde retten til at lede og fordele arbejdet. Med Septemberforliget i 1899 blev Danmark det første land i verden, hvor der blev indgået et nationalt kompromis om indfl ydelse. Arbejdsgiverne vandt ledelsesretten, og fagforeningerne vandt retten til at forhandle løn og arbejdsvilkår og indgå overenskomster om disse spørgsmål (Galeson, 1955, Christensen et al., 2015). Dermed var spørgsmålet om indflydelse dog langt fra afklaret. I årtierne efter Septemberforliget tilkæmpede fagforeningerne sig gradvist mere indflydelse. Indflydelsen på løn og arbejdsforhold blev udbygget. Overenskomstsystemet blev mere omfattende. Medarbejderne fik ret til at vælge tillidsrepræsentanter, som arbejdsgiverne havde pligt til at inddrage i beslutningsprocesserne. Samarbejdsudvalg og senere arbejdsmiljøudvalg blev etableret. Endelig fik medarbejdere i aktieselskaber ret til at vælge repræsentanter til selskabernes bestyrelser. I tiden efter 2. verdenskrig voksede en anden mere konsensusorienteret strømning til udvikling af indflydelse i arbejdet frem. Denne strømning har siden forgrenet sig i variationer og antaget mange forskellige navne, bl.a. Human Relations og Human Resources, Socioteknik, High Performance Work Systems samt Workplace Innovation. En af grundlæggerne var Kurt Lewin, som allerede i 1940erne blev kendt for sin formulering af principper for en involverende arbejdsorganisation og demokratisk ledelse (Lewin et al., 1939). Socioteknikken, som blev udviklet i 1950erne, havde som målsætning at udvikle en arbejdsorganisation, hvor hensynet til det sociale system og det teknisk/økonomiske system var ligeværdigt (Trist & Bamforth, 1951). En sådan ligeværdighed kunne etableres i en arbejdsorganisation, hvor medarbejderne havde en stor grad af autonomi i et gruppeorganiseret arbejde, samtidig med at medarbejderne havde ansvar overfor virksomheden

Action needed for the EU Common Agricultural Policy to address sustainability challenges

Abstract Making agriculture sustainable is a global challenge. In the European Union (EU), the Common Agricultural Policy (CAP) is failing with respect to biodiversity, climate, soil, land degradation as well as socio-economic challenges. The European Commission's proposal for a CAP post-2020 provides a scope for enhanced sustainability. However, it also allows Member States to choose low-ambition implementation pathways. It therefore remains essential to address citizens' demands for sustainable agriculture and rectify systemic weaknesses in the CAP, using the full breadth of available scientific evidence and knowledge. Concerned about current attempts to dilute the environmental ambition of the future CAP, and the lack of concrete proposals for improving the CAP in the draft of the European Green Deal, we call on the European Parliament, Council and Commission to adopt 10 urgent action points for delivering sustainable food production, biodiversity conservation and climate mitigation. Knowledge is available to help moving towards evidence-based, sustainable European agriculture that can benefit people, nature and their joint futures. The statements made in this article have the broad support of the scientific community, as expressed by above 3,600 signatories to the preprint version of this manuscript. The list can be found here (https://doi.org/10.5281/zenodo.3685632). A free Plain Language Summary can be found within the Supporting Information of this article.Peer reviewe