Biology subjects

Olsthoorn, R.

Publications and source records attributed to Olsthoorn, R..

3 recordsLinked to original sources

bioRxiv2024

High-throughput competitive binding assay for targeting RNA with small molecules: discovery of new PreQ1 riboswitch ligands

In the evolving landscape of RNA targeting, there is an indisputable need for new screening methodologies to find small molecules targeting relevant tertiary RNA structures, like viral pseudoknots or bacterial riboswitches. Here, we developed a competitive binding high-throughput screening assay to identify ligands for the bacterial PreQ1-I riboswitch. In this assay, ligands compete with a rationally designed quencherlabeled antisense for binding to the riboswitch. The method is validated for the Fusobacterium nucleatum (Fnu), Thermoanaerobacter tengcongensis (Tte), Bacillus subtilis (Bsu) and Enterococcus faecalis (Efa) PreQ1 riboswitches, using the natural riboswitch ligand PreQ1 and various analogues. A commercial RNA-focused library consisting of [~]15,000 compounds was then screened against the Fnu riboswitch, leading to the identification of 4 hits exhibiting competitive binding activity. These hits were evaluated in in vitro translation assays against several PreQ1 riboswitches. The most promising hit 4494 showed competitive binding activity to the Fnu, Tte, Bsu and Efa riboswitches, and was able to inhibit translation of a Tte riboswitch-regulated reporter gene, making it an interesting starting point for the development of new antibiotics. In essence, this HTS assay has the potential to discover highly sought-after RNA targeting small molecules for complex and clinically relevant RNA structures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/595132v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d7af01org.highwire.dtl.DTLVardef@227e93org.highwire.dtl.DTLVardef@a3f24corg.highwire.dtl.DTLVardef@17883e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Replication of alphaviruses requires a pseudoknot that involves the polyA tail

Alphaviruses, such as Sindbis virus and Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates including humans. A conserved sequence element (CSE) of [~]19 nucleotides (nts) in the 3 non-coding region is important for replication. Despite extensive mutational analysis of the CSE no comprehensive model of this element exists to date. Here it is shown that the CSE can form an RNA pseudoknot with part of the polyA tail and is similar to the human telomerase peudoknot with which it shares 17 nts. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE.

microbiology↗

bioRxiv2019

A widespread Xrn1-resistant RNA motif composed of two short hairpins

Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3 untranslated region of several beny-and cucumovirus RNAs harbors a so-called coremin motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence, to deduce putative structural features responsible for Xrn1-stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5 proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 9-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn-stalling motifs are more widespread among RNA viruses than previously realized.

biochemistry↗