Biology subjects

Sigel, R. K. O.

Publications and source records attributed to Sigel, R. K. O..

6 recordsLinked to original sources

bioRxiv2023

FRET-guided modeling of nucleic acids

The functional diversity of RNA is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new, attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule FRET measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling we recreate FRET experiments in silico, visualize the underlying structural dynamics and quantify the simulated reaction coordinates. Using multiple accessible-contact volumes (multi-ACV) as a post-hoc scoring method for fragment-assembly in Rosetta, we demonstrate that FRET effectively refines de novo RNA structure prediction without the need of explicit dye labeling. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch with sub-helical accuracy to the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions. Graphical abstractSchematic workflow of integrative FRET modeling using all-atom fluorophores or an accessible-contact volume dye model. All-atom molecular dynamics track the dye coordinate explicitly as part of the simulation while multi-ACV infer mean dye positions post hoc. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/552238v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@122bdf7org.highwire.dtl.DTLVardef@1e5c06forg.highwire.dtl.DTLVardef@bcc3aaorg.highwire.dtl.DTLVardef@d205ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Discovery of Theta Ribozymes in Gut Phages-Implications for tRNA and Alternative Genetic Coding

Ribozymes, relics of the "RNA world", are essential across all domains of life. Nonetheless, the functions and genomic contexts of recently discovered small ribozymes, such as minimal hepatitis delta virus (HDV)-like ribozymes, remain elusive. Using bioinformatic analyses, we identified a novel subfamily of minimal HDV-like ribozymes, coined theta ribozymes. Hundreds of unique examples were found adjacent to viral tRNAs within Caudoviricetes bacteriophages of the mammalian gut virome. In vitro experiments confirm site-specific self-scission activity, suggesting their involvement in processing tRNA 3-trailers. Intriguingly, a significant fraction of theta ribozymes is associated with viral suppressor tRNAs, potentially regulating the late-stage assembly of recoded bacteriophages. These findings advance the understanding of RNA-based mechanisms underlying the intricate interplay between the bacterial and viral parts of the mammalian gut microbiome. One-Sentence SummaryNewly unveiled theta ribozymes associate with suppressor tRNAs of alternatively coded gut phages: a potential lytic switch.

bioinformatics↗

bioRxiv2023

Organometallic Pillarplexes that bind DNA 4-way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination and repair) and are dynamic structures which adopt either open or closed conformations, with the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core giving them an ideal structure to interact with the central cavities of open DNA junctions. Combining experimental studies and MD simulations we show that an Au pillarplex can bind DNA 4-way junctions (Holliday junctions) in their open form, a binding mode not accessed by synthetic agents before. The Au pillarplexes can bind designed 3-way junctions too but their large size leads them to open up and expand that junction, disrupting the base pairing which manifests in an increase in hydrodynamic size and a lower junction thermal stability. At high loading they re-arrange both 4-way and 3-way junctions into Y-shaped DNA forks to increase the available junction-like binding sites. The structurally related Ag pillarplexes show similar DNA junction binding behaviour, but a lower solution stability. This pillarplex binding contrasts with (but complements) that of the metallo-supramolecular cylinders, which prefer 3-way junctions and we show can rearrange 4-way junctions into 3-way junction structures. The pillarplexes ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures where they are key interconnecting components. Studies in human cells, confirm that the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction-binders into organometallic chemistry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/522759v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@10fac36org.highwire.dtl.DTLVardef@1f32f9forg.highwire.dtl.DTLVardef@bb8d72org.highwire.dtl.DTLVardef@1433de5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Anticodon-like loop-mediated dimerization in the crystal structures of HdV- like CPEB3 ribozymes

Cytoplasmic polyadenylation element-binding (CPEB) proteins are involved in many cellular processes, including cell division, synaptic plasticity, learning, and memory. A highly conserved, short mammalian ribozyme has been found within the second intron of the CPEB3 gene. Based on its cleavage mechanism and structural features, this ribozyme belongs to the hepatitis delta virus (HDV)-like ribozyme family. Here, we present the first crystallographic structures of human and chimpanzee CPEB3 ribozymes, both confirming the general topology of the HDV ribozyme with two parallel coaxial helical stacks. However, the residues involved in forming the P1.1 mini-helix, which is an integral part of the characteristic nested double pseudoknot involving P1, P2, and P3, instead participate in a seven nucleotides loop with a conformation similar to the one from the anticodon (AC) loop of tRNAs when interacting with the mRNA codon. The conformation of the loop supports the formation of a four-base pair helix by interacting with the AC-like loop from a symmetry-related ribozyme leading to ribozyme dimer formation. The present crystal structures link for the first time the sequence specificities of the CPEB3 and the HDV (genomic and antigenomic) ribozymes to their different structural features. This work corroborates the hypothesis made by Szostak that HDV ribozymes may have evolved from the CPEB3 ribozyme.

biophysics↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗

bioRxiv2020

Screening for New Interaction Partners for a Group IIB Intron Ribozyme with Surface Plasmon Resonance Imaging Coupled to MALDI Mass Spectrometry

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5{gamma}) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5{gamma} (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/082776v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@50a09borg.highwire.dtl.DTLVardef@1602253org.highwire.dtl.DTLVardef@6d9cc7org.highwire.dtl.DTLVardef@4ff0f0_HPS_FORMAT_FIGEXP M_FIG C_FIG Author contributionUlrike Anders and Maya Gulotti-Georgieva contributed equally to this work.

molecular biology↗