Biology subjects

Birkedal, V.

Publications and source records attributed to Birkedal, V..

4 recordsLinked to original sources

bioRxiv2026

Fluorinated RNA origami enables serum-stable nanodevices for sensing and targeting

Chemically modified RNAs with increased stability and reduced immunogenicity have transformed RNA therapeutics. Rational RNA design methods, including RNA origami, seek to further extend RNA medicine and biotechnology by encoding advanced functions such as signalling, targeting, and controlled release within the RNA polymer. However, current design methods lack the ability to integrate chemical modification or predict how it shapes the structure of large RNA assemblies inhibiting its use in RNA therapeutics. Here we demonstrate that 2-fluoro pyrimidine RNA (FY-RNA) origami structures can be co-transcriptionally folded to generate serum-stable nanodevices. Cryogenic electron microscopy reveals that FY-RNA can alter folding pathways and perturb tertiary motifs, while molecular dynamics simulations show how 2-fluoro modification affects hydrogen bonding, sugar pucker, and helix-helix interactions. Despite these structural perturbations, fluorogenic aptamers embedded within RNA origami retain partial activity and enable logic-based molecular sensing in human serum. Finally, we use an FY-RNA scaffold to determine the structure of an FY-RNA anti-Spike aptamer bound to the Spike protein at 3.4 [A] resolution, uncovering fluorine-specific structural motifs and protein interactions. Together, our results establish design principles for nuclease-resistant RNA architectures and position FY-RNA as a versatile polymer for constructing medical nanodevices and environmental sensors. More broadly, this work provides a framework for systematically exploring the folding landscape of chemically modified RNAs, expanding the chemical and functional diversity accessible to nucleic acid nanotechnology and RNA medicine.

biochemistry↗

bioRxiv2026

Slow diffusion limits phosphorylation in a biomolecular condensate

Biomolecular condensates form dynamic compartments that regulate biochemical reactions in cells. Condensates recruit many kinases and regulate their enzymatic activity. Condensates alter the rate of enzymatic reactions through several opposing effects, so it is unclear whether these mostly enhance or retard phosphorylation. Here, we use a synthetic condensate formed by intrinsically disordered proteins to show that slow diffusion in the condensate controls phosphorylation kinetics in the dense phase. We vary the length of substrates by appending phase-separating repeat proteins of different lengths, in order to study how phosphorylation depends on partitioning, diffusion and volume fraction across substrate motifs with different intrinsic kinetics. The condensate environment is generally inhibitory to phosphorylation, although the enzyme remains intact. This inhibition is partially offset by an enhanced reaction rate in the dilute phase, likely due to soluble nanoclusters. Phosphorylation rates are strongly correlated to diffusion coefficients of substrates in the condensate, suggesting mass-transport limitation. Our results suggest that condensates can modify the substrate usage of a kinase via different trade-offs between diffusion and partitioning. We suggest that diffusion limitations are likely a common feature of many macromolecular reactions in condensates, and that high fluidity is crucial for condensates to act as reaction crucibles.

biophysics↗

bioRxiv2025

Secret life of prophages: template-directed synthesis of DNA superstructures via prophage activation and rolling circle replication in bacterial biofilms

Extracellular DNA (eDNA) plays crucial roles in biofilm formation and function, yet the role of bacteriophages (phages) in controlling eDNA synthesis, structure and activity remains obscure. Here, we demonstrate that phages harbored by environmental bacteria can be exploited for programmable synthesis of functional eDNA superstructures. We designed a 112-nucleotide circular template (T1) and used it to direct rolling circle replication (RCR) of G-quadruplex (GQ) motifs in Shewanella oneidensis and Bacillus subtilis. Under nutrient-limiting conditions, prophage activation triggered cell lysis and subsequent extracellular DNA synthesis, producing multimeric GQ concatemers that self-assembled into distinct morphologies: spherical structures ([≤]10 m) in S. oneidensis and wire-like structures (>50 m) in B. subtilis. Real-time monitoring using fluorescent reporter strains revealed that DNA synthesis occurred predominantly after bacterial lysis, coinciding with prophage replication. The resulting DNA superstructures exhibited peroxidase activity through GQ-hemin DNAzyme formation and enhanced the electrochemical properties of S. oneidensis biofilms, showing a 3-fold increase in current density. This work unveils a previously unknown mechanism by which prophages contribute to biofilm architecture and establishes a biotechnological platform for engineering functional DNA materials in living bacterial communities, with potential applications in biotechnology and synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/691978v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@aec065org.highwire.dtl.DTLVardef@d94709org.highwire.dtl.DTLVardef@c6c228org.highwire.dtl.DTLVardef@f4f465_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Extracellular G-quadruplex and Z-DNA protect biofilms from DNase I and forms a DNAzyme with peroxidase activity

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often arises from lysed cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplex formation might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media amended with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structure in biofilms grown in vitro and in vivo using a murine implant-associated osteomyelitis model. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt, and we confirmed that G-quadruplex DNA and Z-DNA was also present in biofilms from infected implants. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/541711v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@c32012org.highwire.dtl.DTLVardef@6e8c7eorg.highwire.dtl.DTLVardef@1c9cc6corg.highwire.dtl.DTLVardef@18be51e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗