Biology subjects

Kristoffersen, E. L.

Publications and source records attributed to Kristoffersen, E. L..

5 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↗

bioRxiv2025

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

bioRxiv2024

Roles of dimeric intermediates in RNA-catalyzed rolling circle synthesis

The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have never been explored. Here we have used cryogenic electron microscopy (cryo-EM) to characterize products and intermediates relevant for RCS replication and find that these form an unexpectedly diverse group of RNA nanostructures. The main structural motif observed is a fully hybridized dimeric complex composed of two scRNAs and their complement strands resolved to 5.3 [A]. Cryo-EM also reveals higher order dimer filaments and dimer assembly intermediates suggesting a mechanism for assembly of the observed complexes. We show that the dimer complexes are stable and inhibit RNA-catalyzed RCS, but can be reactivated by addition of more scRNA templates. We propose that dimer formation may be a general property of RCS replication and speculate that the observed dimers might have benefited a primordial RNA genetic system by providing a stable "storage" form of RNA replication products and by coordinating RNA replication on both scRNA template strands.

biochemistry↗

bioRxiv2022

Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) including a variant that uses trinucleotide triphosphates (triplets) as substrates have been created by in vitro evolution and are the closest functional analogues of the replicase but the structural basis for their function is poorly understood. Here, we leverage single-particle cryo-EM and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The TPR cryo-EM structure at 5-[A] resolution reveals an RNA heterodimer comprising a catalytic and an inactive accessory subunit, where the complex resembles a left hand with thumb and fingers at a 70{degrees} angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme whereby heterodimer formation and KL interactions preorganize the TPR for optimal template binding and templated RNA synthesis activity. These results provide a foundation for a better understanding RNAs potential for self-replication.

biochemistry↗

bioRxiv2021

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of lifes first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5 and 3 ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

biochemistry↗