Biology subjects

Kostiainen, M. A.

Publications and source records attributed to Kostiainen, M. A..

7 recordsLinked to original sources

bioRxiv2026

Impact of coding region accessibility and uridine chemistry on translation in mRNA-DNA hybrid origami

Messenger RNA (mRNA) is a prerequisite for programmable protein expression, but its therapeutic and synthetic-biology applications are limited by instability and susceptibility to degradation. Hybridizing mRNA to short DNA strands can fold it into a compact origami nanostructure, protecting it from degradation but impeding ribosome access. However, how such a folded mRNA is translated, and which parts must be left unpaired, remain unclear. Here we fold an EGFP-encoding mRNA into a six-helix bundle and leave defined regions of the coding sequence unpaired to examine what the ribosome requires. We find that the start of the coding sequence must be accessible for translation, whereas leaving the far end unpaired makes no difference. Counterintuitively, leaving more of the coding sequence unpaired does not help: translation first falls and then partially recovers as the unpaired region lengthens, a reproducible pattern set by how that region folds rather than by its length. Modified mRNAs carrying 5-methoxyuridine or N1-methylpseudouridine still fold correctly into the six-helix bundle and show the non-monotonic translation pattern; the modification only shifts the overall level of protein produced, with N1-methylpseudouridine giving the most. Together these results begin to define how a folded mRNA can be made both stable and efficiently translated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/734245v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@a54ac6org.highwire.dtl.DTLVardef@1d02202org.highwire.dtl.DTLVardef@168f0cforg.highwire.dtl.DTLVardef@99b51e_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Folding of mRNA-DNA origami for controlled translation and viral vector packaging

mRNA is an important molecule in vaccine development and treatment of genetic disorders. Its capability to hybridize with DNA oligonucleotides in a programmable manner facilitates the formation of RNA-DNA origami structures, which can possess a well-defined morphology and serve as rigid supports for mRNA delivery. However, to date, compre- hensive studies on the requirements for efficient folding of mRNA into distinct mRNA-DNA structures while preserving its translation func- tionality remain elusive. Here, we systematically investigate the impact of design parameters on the folding of protein-encoding mRNA into mRNA-DNA origami structures and demonstrate the importance of the availability of ribosome-binding sequences on the translation effi- ciency. Furthermore, these hybrid structures can be encapsulated inside virus capsids for protecting them against nuclease degradation and also for enhancing their cellular uptake. This multicomponent system therefore showcases a modular and versatile nanocarrier. Our work pro- vides valuable insight into the design of mRNA-DNA origami structures contributing to the development of mRNA-based gene delivery platforms.

bioengineering↗

bioRxiv2023

Supercharged Fluorescent Protein-Apoferritin Cocrystals for Lighting Applications

The design of lighting sources based on fluorescent proteins (FPs) has been limited by the lack of protocols to stabilize FPs under preparation (deposition techniques, organic solvents, etc.) and working (temperature, irradiation, etc.) conditions. As a critical bottleneck, photo-induced heat generation due to FP motion and quick heat transfer leads to working device temperatures of ca. 70 {degrees}C, resulting in a quick FP-denaturation and, in turn, a quick loss of the device performance. Herein, we showcase FP stabilization for lighting devices with an electrostatically self-assembled FP-apoferritin cocrystals embedded in a silicone-based color down-converting filter. This strategy highlights three major advances: i) engineering of positively supercharged FPs (+22) without losing photoluminescence and thermal stability compared to its native form, ii) a crystallization protocol resulting in highly emissive cocrystals keeping the photoluminescence features of the FPs, and iii) a 40-fold increase of the lighting device stability compared to reference devices due to the reduction of the device working temperatures to 40 {degrees}C. Thus, the success of this multidisciplinary approach contributes toward developing stable energy-related protein-based optoelectronics.

bioengineering↗

bioRxiv2023

Repurposing host-guest chemistry to sequester virulence and eradicate biofilms in multidrug resistant Pseudomonas aeruginosa and Acinetobacter baumannii

The limited diversity in targets of available antibiotic therapies has put tremendous pressure on the treatment of bacterial pathogens, where numerous resistance mechanisms that counteract their function are becoming increasingly prevalent. Here, we utilize an unconventional anti-virulence screen of host-guest interacting macrocycles, and identify a water-soluble synthetic macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a mechanism of action that involves binding to both homoserine lactones and lipopolysaccharides, key virulence factors in Gram-negative pathogens. Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-generation cephalosporin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and increasing the penetration and efficacy of standard-of-care antibiotics in combined administrations. The binding of homoserine lactones and lipopolysaccharides also sequesters their direct effects as toxins on eukatryotic membranes, neutralizing key tools that promote bacterial colonization and impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both existing antibiotic resistance mechanisms, as well as the build-up of rapid tolerance/resistance. The versatility of macrocyclic host-guest chemistry provides ample strategies for tailored targeting of virulence in a wide range of Gram-negative infectious diseases.

biochemistry↗

bioRxiv2023

Reconfigurable pH-Responsive DNA Origami Lattices

DNA nanotechnology enables straightforward fabrication of user-defined and nano-meter-precise templates for a cornucopia of different uses. To date, most of these DNA assemblies have been static, but dynamic structures are increasingly coming into view. The programmability of DNA not only allows encoding of the DNA object shape, but it may be equally used in defining the mechanism of action and the type of stimuli-responsiveness of the dynamic structures. However, these "robotic" features of DNA nanostructures are usually demonstrated for only small, discrete and device-like objects rather than for collectively behaving higher-order systems. Here, we show how a large-scale, two-dimensional (2D) and pH-responsive DNA origami -based lattice can be assembled on a mica substrate and further reversibly switched between two distinct states upon the pH change of the surrounding solution. The control over these two configurations is achieved by equipping the arms of the lattice-forming DNA origami units with "pH-latches" that form Hoogsteen-type triplexes at low pH. In a nutshell, we demonstrate how the electrostatic control over the adhesion and mobility of the DNA origami units on the surface can be used both in the large lattice formation (with the help of directed polymerization) and in the conformational switching of the whole lattice on the substrate. To further emphasize the feasibility of the method, we also demonstrate the formation of reconfigurable 2D gold nanoparticle lattices. We believe this work serves as an important milestone in bridging the nanometer-precise DNA origami templates and higher-order large-scale systems with the stimuli-induced dynamicity.

bioengineering↗

bioRxiv2022

DNA origami directed virus capsid polymorphism

Most known viruses protect their genome by encapsulating it inside a protein capsid. Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. The assembly process of native capsids is highly cooperative and governed by the protein geometry, protein-protein as well as protein-nucleic acid interactions. Importantly, the absolute control over the size and shape of virus capsids would have imminent advantages in the development of new vaccines and delivery systems. However, tools to direct the assembly process in a programmable manner are exceedingly elusive or strictly limited to specific structures. Here, we introduce a modular approach by demonstrating DNA origami directed polymorphism of single protein subunit capsids. We achieve control over the capsid shape, size, and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms for the capsid proteins. Binding assays and single-particle cryo-electron microscopy reconstruction show that the DNA origami nanoshapes are efficiently encapsulated within the capsid. Further, we observe that helical arrangement of hexameric capsomers is the preferred mode of packing, while a negative curvature of the origami structure is not well tolerated. The capsid proteins assemble on DNA origami in single or double layer configurations depending on the applied stoichiometry. In addition, the obtained viral capsid coatings are able to efficiently shield the encapsulated DNA origami from nuclease degradation and prevent the structures from aggregation. Therefore, these findings may in addition find direct implementations in DNA nanotechnology-based bioengineering by paving the way for the next-generation cargo protection and targeting strategies.

bioengineering↗

bioRxiv2020

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release

Doxorubicin (DOX) is a common drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of DOX-loaded DNA-carriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures (DONs). In our experimental conditions, all DONs show similar DOX binding capacities (one DOX molecule per two to three base pairs), and the binding equilibrium is reached within seconds, remarkably faster than previously acknowledged. To characterize drug release profiles, DON degradation and DOX release from the complexes upon DNase I digestion was studied. For the employed DONs, the relative doses (DOX molecules released per unit time) may vary by two orders of magnitude depending on the DON superstructure. In addition, we identify DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration. These features have been largely ignored in experimenting with DNA nanostructures, but are probably the major sources of the incoherence of the experimental results so far. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA-carriers.

biochemistry↗