Biology subjects

Linko, V.

Publications and source records attributed to Linko, V..

6 recordsLinked to original sources

bioRxiv2026

DNA origami uptake in Y-79 retinoblastoma cells driven by oligolysine coating

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG-oligolysine block-copolymers like PEG5K-K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K-K10 coatings and PL3 targeting peptides to improve uptake of 24-helix bundle (24HB) DONs into Y-79 retinoblastoma cells. Uptake studies revealed that PEG5K-K10 was essential for DON uptake in Y-79 cells, as uptake only occurred upon exceeding a distinct PEG5K-K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K-K10 reduced cell viability at higher concentrations (IC50 36.8 {micro}M), coated DONs were well-tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K-K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous, and support further optimization of DONs for ocular drug delivery.

biochemistry↗

bioRxiv2024

Fluorescence Anisotropy Analysis of the Interaction between Doxorubicin and DNA Origami Nanostructures

Owing to doxorubicins high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing non-destructively the binding between DOX and DONs, and the purity of formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs > 0.5, homo-Forster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios > 1. For loading ratios > 0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios > 1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.

biochemistry↗

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

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↗