KINETICS OF NUCLEIC ACIDS HYBRIDIZATION AND OF COMPLEX DNA STRUCTURES FORMATION ON A BIOSENSING SURFACE
Creators: name: VANJUR, LUKA
Publisher: name: Universit� degli Studi di Milano
Year: 2021

description: Use of DNA-based molecular probes on a biosensing surface enables unprecedented designs, with nely tuned responsive structures. Furthermore, cost and production of DNA, in addition to its overall ability to interact with various biological molecules, make DNA the ideal candidate for biosensing surface functionalization. However, all DNA based biosensing relies at some point on the pairing of to single stranded DNA molecules, namely DNA hybridization. I utilized a label-free, optical, multiplexed, biosensing platform to study probing capabilities of surface grafted DNA, from simple short sequences to highly ordered complex nanostructures. My PhD research started with the study of kinetics of simple oligo hybridization on surface. DNA hybridization on surface is usually treated under the well established Langmuir model, used to treat majority of surface adsorbtion phenomena. However, electrostatics of DNA phosphate backbone in addition to high denstiy of DNA monolayer on sensing surface, prevents this model to be applied in all it's potency. We observed strong suppression in binding kinetics even at concentrations below the KD. Moreover, this suppression correlated positively with DNA probe density, indicating a possible electrostatic in uence. Based on the electrostatic theory of DNA monolayers I developed a simple model accounting for the electrostatic penalty associated with entry of DNA molecule into charged DNA monolayer. Moreover, in addition to electrostatic repulsion hampering, steric eects arising from such high density areas also aect the overall sensitivity and rapidity of surface nucleic acids sensing with DNA. We proposed dierent strategies to combat this obstacles, including varying salt concentrations to counter electrostatic repulsion, grafting DNA probes on hydrogel, eectively reducing charge density. Use of multiple strands was also proposed considering the observed high anity towards DNA hybridization to partially double stranded probes, that provide further stacking stabilization, and decrease the dissociation events. DNA as a sensing probe was also successfully utilized in sensing micro-RNA (miRNA), a widely acknowledged and used biomarker for early-disease diagnostics. MiRNA molecules, like any other RNA molecule, readily hybridizes with DNA, forming a RNA/DNA hybrid, however, miRNAs specically are very scarcely and non-uniformly distributed in sera, blood and tissue, this inhibits the availability of miRNAs, despite their potential as biomarkers. We grafted various DNA strands with complementary sequences to 5 dierent known miRNAs. Our multiplexed assay was able to detect as low as 0.5 pM miRNA while yielding 30x fold mass amplication 90 minutes after the sample injection. Amplication was achieved by a specic antibody (Ab1) targeting DNA-RNA hybrids, polyclonal secondary antibody (Ab2) targeting primary antibody and careful system optimization. A simple numeric model was developed accounting the formation of DNA/RNA hybrids, Ab1 binding to hybrid and formation of Ab1-Ab2. Last interaction was treated as a competitive system, since the Ab pairs also tend to form in solution as well as on surface. System was optimized for amplication factor while keeping the miRNA concentration and total assay duration xed. The amplication signal was shown to be sequence dependent, since the kinetics of DNA/RNA hybridization depends on the sequence and predictive models are not available. DNA microarrays are often employed to study the binding and kinetics of various transcription factors. We investigated binding yeast gene regulator Gal4, on spots containing consensus and non-consensus sequence both in the form of simple DNA strand and DNA hairpin structure. Through the experimental observations, we found that the initial binding step is charge mediated and can be therefore ne tuned through ionic conditions. A Two-step nested well model was built, to explain these observations. First encounter is a non-specic interaction, followed by conformational adjustment until the consensus sequence is reached. Finally, potential of DNA to self-assemble into structure with higher complexity and perform specic functions was explored. Hybridization Chain Reaction (HCR) is nowadays well established isothermal reaction based on self-assembly properties of DNA. This nucleic acid triggered isothermal reaction shows great promise in biosensing applications, primarily as a means for enzyme-free signal amplication. We observed the formation of HCR laments in real time by grafting trigger DNA sequence on surface and releasing interacting hairpins in solution. Observed binding curves were cleary dierent from simple DNA hybridization, indicating multiple reaction occuring on the surface. We modelled this behaviour similarly to protein-DNA binding. Binding of rst hairpin is fast, due to oligo-like hybridization by the hairpin overhang. This recognition step is followed by slow hairpin opening which ends in exposition of overhang, or binding site for second hairpin. Two relaxation model thus accounts for rst hairpin binding to trigger strand on surface and to hairpin unzipping. Fluorescence confocal microscopy investigation was performed on HCR spots with dye-conjugated rst hairpin. Direct visual comparison with DNA monolayer shows dramatic dierence in intensity prole of the spot, which conrms the presence of HCR laments on surface. In the last part of the thesis work I explored the functionalization of the biosensor surface with large-scale a complex structure enabling control of probes with nanometer precision. Highly ordered DNA origami rectangles were developed and functionalized with sticky ends (tethers) for adsorbtion on DNA grafted surface. Investigation was carried out on the number of 40bp long sticky ends required to bring the structure to the surface, as well the kinetics based on the number of tethers. For this purpose, 2, 4 and 6 legged rectangles were produced, their stability and proper folding was veried directly via AFM. Observations of origami binding revealed: despite the large size and net charge of the origami, rectangles with at least 4 tethers complementary to DNA probes readily bind to the biosensor surface; number of tethers primarily aects the stability through lowering the dissociation rate, which was interpreted as probability of all available tethers being simultaneously detached - which directly correlates with number of tethers.