Monitoring DNA conformational changes using an acoustic biosensor

Στη δουλειά αυτή παρουσιάζεται διεξοδικά η ικανότητα των ακουστικών βιοαισθητήρων να ανιχνεύουν μεταβολές που σχετίζονται με τη διαμόρφωση του ακινητοποιημένου πάνω στην επιφάνεια DNA. Ο τρόπος ακινητοποίησης, η εγγενής κυρτότητα, η δημιουργία τριπλών ελίκων αλλά και οι ηλεκτροστατικές αλληλεπιδράσεις με θετικά φορτισμένες πρωτεΐνες αποτέλεσαν τις πηγές προέλευσης αυτών των μεταβολών. Σε κάθε μία από τις περιπτώσεις αυτές οι αλλαγές στο πλάτος και στη φάση του ακουστικού κύματος συνδυάστηκαν για να περιγράψουν ποιοτικά αλλά και να συγκρίνουν ποσοτικά τις διαφορετικές διαμορφώσεις. Μάλιστα, η εφαρμογή της θεωρίας του ιξώδους για διαλύματα κατάφερε μέσω των ακουστικών μετρήσεων να ερμηνεύσει θεωρητικά και να ποσοτικοποιήσει, για πρώτη φορά, τις δομικές αλλαγές του DNA πάνω στην επιφάνεια της μικροσυσκευής. Η επιτυχημένη αυτή συλλογή δεδομένων οφείλεται, σε ένα βαθμό, στις μοναδικές ιδιότητες που διαθέτει το DNA συγκριτικά με τα άλλα πολυμερή αλλά και στην ύπαρξη μοριακών τεχνικών που επιτρέπουν τη δημιουργία μορίων με ελεγχόμενα μεγέθη και προκαθορισμένα χαρακτηριστικά.Duplex DNA can be analyzed by a variety of techniques in order to characterize DNA-protein interactions; parameters assessed during the binding interaction include intrinsic properties of the DNA, such as length and curvature, the sequence specificity of the proteins and charge effects. A primary goal of this work was to explore the ability to use acoustic biosensors for monitoring conformational changes of surface-attached biomolecules during binding interactions; a task that is challenging and of significance to biotechnology. A second goal was to use experimental data to test the validity of a novel viscosity-based theoretical model, which uses acoustic data to provide quantitative information related to DNA conformation and bending processes. In a first step, the parameters examined for surface-attached DNA in solution were the length of the molecules, their shape and their attachment mode. "Straight" ds-DNA molecules of various lengths, as well as "bent" and "triangle" DNA molecules were constructed and attached to the device surface; results clearly showed that the acoustic response could probe variations in the conformation of those molecules. Furthermore, the excellent agreement found between theoretical predictions and experimental data verified that the acoustic signal can be used as a measure of the intrinsic viscosity of the attached biomolecules at the device surface/liquid interface. A second set of experiments was performed in order to investigate nonspecific protein-DNA interactions. Electrostatic binding of a positively charged histone protein on the negatively charged backbone of the surface immobilized DNA was examined as a function of surface charge, DNA length, shape and surface coverage. Acoustic measurements reflected on the viscoelastic behavior of the adsorbed biomolecules suggesting the formation of a flat, collapsed DNA film, cross-linked with histone proteins. This explanation was further validated by AFM imaging of histone-DNA complex; AFM images verified the interpretation that non-specific charge interactions lead to the formation of rigid, compact biomolecular layers. Last but not least, sequence specific DNA bending was investigated as a third parameter that, in addition to intrinsic shape and charge, affects protein-DNA interactions. The method employed for that purpose was the oligonucleotidedirected triple helix formation, a general method for the sequence-specific recognition of double helical DNA. Using triple helix formation for DNA recognition in the major groove, a bifunctional oligonucleotide was designed to mimic a DNA bending protein. Acoustic measurements were again able to discriminate the various conformations adopted by the DNA molecules and were correlated to published values. The significance of these results is demonstrated by the fact that for the first time, acoustic data can be combined with viscosity theory to provide not just qualitative but quantitative information on the relatively complex phenomenon of induced DNA bending