HIGH-RESOLUTION NUCLEIC ACID ANALYSIS WITH A DNA NANOTECHNOLOGY APPROACH

The goal of my research program is to develop a DNA-based nanosensor for nucleic acids analysis. I plan to use DNA Origami nanostructures that are formed by a-few-thousand-nucleotides-long, circular, single stranded (ss)DNA “scaffold” folded to form a specific shape by the action of a few hundreds of short (approx. 30 nucleotides) ssDNA “staples”, which hybridize over non-consecutive regions of the scaffold. Staples can be incorporated within the structure with well-defined stoichiometry and some of them can be designed to serve as highly-specific receptor for short nucleic acids sequences. I plan to introduce a restriction site within staples adjacent to such probes to permit their steric protection from enzymatic degradation as a consequence of a probe-target recognition event in their vicinity. The restriction reaction, therefore, “writes” the amount of target molecules captured on the nanosensors by permanently modifying certain target-specific staples within the DNA nanostructure. In turn, the amount of such modified staples is associated with the amount of target molecules captured on the nanosensor surface from the solution, and can be subsequently analyzed with standard DNA quantification techniques such as quantitative PCR (qPCR), or high-throughput DNA sequencing. This research plan is based on recent results obtained in our laboratory showing that, in self-assembled DNA structures, restriction enzymatic reactions are steric-regulated in a step-wise fashion. Therefore, one goal of this PhD thesis is the development of a quantitative assay to evaluate the efficiency of enzymatic reactions within such nanostructures, as well as staples incorporation and stability for aiding fundamental studies of enzymatic reactions in DNA nanostructures. Specifically, the restriction quantification method proposed is based on a linear PCR (L-PCR) amplification reaction that involves staple-specific carriers (70-nucleotide-long ssDNA) that can fully hybridize to staple fragments produced by the enzymatic cutting. The polymerase action leads to the formation of a duplex DNA fragment 70 base pairs (bp) long for each cleaved staple, whereas the hybridization of un-cleaved staples on the carrier prevents such polymerase reaction. To study staples incorporation efficiency, the same protocol can be used, but DNA carriers are designed to hybridize the full length of the DNA staple sequence. I prepared L-PCR samples to evaluate next-generation sequencing (NGS) quantification accuracy and L-PCR efficiency. As a proof-of-concept, I analyzed 5 staples of a triangular DNA nanostructure and obtained information on their incorporation efficiency or cleavage. This thesis also describes the design and development of a DNA based nanosensor for improving the accuracy of short nucleic acid quantification with qPCR. The work aims at coupling qPCR with a self-assembled nanosensor, which can help overcome amplification and retro-transcription reaction bias, and circumvent the detection threshold of 2-fold concentration variation, without requiring updates to traditional qPCR instrumentation. Components of such sensor are three consecutive “foot-loop” DNA probes each carrying a target-complementary sequence in the loop. Probes are assembled over a common scaffold that joins their “feet”. Each “foot” carries a restriction site and upon hybridization of three copies of the same target molecule on the respective loops, the site of each foot is destabilized (termed “bingo” configuration). Only in this case, the whole scaffold is protected from enzymatic cleavage and can be amplified with PCR. Target and bingo-scaffold concentrations are correlated by power function of 3. The results obtained demonstrate the increased accuracy of the Bingo-qPCR assay with respect to standard qPCR in evaluating small variation of enzymatic activity, and prove the feasibility of the target detection switch-based reaction.