Regulation Of Nucleic Acids Self-assembly By Molecular Chirality And Its Application In Biosensors

Chirality is one of the fundamental biochemical properties in a living system. Most of the biological molecules (proteins, RNA, DNA, etc.) are composed of small chiral biomolecules (e.g., L-amino acids, D-sugars, L-phospholipids, etc.). As a result, a lot of biological and physiological processes were greatly influenced by the chirality of molecules. In order to reveal the mystery of chiral selectivity in living system and understand the essence of chiral phenomenon in nature. We regulated the nucleic acid self-assembly by coupling the chirality with nucleic acid self-assembly, and applied this result on biosensor. This result may open a new direction for developing new nano biomaterials and devices, and provide a new idea for clinical diagnostics. The main contents of the dissertation were as follows:(1) Taking thiolated methylene blue (MB) modified DNA as a model molecule, we investigated the nucleic acid self-assembly on N-isobutyryl-L(D)-cysteine (NIBC) modified gold surface. We showed that the peak current of the L-NIBC modified gold surface (L-surface) is larger than that of D-surface due to a stronger interaction between short-chain DNA and L-surface; however, D-surface has higher hybridization efficiency than L-surface. Moreover, we applied this result to actual application by choosing an electrochemical DNA (E-DNA) sensor as a potential platform. More importantly, our findings were successfully employed to program the sensitivity and limit of detection. Chirality was ubiquitous in living systems and very important in biochemical processes; thus, this result is significant for clinical diagnostics, gene therapies, and relevant studies. On the other hand, owing to the ubiquity of the interaction between biomolecules in the living world, this result can help us to better understand the origin of chiral preference in nature.(2) We regulated the construction of supersandwich and traditonal sandwich by combining the sandwich structure DNA and molecular chirality. The results showed that the D-NIBC modified supersandwich structure DNA has higher hybridization efficiency than L-NIBC modified one, and the limit of detection obtained from D-NIBC modified supersandwich is 1 pM, remarkblely lower than that on L-NIBC modified supersandwich by about 2 orders of magnitude. Supersandwich assay has an ability of amplifying the difference of hybridization efficiency even perform in 10%blood serum. In addition, the amplification ability of the supersandwich assay compared to the traditional sandwich assay can be finely edited by molecular chirality. This result showed that the chirality may be a potential method for regulating nucleic acid hybridization.(3) We demonstrated a stereochemistry guided SNPs detection by using electrochemical method. We observed that the signal gain of L-Trp incubated sensor is lower than the corresponding D-Trp treated one in the presence of perfectly matched (PM) target. However, the D-value of signal gain was not significant when sensors challenged with 1-mismatched (1-MM) target. We quantitated the specificity of the sensor by defining the ratio of D-value of signal gain in PM targets to that value in 1MM targets as discrimination factor (DF), and obtain the higher DF of 7.21. Furthermore, we further enhanced the specificity by using supersandwich assay, and the DF of SNPs was enlarges to 38.71. Supersandwich assay thus improved the sensitivity and enhances the specificity. We not only regulated the DNA hybridization through molecular chirality, but also obtained an outstanding specificity. Chirality is one of the fundamental biochemical properties in the living world, thus this result is helpful for developing new biodevices, and provide a useful method toward highly multiplexed clinical diagnostics at the point-of-cares.(4) We described universal strategies to engineer electrochemical aptamers-based sensors with dual programmable dynamic ranges. Using the adenosine triphosphate (ATP) binding aptamer as a model receptor, we designed a dual "signal-on" and "signal-off" electrochemical aptasensor that use a surface bound, methylene-blue labeled DNA strand complementary to a ferrocene-labeled ATP aptamer. We first showed that base mutations to the surface bound complementary DNA reduce the affinity and thus arbitrarily shift the dynamic range of this dual signaling sensor to lower ATP concentrations. We then showed that the dynamic range of these sensors can be extended by up to 6400-fold by using a combination of surface bound cDNA with varying affinity for the ferrocene-labeled ATP aptamer. Simultaneously, we can narrowed the dynamic range (to 10-fold) of this dual signaling sensor through a sequestration mechanism and the addition of a non-signaling, DNA sequence on the sensor head that display an even high affinity for the aptamers. Finally, by combining these strategies, we also engineered a sensor that simultaneously provides a "signal-off" readout with an extended dynamic range and ultrasensitive "signal-on" readout for high precision measurements. These strategies broaden the application of biosensors by providing sensors with both extended and highly precise dynamic ranges.