Metal Ions-triggered DNA Folding&Controlling Metallo-toehold Mediated DNA Strand Displacement

Thymine-cytosine-rich DNA is a kind of strongly flexible biopolymer. It can specifically bind with Hg(Ⅱ) or Ag(Ⅰ) ions to generate metal-mediated base pairs (T-Hg-T and C-Ag-C) in hairpin-like structure from a random coil structure. Isothermal titration calorimetry experiments were performed to reveal the detail of whole binding process. The observed negative ΔH was favorable for the specific binding between the Hg(Ⅱ) ion and the T:T mismatched base pair, while negative AS values demonstrate that the oligonucleotide conformation changes from a random coil to a regular, stable hairpin when Hg(Ⅱ) ions are added. Moreover, we found that in the process of hairpin folding from random coil induced by external environment, DNA tended to make the link ring of hairpin a segment of four or five bases.Furthermore, we reported the mechanism of the formation of hairpin structure of thymine-cytosine-rich oligonucleotides induced by Hg(Ⅱ) and Ag(Ⅰ) ions. The study also confirmed and extended our understanding of the nature of metal-DNA adducts. Designed thymine-cytosine-rich oligonucleotides can significantly change in structure upon the addition of Hg(Ⅱ) ions, which converts the random coil single-strand to an anti-parallel hairpin-like folded structure. The ITC-derived thermodynamic parameters exhibited two possible pathways:one with the binding in the inner hairpin and another with the binding in the terminal hairpin. These two different binding pathways resulted in identical final products. Obvious results were observed when Ag(Ⅰ) ions were then added, confirming the existence of the spacing C-base loop of the hairpin-like formation of the Hg-DNA complex as well as the interactions between the Ag(Ⅰ) ions and the C:C mismatched base pairs that highly strengthened the structure. The result demonstrates that isothermal titration calorimetry is a powerful tool to study mechanism of DNA folding, induced by ions as well. Excellent agreement was found in coupled CD and UV measurements.DNA folding is a very complex process. The same ion, different order will make different results in DNA folding. Under the condition of silver ions, DNA had adopted a new found metallo-basepair, T-Ag-C, which is totally different from the traditional C-Ag-C pair. T-Ag-C formed more metallo-basepairs, which also blocked most of the T bases of the DNA. It greatly promoted the ability of silver ions in combination with DNA, but at the same time, reduced reactivity of DNA on mercury ions. The hairpin structure is not perfect but it is stable.In the last chapter, we present for the first time a new concept:metallo-toeholds. We have discovered that metal ions (Hg2+) that specifically interact with mismatched base pairs (T:T) can be employed with metallo-toeholds to intentionally trigger strand displacement in DNA devices. Using this concept, we have developed a mechanism that allows increased control over the kinetics of strand displacement. Using metal ions (Hg2+) as a regulatory factor, the metallo-toeholds allowed effective tuning of the rate of strand displacement. Through additional design of the sequence of the toeholds, we could also regulate the range of reaction rates. Mismatched base pairs between the toehold and displacement domains induced an obstacle between toehold binding and strand displacement, slowing down the reaction. The insertion of Hg2+ions provided the metallo-toeholds with perfect complementary, driving the strand displacement successfully. Too many Hg2+ions presumably led to blocking of the T sites on the toeholds, impeding the reaction. But the blocking system could be emancipated by dithiothreitol (DTT), which can sequestrate Hg2+ions in solution. Strand displacement has already been used as the basis for the operation of many synthetic molecular machines. We expect that this novel concept might be applicable to strategies for the design of molecular machines that function based on toehold-mediated strand displacement reactions in the presence of metal ions.