The Design Of Biosensors For Nucleic Acid, Lead Ion And Mercury Ion Based On G-quadruplex

G-quadruplex is a kind of special structure DNA formed by G rich nucleic acid sequences. It possesses fluorescence characteristics when combining with relevant materials, so it has been applied to the design of a variety of biosensors, such as the detection for drugs related with life activities, nucleic acids, proteins, enzymes, heavy metal ions and small molecules. In this work, we have designed three kinds of biosensors to detect DNA, Pb2+ and Hg2+ respectively based on G-quadruplex with protoporphyrin IX(PPIX) as a fluorescence signal reporter. This thesis is divided into thtee parts:Part 1, we have designed a label-free hairpin structure of oligonucleotide probe, which mainly consists of three parts: stem portion, loop portion(specifically bind with target DNA) and G-rich area. In the absence of target DNA(T-DNA), probe DNA forms hairpin structure. In the meanwhile, part of G-rich bases is caged in the stem-loop structure and thus G-rich bases cannot form into G-quadruplex structure, so there is no fluorescence signal. In the presence of T-DNA, the hairpin structure is open and releases the G-rich area to fold into G-quadruplex structure through complementary base pairing with T-DNA, thus forming G-quadruplex-PPIX complex with strong fluorescence signal to achieve the goal of detection of target DNA. The limit of detection is 3.5 nM. In addition, when the target DNA has single, two or three base mutation, this method can also be fit for detection easily.Part 2, the sequences as same as G-rich region of DNA that used in the detection of DNA are employed. In the absence of Pb2+, G-quadruplex-PPIX complex has strong fluorescence signal. On addition of Pb2+, Pb2+ competitively binds to K+-stabilized G-quadruplex structure due to higher efficiency at stabilizing G-quadruplex than K+, resulting in structure change, release of PPIX combined with G-quadruplex and fluorescence intensity decrease, thus achieving the goal of detection of Pb2+ ultimately. This method has good sensitivity and specificity with detection limit of 2.6 nM.Part 3, we have designed two label-free DNA sequences consisted of G-rich sequences and three T-base mismatch area. In the absence of Hg2+, two oligonucleotide chains form unstable mismatch structure, and G-rich sequences is difficult to form G-quadruplex, so the fluorescent signal is very weak. With the existence of Hg2+, the two oligonucleotides hybridize to each other to form a duplex, in which T-T mismatches are stabilized by T-Hg2+-T base pair. As a result, the G-rich sequences of the two oligonucleotides can associate to form a split G-quadruplex, which is able to bind PPIX to form G-quadruplex-PPIX complex with strong fluorescence signal. The "turn-on" method(signal increases with increasing concentration of Hg2+) can avoid the false positive results in detection, and then be more reliable comparing with general "turn-off" ways(signal decreases with increasing concentration of Hg2+). In addition, this method with no chlorinated hemoglobin(Hemin) and H2O2 as co-factors eliminates the complex procedure to make the operation simpler. The detection limit of this method is 4.6 nM.