Functional Fluorescent Nucleic Acid Probes For The Detection Of Adenosine And PH

Adenosine is a purine nucleoside comprising a molecule of adenine attached to aribose sugar molecule moiety via a glycosidic bond, it plays an important role inbiochemical processes. Intracellular pH homeostasis is also crucial in variousbiological processes, disorder of intracellular pH can also affect human physiology.So, detetion with high sensitivity of adenosine and intracellular pH values is of greatimportance.Functional nucleic acids (FNAs) are nucleic acids whose functions are beyondthe conventional genetic roles, including aptamers, nucleic acid enzyms (NAEs) andaptazymes. In this research paper, we construct three FNA-based fluorescence probesto detect adenosine and intracellular pH, the contents of the thesis are as follows:1. Combining the elements of split aptazyme and catalytical molecularbeacon(CMB), we developed a versatile sensing strategy for amplified detection ofbiotarget adenosine. The aptazyme, composed with8-17DNAzyme andadenosine-specific aptamer, is cut into two separate fragments. In the absence oftarget adenosine, the two split aptazyme are inactive to the CMB substrate, thusproviding a much lower background. However, the presence of target adenosine canbind the two split aptazyme to form a “sandwich” complex, which activates thecleavage activity of the aptazyme. The CMB substrate thus can be cleaved with thehelp of the cofactor metal ions, causing the quenched CMB fluorophore/quencher pairto be separated, thereby producing a dramatic increase of fluorescent signal. Theresults showed that the detection method has a dynamic range from1M to1mM,with a detection limit of1mol/L.2. Combining the inhibited aptazyme and catalytical molecular beacon, wedeveloped a versatile sensing strategy for amplified detection of adenosine. In thisstrategy, the adenosine aptamer links to the8-17DNAzyme to form an aptazyme. Ashort sequence, denoted as inhibitor, is designed to form a duplex spanning theaptamer–DNAzyme junction, which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine, the aptamer binds to aden osine, thus theinhibitor dissociates from the aptamer portion of the aptazyme and can no longer formthe stable duplex required to inhibit the catalytic activity of the aptazyme. Thereleased DNAzyme domain will hybridize to the CMB and catalyze the cleavage inthe presence of Zn2+, making the fluorophore separate from the quencher and resulting in fluorescence signal. The results showed that the detection method has a dynamicrange from10nM to1mM, with a detection limit of10nmol/L.3. Combining i-motif and gold nanoparticle (AuNP), we developed a nanoprobeto monitor pH changes in Hela cells. In our study, fluorophore-labeled sequence bindsto the i-motif sequence that is attached to the surface of AuNP. In the neutral pH state,the fluorescence of the probe is quenched by the AuNP. When the pH decreased, theconformational change of the i-motif disrupts the Watson-Crick base-pairing betweeni-motif and fluorophore-labeled sequence, which causes fluorophore-labeled sequenceto be liberated with an increase in the fluorescence due to the great distance of thefluorophore from the AuNP. These nanoprobes are readily taken by cells, thus thesignal intensity can be used to sense intracellular pH change. Additionally, the resultsshowed that the nanoprobes, which have a dynamic range from pH5.0to pH7.5,resist nuclease degradation.