Recognition And Application Of Nucleic Acid Depending On Isoquinoline Alkaloid

Most diseases are related to genes, so the study of alkaloids interaction with DNA has caused great attention. Natural isoquinoline alkaloids, especially in the group of protoberberines and benzophenanthridines, are abundant in medicinal plant species and have important biological and therapeutic activities including antimicrobial, anti-inflammatory, anti-oxidation, and anticancer. Their activities are believed to partially originate from their binding with nucleic acids. Although the alkaloid structure-dependent interactions with DNAs (ds-DNA, abasic site DNA, G-quadruplex, triplex) and RNAs have been thoroughly investigated, no nucleic acid has been reported for a highly specific and tight binding to these biologically important isoquinoline alkaloids. Because these isoquinoline alkaloids (especially protoberberine groups) are almost non-fluorescent and share similar molecular weight and structural frame, a reliable method is necessary to identify DNA that can selectively target one of these isoquinoline alkaloids, especially by a label-free fluorescence technique. Based on this, the thesis choose isoquinoline alkaloids as fluorescent probes to identify human telomeric G-quadruplex, abasic site DNA, triplex DNA. The methods used in the experiment are rapid, sensitive, selective, and low background. The main contents include:1. Selective lighting up of epiberberine alkaloid fluorescence by fluorophore-switching aptamer and stoichiometric targeting of human telomeric DNA G-quadruplex multimerHuman telomeric G-quadruplex can serve as a potential fluorophore-switching aptamer to target natural isoquinoline alkaloids. We found that among the G-quadruplexes studied here and the various structurally-similar alkaloids, only the human telomeric DNA G-quadruplex, especially with a 5’-TA-3’residue at the 5’end of the G-quadruplex tetrad (5’-TAG3(TTAG3)3-3’, TA[Q]) as the minimal sequence, is the most efficient fluorophore-switching aptamer to selectively light up the epiberberine(EPI) fluorescence. Compared to the 5’end flanking sequences, the 3’end flanking sequences of the tetrad contribute significantly less to the recognition of EPI. The binding affinity of EPI to TA[Q] (with a disassociation constant of 37 nM) is at least 20 times tighter than those of the other alkaloids. The steady-state absorption, steady-state and time-resolved fluorescence, and NMR studies demonstrate that EPI most likely interact with the 5’end flanking sequences beyond the core [Q] and the G-quadruplex tetrad in a much more specific manner than the other alkaloids. The highly selective and tight binding of EPI with the fluorophore-switching aptamer and large fluorescence response upon DNA binding suggest the potential development of a selective EPI sensor. More importantly, EPI, as the brightest fluorophore-switching aptamer emitter among the alkaloids, can also serve as an efficient conformation probe for human telomeric DNA G-quadruplex and discriminate the DNA G-quadruplex from the RNA counterpart. Furthermore, EPI can bind stoichiometrically to each G-quadruplex unit of long telomeric DNA G-quadruplex multimer with the most significant fluorescence enhancement, suggesting the potential use of EPI as a bioimaging probe and a therapeutic DNA binder.2. Special recognition abasic DNA with isoquinoline alkaloidsIsoquinoline alkaloids can specifically recognize abasic site DNA(AP DNA). Large fluorescence response upon DNA binding can achieve recognition DNA with a label-free fluorescence technique. When the abasic site’s flanking bases is adenine, while the opposite base is cytosine, fluorescence enhancement of the system is obvious. Therefore, on the basis of placing an AP site opposite the target base, we can use isoquinoline alkaloids as fluorescent probes for developing a new method of cytosine detection.3. Recognition triplex DNA with isoquinoline alkaloidsIsoquinoline alkaloids can identify triplex DNA from double-stranded DNA, the formation of triplex DNA confirmed by Tm tests. Then we found that fluoresence response of chelerythrine (CHE) to triplex DNA is higher than to double-stranded DNA, and the background for CHE is low at the same time. Large fluorescence response upon DNA binding suggest specific binding of CHE with triplex DNA.