Research On The Evaluation Of Interaction Between Several Protoberberine Alkaloids And G-quadruplex

G-rich nucleic acid sequences are capable of forming four-stranded helicalstructures, called G-quadruplex. A diversity of G-quadruplex structures exist withdifferent G-tetrad cores and loop arrangements. Many biologically related nucleic acidsequences can fold into G-quadruplex structure, such as telomeres and promoterregions of many oncogenes, which may influence a wide range of biological activities,including apoptosis, cellular proliferation and tumorigenesis. The activity oftelomerase and the expression of oncogenes could be inhibited by the formation ofG-quadruplex. Hence, the ligands that could facilitate the formation and stabilizationof G-quadruplex structure have attracted more and more attention as a new class ofpotentially selective antitumor drugs. In particular, some synthetic short G-richoligonucleotides are found to have the ability to inhibit HIV-1replication in vitro.They can inhibit HIV1-IN activity at the nanomolar concentration level by formingthe G-quadruplex structure. The ligands that can bind to the folded G-quadruplexstructure is extremely important in HIV-1integrase inhibition strategy.Alkaloids, as one important class of small natural compounds, have significantbiological activities. Protoberberine alkaloids are isoquinoline alkaloid and the mostrepresentative one is berberine with antibacterial, analgesic and hypoglycemic effects.Recent studies have found that berberine is likely to play a role in the treatment ofcancer and AIDS.In this thesis, we have mainly selected four protoberberine alkaloids (berberine,jatrorrhizine, palmatine and tetrahydropalmatine) and investigated the interactionbetween alkaloids and a series of important G-quadruplex structures employing CDand ESI-MS. The main contents and results are shown as follows:1. The conditions of electrospray ionization mass spectrometry for detection ofG-quadruplex were optimized and the optimum Dry Gas temperature and CapillaryVoltage was2.5KV and200℃; The influence of pH on the G-quadruplex formationwas studied and the results indicated that a neutral pH was more conducive to theformation of G-quadruplex structures; The influence of methanol as auxiliary solventto aid desolvation of the sample was studied and the results indicated that addingmethanol will not affect the authenticity of our experimental results.2. The interaction of natural alkaloids with the telomere G-quadruplex was investigated. The results showed that berberine, jatrorrhizine and palmatine had goodaffinities and selectivities to telomere G-quadruplex, however, tetrahydropalmatinecan not interact with the telomeric G-quadruplex. Phosphorothioation telomericsequences did not have a great impact on the formation of G-quadruplex structure, butphosphorothioate modification could reduce the binding affinity of alkaloid towardtelomere G-quadruplex to a certain degree.3. The interaction of natural alkaloids with the G-quadruplex in the promoter ofoncogene was investigated. The results indicate that jatrorrhizine can induce Pu18andc-kit87up to form mixed-type and parallel G-quadruplex respectively; Chelerythrinecan induce Pu18and ckit87up to form parallel and antiparallel G-quadruplexrespectively. The addition of jatrorrhizine and chelerythrine can induce the switch ofPu18G-quadruplex in the presence of K+from mixed-type to anti-parallel and parallelrespectively. However, jatrorrhizine and chelerythrine can not change theconformation of c-kit87up G-quadruplex in the presence of K+. The affinityexperiments indicated that berberine, jatrorrhizine and palmatine showed goodbinding affinities toward all the five kinds of G-quadruplexes in the promoters ofoncogene. The selectivity experiments showed that berberine and palmatine had goodselectivities toward G-quadruplexes in the promoters of oncogene.4. The interaction of natural alkaloids with synthetic G-rich oligonucleotides wasinvestigated. The inducing results indicated that berberine and sanguinarine caninduce T30695and T30177to form parallel G-quadruplex. ESI-MS data indicatedthat the formation of intramolecular G-quadruplex was predominant at lowerconcentration of NH4OAc, while increasing the concentration to some degree allowedthe intramolecular G-quadruplex to completely transform to the dimeric G-quadruplex.The affinity experiments indicated that berberine, jatrorrhizine and palmatine allshowed good binding affinities toward intramolecular G-quadruplex and dimericG-quadruplex in addition to tetrahydropalmatine. And the binding affinities ofberberine, jatrorrhizine and palmatine toward dimeric G-quadruplex were higher thanintramolecular G-quadruplex.