Li-Ping Bai

Ligand Binding to Tandem G Quadruplexes from Human Telomeric DNA

Ligand-induced stabilization of intramolecular telomeric G quadruplexes produced in the single-stranded overhang of the human telomere has become an attractive strategy for the development of anticancer drugs. Several distinct solution conformations of human telomeric G quadruplexes have been elucidated in the presence of sodium and potassium cations. The K-form, hybrid-type G-quadruplex structure has been considered to be a physiologically relevant conformation of the human telomeric sequence, and thus, can be specifically targeted by G-quadruplex-interactive, small-molecule ACHTUNGTRENNUNGdrugs. Recently, a beads-on-a-string model was proposed for the telomeric overhang, in which every four consecutive Grich repeats adopt an individual G-quadruplex structure, and two G-quadruplex units are connected by one TTA linker. c, 4] Ligand binding to the G quadruplex has mostly been investigated on telomere sequences producing a single G quadruplex, but few studies of ligand binding to beads-on-a-string G quadruplexes have been reported. To gain insight into the beads-on-a-string model and the nature of ligand binding, we undertook the polymerase stop assay on human telomere sequences of three to eight repeats (Table S1 in the Supporting Information) with TMPyP4, a G-quadruplex-interactive ligand, and sanguinarine (Scheme 1), a natural isoquinoline alkaloid. The results described in this paper confirm the beads-on-astring structure of telomeric overhang and suggest a mode of ligand binding between tandem G-quadruplex beads. These observations should be taken into account for structure-based design of anticancer drugs targeting human telomeric DNA. Sanguinarine is a natural isoquinoline alkaloid isolated from the North American herb bloodroot (Sanguinaria canadensis). It was approved by the FDA in 2003 to be added to oral cleansing products as an antibacterial agent. Sanguinarine also possesses potent anticancer activity. We have previously reported its DNA-binding activity and distinct sequence selectivity to double-stranded DNA, which was proposed to be one of the molecular mechanisms of its anticancer activity. The structural similarity of sanguinarine with berberine, another isoquinoline alkaloid possessing G-quadruplex-binding activity, prompted us to speculate that sanguinarine is probably a G-quadruplex binder. In this communication, we report its binding to the ACHTUNGTRENNUNGtelomeric overhang using DNA polymerase stop assays. DNA templates Tem-3 and Tem-4 (Table S1), which contains three and four human telomeric repeats d ACHTUNGTRENNUNG(TTAGGG), respectively, were employed, and TMPyP4 was used as the reference compound in the assay. Neither TMPyP4 nor sanguinarine blocked DNA synthesis on Tem-3, because an intramolecular Gquadruplex structure could not form with three human telomeric repeats on Tem-3 (Figure 1). In contrast, both sanguinarine and TMPyP4 produced paused bands in DNA synthesis on Tem-4. The position of the paused bands was the same for the two ligands. For each ligand, a series of concentration-dependent paused bands appeared at the beginning of the G-quadruplex-forming site, that is, the first site of G-rich repeats in Tem-4 (from 3’ to 5’). In the presence of 3 mm TMPyP4, the polymerase reaction was totally suppressed to give no elongation of the primer. The tight binding of sanguinarine and TMPyP4 to the K-form hybrid-type G-quadruplex structure was clearly indicated from the large increase in the melting temperature (DTm) of dAGGG ACHTUNGTRENNUNG(TTAGGG)3 (11.4 8C for sanguinarine and 9.5 8C for TMPyP4 in a 2:1 ligand/DNA molar ratio). ACHTUNGTRENNUNGFurthermore, a broad negative induced band at 351 nm in the CD spectra confirmed the interaction between sanguinarine and the K-form G quadruplex formed by dAGGG ACHTUNGTRENNUNG(TTAGGG)3 (Figure 2). Regarding the mode of ligand binding to the G quadruplex, NMR, 10a,11] fibre diffraction, and single-crystal X-ray crystallographic analyses have confirmed that a variety of planar aromatic binders including TMPyP4 interact with the G quadruplex by end stacking rather than intercalation between G tetrads. TMPyP4 was also found to stack onto the external loop of a bimolecular G quadruplex in a crystal structure. Recently, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) has been used to analyze complexes of G quadruplexes with ligands, since G quadruplexes can maintain their structure in the gas phase in the presence of a suitable cation such as NH4 + . ESI-MS showed the ammonium ion to be loScheme 1. Chemical structures of sanguinarine and TMPyP4.

Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA

AbstractSpectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. FigureAssociation constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis

Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes

A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K+-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na+-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrines binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K+-form human telomeric DNA G-quadruplex over the Na+-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands.

Authentication of the 31 species of toxic and potent Chinese Materia Medica by light microscopy, Part 3: two species of T/PCMM from flowers and their common adulterants.

Toxic and potent Chinese Materia Medica (T/PCMM) has become a hot and sensitive topic as more and more people around the world are interested in the safety of herbal medicines. T/PCMM is irreplaceable in treating some diseases; but it can easily cause serious problems if confused with other herbal medicines. Accurate identification is essential to ensure their safe use, but up to now, the literature on the authentication of T/PCMM is scant. Thus, we are undertaking a study of 31 T/PCMM originating from plants, animals, minerals, and secreta. Our previous study established microscopic observation as a simple, fast, accurate, and convenient method for identifying and authenticating animal and seed T/PCMM. This study focused on the authentication of flower T/PCMM as a part of the whole study. The flower T/PCMM studies were derived from two species, Datura metel L. (Flos Daturae) and Rhododendron molle G. Don (Flos Rhododendri Mollis). Other species easily confused with these two were also examined and characterized. Using the microscope camera, normal light and polarized light microscopy, we determined the macroscopic and microscopic features of the flowers; in addition, the oil immersion lens was used to study the pollen grain characteristics. The results demonstrated that flower T/PCMM can be identified and authenticated using a light microscope equipped with an oil immersion lens. This same equipment can be easily used to characterize other herbal flower medicines. Microsc. Res. Tech., 2009.

Authentication of the 31 species of Toxic and Potent Chinese Materia Medica (T/PCMM) by microscopic technique, part 2: Three species of seed T/PCMM.

Toxic and Potent Chinese Materia Medica (T/PCMM) are being used more and more in the treatment of various diseases. In view of their toxic side effects and to ensure their safe use, accurate and reliable authentication is indispensable. However, identifying characteristics of T/PCMM are seldom reported, even though modern microscopy can provide ample, unique identifying characteristics from cells found in transverse sections and powders. In particular, no systematic authentication studies on seed T/PCMM have been conducted. In the course of our study on 31 T/PCMM originating from plants, animals, minerals, and secreta, an accurate and convenient method, based on microscopic techniques, has been developed and reported for the authentication of animal T/PCMM. The present study deals with detailed investigations on three species of seed T/PCMM, namely Semen Hyoscyami (Hyoscyamus niger L.), Semen Euphorbiae (Euphorbia lathyris L.), and Semen Strychni (Strychnos nux‐vomica L.). The macroscopic characters are here described in detail, and the microscopic characters were conclusively determined by common and polarized light microscopy. Results showed that these three T/PCMM can be easily identified by the present method even when powdered and combined. Thus, the microscopic method is applicable for authentication of the earlier three T/PCMM, and the morphological and microscopic characteristics described here are proposed as parameters to establish the authenticity of these three T/PCMM. Microsc. Res. Tech., 2008.

A Ligand That Targets CUG Trinucleotide Repeats

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9-diaminoalkyl-substituted 1,10-phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (Tm ) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time-of-flight (ESI-TOF) mass spectrometry showed that DAP bound to r(CUG)9 but not to r(CAG)9 and r(CGG)9 . The dual luciferase assay clearly indicated DAP bound to the r(CUG)n repeat by affecting the translation in vitro.

Naphthyridine-Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA.

The expansion of CAG repeats in the human genome causes the neurological disorder Huntingtons disease. The small-molecule naphthyridine-azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA-binding to the CAG repeat DNA and RNA, we conducted systematic structure-binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA, modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)9 and r(CAG)9 . NBzA binding to d(CAG)9 was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time-of-flight (ESI-TOF) mass spectrometry. For the binding to r(CAG)9 , both NA and NBzA showed stepwise binding in ESI-TOF MS, and NBzA-binding to r(CAG)9 induced more extensive conformational change than NA-binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.