Double-chain Reversal Loops Research Articles

Abstract 5225: Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter

Abstract The human BCL-2 gene contains a 39-bp GC-rich region upstream of its P1 promoter which has been shown to be critical for BCL-2 transcriptional regulation. Therapeutic inhibition of BCL-2 expression can reduce cell proliferation and the apoptotic threshold of tumorigenic cells. Previously, we have reported that the major G-quadruplex (G4) formed in the Pu39 G-rich sequence adopts a stable, parallel-stranded structure with two 1-nt short loops and a unique 13-nt long central loop. Parallel G-quadruplexes have been found to be prevalent in promoter sequences; the 13-nt loop in BCL-2 Pu39G4 is the longest in this prototype parallel G-quadruplexes identified in human gene promoters. The structure of this G4 with such a long loop is important because it can enable specific small molecule recognition of this BCL-2 G-quadruplex over other G-quadruplexes and provide a potential basis for rational drug design targeting BCL-2 transcription. Thus, the objective of our study is to determine the molecular structure of the BCL-2 Pu39 G-quadruplex and the molecular interactions of this long loop in physiologically relevant K+ solution by Nuclear Magnetic Resonance (NMR) spectroscopy. Our NMR study shows that the BCL-2 Pu39G4 adopts a well-defined parallel-stranded G-quadruplex structure which contains three G-tetrads stabilized by Hoogsteen hydrogen bonding and three double-chain reversal loops containing 1:13:1 bases. The two 1-nt double-chain reversal loops on two edges of the tetrad provide overall stability of the BCL-2 Pu39G4. The central 13-nt double-chain reversal long loop is more dynamic in conformation. Specific interactions of the long loop are observed at both the 5’ and 3’ end with external tetrads as well as the flanking residues, which may also contribute the stable formation of this G4. The middle region of the 13-nt central long loop is highly dynamic and doesn’t show much interaction with the G-quadruplex core. The specific molecular interactions of the unique long central loop are specific to the BCL-2 Pu39G4 sequence/structure and significantly differ from those in other parallel-stranded structures, e.g. c-myc and VEGF. The specific interactions are supported by our mutational analyses. The results are also supported by the DMS footprinting results which show that the guanines involved in the tetrad formation in this major G-quadruplex are well-protected against DMS methylation. Therefore, the knowledge about the molecular structure of this non B-DNA conformation of the BCL-2 promoter region is essential for using the G-quadruplex as a target for anticancer drugs, which could be a novel approach to anti-BCL-2 drug discovery in cancer therapy. Citation Format: Guanhui Wu, Clement Lin, Danzhou Yang. Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2017-5225

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Interactions of two cytotoxic organoruthenium(II) complexes with G-quadruplex

Two previously isolated cytotoxic complexes [(η(6)-p-cymene)Ru(κ(2)-CF3COCHCOC5H3O)L](n+) (L=Cl (1); n=0, pta (2) (pta=1,3,5-triaza-7-phosphaadamantane); n=1) were investigated for their selectivity and ability to interact with DNA G-quadruplex adopted by d[G3ATG3ACACAG4ACG3] (3) whose topology exhibits diagonal, edge-type and double-chain reversal loops. Structural changes were followed using high-resolution NMR techniques in the presence of 1 and 2. Results showed weak interaction between the organoruthenium complexes and G-quadruplex. Moreover, no significant changes in thermal stability were confirmed by a UV-melting assay for both 1 and 2. These findings emphasize that anticancer activity of Ru(II) complexes may not be correlated with binding to nucleic acid like G-quadruplex.

Single-molecule analysis of thymine dimer-containing G-quadruplexes formed from the human telomere sequence.

The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5′-TTAGGG-3′ human telomere sequence can fold into G-quadruplexes that adopt the hybrid, basket, or propeller fold. The telomere sequence is hypersensitive to UV-induced thymine dimer (T=T) formation, yet it does not cause telomere shortening. In this work, the potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand why this damage is tolerated in telomeres. First, established methods, such as thermal melting measurements, electrophoretic mobility shift assays, and circular dichroism spectroscopy, were utilized to determine the effects of the damage on these structures. Second, a single-molecule ion channel recording technique using α-hemolysin (α-HL) was employed to examine further the structural differences between the damaged sequences. It was observed that the damage caused slightly lower thermal stabilities and subtle changes in the circular dichroism spectra for hybrid and basket folds. The α-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are tolerated in edgewise and diagonal loops. The largest change was observed for the T=T-containing natural telomere sequence when the propeller fold (all double-chain reversal loops) was studied. On the basis of the α-HL experiments, it was determined that a triplexlike structure exists under conditions that favor a propeller structure. The biological significance of these observations is discussed.

RecA-Binding pilE G4 Sequence Essential for Pilin Antigenic Variation Forms Monomeric and 5′ End-Stacked Dimeric Parallel G-Quadruplexes

Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence(5'-G(3)TG(3)TTG(3)TG(3)) located upstream of the N.gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system. We have determined NMR-based structures of the all parallel-stranded monomeric and5' end-stacked dimeric pilE G-quadruplexes in monovalent cation-containing solutions. We demonstrate that the three-layered all parallel-stranded monomeric pilE G-quadruplex containing single-residue double-chain reversal loops, which can be modeled without steric clashes into the 3 nt DNA-binding site of RecA, binds and promotes E.coli RecA-mediated strand exchange invitro. We discuss how interactions between RecA and monomeric pilE G-quadruplex could facilitate the specialized recombination reactions leading to pilin diversification.

Solution structure of all parallel G-quadruplex formed by the oncogene RET promoter sequence.

RET protein functions as a receptor-type tyrosine kinase and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the promoter plays an important role in the transcriptional regulation of RET. Here, we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. The overall G-quadruplex is composed of three stacked G-tetrad and four syn guanines, which shows distinct features for all parallel-stranded folding topology. The core structure contains one G-tetrad with all syn guanines and two other with all anti-guanines. There are three double-chain reversal loops: the first and the third loops are made of 3 nt G-C-G segments, while the second one contains only 1 nt C10. These loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure and their conformations are in accord with the experimental mutations. The distinct RET promoter G-quadruplex structure suggests that it can be specifically involved in gene regulation and can be an attractive target for pathway-specific drug design.

Abstract 5134: G-Quadruplex structure formation in the proximal promoter region of the human vascular endothelial growth factor gene

Abstract The human vascular endothelial growth factor gene, VEGF, is a key regulator of angiogenesis. It plays an important role in tumor survival, growth and metastasis. It is over-expressed in many types of human cancers including glioma and renal cell carcinoma, ovarian and pancreatic cell carcinomas. The VEGF proximal promoter region contains a poly G/poly C rich tract that is essential for basal and inducible VEGF expression. The guanine-rich (G-rich) strand on this tract is shown to form a specific secondary structure, the G-quadruplex. Here we have elucidated the solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution by proton Nuclear Magnetic Resonance (NMR) spectroscopy followed by the structural calculation of the G-quadruplex using Distance Geometry Simulated Annealing (DGSA) module of XPLOR-NIH software. It is inferred that this strand adopts a well-defined parallel-stranded G-quadruplex structure, which contains three G-tetrads stabilized by Hoogsteen hydrogen bonding, as well as three double-chain reversal loops containing 1:4:1 bases, a feature important for the formation of the most thermodynamically stable conformation. The size of the 4-base loop plays a critical role in determining the most favored folded pattern of a G-quadruplex. These 4-base loop residues interact with the residues of the 3’ flanking region of VEGF sequence; also, the 1-base double-chain reversal loop on two edges of the tetrad are very stable, which overall contribute in stabilizing the 1:4:1 conformer. We have found that the two cytosines in this central 4-base loop are involved in hydrogen bonding with the 3’ terminal end residues of VEGF, which is a salient feature as this interaction is sequence-specific and differs from that in other parallel-stranded structures, e.g. c-myc, significantly. These results are also supported by the DMS footprinting results by Guo et al. (2008) which show that the guanines involved in the tetrad formation in this major G-quadruplex loop isomer 1:4:1 are well-protected against DMS methylation. Therefore, the knowledge about the secondary structure of this non B-DNA conformation of the VEGF promoter region is essential for using the G- quadruplex as a target for anticancer drugs, which could be a novel approach to anti-angiogenesis drug discovery in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5134. doi:10.1158/1538-7445.AM2011-5134

Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene

A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br2 footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C–C+ base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations.

Sequence effects of single base loops in intramolecular quadruplex DNA

We have examined the properties of intramolecular G-quadruplexes in which the G3 tracts are separated by single base loops. The most stable complex contained 1′,2′-dideoxyribose in all three loops, while loops containing T and C were slightly less stable (by about 2 °C). Quadruplexes containing loops with single A residues were less stable by 8 °C for each T to A substitution. These folded sequences display similar CD spectra, which are consistent with the formation of parallel stranded complexes with double-chain reversal loops. These results demonstrate that loop sequence, and not just length, affects quadruplex stability.

Structure of the Biologically Relevant G-Quadruplex in The c-MYC Promoter

The nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter controls up to 80–90% of the transcriptional activity of this gene. We have demonstrated that the guanine-rich strand of the NHE III1 forms a G-quadruplex consisting of a mixture of four biologically relevant loop isomers that function as a silencer element. NMR studies have shown that these G-quadruplexes are propeller-type parallel structures consisting of three stacked G-tetrads and three double-chain reversal loops. An NMR-derived solution structure for this quadruplex provides insight into the unusual stability of the structure. This structure is a target for small molecule inhibitors of c-MYC gene expression.

A Dimeric RNA Quadruplex Architecture Comprised of Two G:G(:A):G:G(:A) Hexads, G:G:G:G Tetrads and UUUU Loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.