Major G-quadruplex Research Articles

Abstract 5775: Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation

Abstract Overexpression of the receptor tyrosine kinase PDGFR-β promotes cancer growth and metastasis. PDGFR-β is a validated cancer target but PDGFR-β kinase inhibitors suffer from lack of selectivity and side effects. G-quadruplexes are an exciting class of non-B DNA secondary structures with functional importance. The G-quadruplex formed in the PDGFR-β promoter is a transcription repressor and targetable by small molecules. Therefore, the PDGFR-β promoter G-quadruplex is an attractive molecular target for anticancer drugs. Structural information of the PDGFR-β promoter G-quadruplex is crucial for understanding its cellular function and rational drug design. Herein, we used nuclear magnetic resonance (NMR) spectroscopy and biophysical methods to determine the structure and dynamics of the major G-quadruplex formed in the human PDGFR-β promoter. Unlike canonical G-quadruplexes formed by four runs of three continuous guanines (G), the major PDGFR-β G-quadruplex adopts a novel broken-strand structure where a two-G segment is continued by a non-adjacent guanine. The broken-strand G-quadruplex has a vacancy-G-tetrad (vG4) which is filled-in intramolecularly by a distal guanine. Interestingly, we discovered that the major PDGFR-β G-quadruplex is a novel mixture of two equilibrating broken-strand G-quadruplexes. Both structures adopt overall parallel-stranded folding with all 1-nt chain-reversal loops. However, a different 3’-filled-in guanine is delivered by a lateral loop, which forms structurally similar hairpin with a unique capping G-G base-pair. The formation of the equilibrating broken-strand G-quadruplexes and unique capping structures are both specific to the PDGFR-β promoter sequence. Intriguingly, the observed dynamic equilibrium between the two co-existing structures with different intramolecular fill-in guanines indicates a novel vG4 intermediate, which could be filled-in by cellular guanine metabolites such as cGMP. Therefore, our study provides critical insights into how the dynamics of the major PDGFR-β G-quadruplex lead to vG4 formation and potential regulation of gene transcription by cellular guanine metabolites. The determined molecular structures of the novel broken-strand G-quadruplexes provide a structural basis for rational design of small molecules to specifically target the major PDGFR-β promoter G-quadruplex for cancer therapeutics. Citation Format: Yichen Han, Jonathan Dickerhoff, Danzhou Yang. Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5775.

Structure of the Major G-Quadruplex in the Human EGFR Oncogene Promoter Adopts a Unique Folding Topology with a Distinctive Snap-Back Loop.

EGFR tyrosine kinase inhibitors have made remarkable success in targeted cancer therapy. However, therapeutic resistance inevitably occurred and EGFR-targeting therapy has been demonstrated to have limited efficacy or utility in glioblastoma, colorectal cancer, and hepatocellular carcinoma. Therefore, there is a high demand for the development of new targets to inhibit EGFR signaling. Herein, we found that the EGFR oncogene proximal promoter sequence forms a unique type of snap-back loop containing G-quadruplex (G4), which can be targeted by small molecules. For the first time, we determined the NMR solution structure of this snap-back EGFR-G4, a three-tetrad-core, parallel-stranded G4 with naturally occurring flanking residues at both the 5'-end and 3'-end. The snap-back loop located at the 3'-end region forms a stable capping structure through two stacked G-triads connected by multiple potential hydrogen bonds. Notably, the flanking residues are consistently absent in reported snap-back G4s, raising the question of whether such structures truly exist under in vivo conditions. The resolved EGFR-G4 structure has eliminated the doubt and showed distinct structural features that distinguish it from the previously reported snap-back G4s, which lack the flanking residues. Furthermore, we found that the snap-back EGFR-G4 structure is highly stable and can form on an elongated DNA template to inhibit DNA polymerase. The unprecedented high-resolution EGFR-G4 structure has thus contributed a promising molecular target for developing alternative EGFR signaling inhibitors in cancer therapeutics. Meanwhile, the two stacked triads may provide an attractive site for specific small-molecule targeting.

DNA G-Quadruplex in Human Telomeres and Oncogene Promoters: Structures, Functions, and Small Molecule Targeting.

DNA G-quadruplex secondary structures formed in guanine-rich human telomeres and oncogene promoters are functionally important and have emerged as a promising new class of cancer-specific drug targets. These globular intramolecular structures are stabilized by K+ or Na+ and form readily under physiological solution conditions. Moreover, G-quadruplexes are epigenetic features and can alter chromatin structure and function together with interactive proteins. Here, we discuss our efforts over the last two decades to understand the structures and functions of DNA G-quadruplexes formed in key oncogene promoters and human telomeres and their interactions with small molecules. Using high-field NMR spectroscopy, we determined the high-resolution structures of physiologically relevant telomeric G-quadruplexes in K+ solution with a major form (hybrid-2) and a minor form (hybrid-1), as well as a two-tetrad intermediate. The intrinsic structural polymorphism of telomeric DNA may be important for the biology of human telomeres, and we proposed a model for the interconversion. More recently, we have worked on G-quadruplexes of MYC, BCL2, PDGFR-β, VEGF, and k-RAS oncogene promoters. We determined the structure of the major G-quadruplex formed in the MYC promoter, a prototype for parallel G-quadruplexes. It is the first example of the parallel-stranded G3NG3 structure motif with a 1-nt loop, which is prevalent in promoter sequences and likely evolutionarily selected to initiate folding. Remarkably, the parallel MYC promoter G-quadruplexes are highly stable. Additionally, we determined the molecular structures of G-quadruplexes formed in human BCL2, VEGF, and PDGFR-β promoters, each adopting a unique structure. For example, the BCL2 promoter contains distinct interchangeable G-quadruplexes in two adjacent regions, suggesting precise regulation by different proteins. The PDGFR-β promoter adopts unique "broken-strand" and vacancy G-quadruplexes, which can be recognized by cellular guanine metabolites for a potential regulatory role.Structural information on G-quadruplexes in complex with small-molecules is critical for understanding specific recognition and structure-based rational drug design. Our studies show that many G-quadruplexes contain unique structural features such as capping and loop structures, allowing specific recognition by drugs and protein. This represents a paradigm shift in understanding DNA as a drug target: Rather than a uniform, nonselective binding site in duplex DNA, the G-quadruplex is being pursued as a new class of selectively targetable drug receptors. We focus on targeting the biologically relevant MYC promoter G-quadruplex (MycG4) with small molecules and have determined its first and additional drug complex structures. Very recently, we have discovered clinically tested indenoisoquinolines as strong MycG4 binders and potent MYC inhibitors. We have also discovered drugs targeting the unique dGMP-bound-vG4 formed in the PDGFR-β promoter. Moreover, we determined the complex structures of the first small molecules that specifically recognize the physiologically relevant human telomeric G-quadruplexes. Unlike the previously recognized dogma that the optimal G-quadruplex ligands are large aromatic or cyclic compounds, our results suggest that smaller asymmetric compounds with appropriate functional groups are better choices to specifically bind G-quadruplexes. This body of work lays a strong foundation for future work aimed at understanding the cellular functions of G-quadruplexes and G-quadruplex-targeted drug design.

Berberine Molecular Recognition of the Parallel MYC G-Quadruplex in Solution.

The medicinal natural product berberine is one of the most actively studied and pursued G-quadruplex (G4)-ligands. The major G-quadruplex formed in the promoter region of the MYC oncogene (MycG4) is an attractive drug target and a prominent example and model structure for parallel G-quadruplexes. G4-targeted berberine derivatives have been actively developed; however, the analogue design was based on a previous crystal structure in which berberine binds as a dimer to a parallel G-quadruplex. Herein, we show that in solution, the binding mode and stoichiometry of berberine are substantially different from the crystal structure: berberine binds as a monomer to MycG4 using a base-recruitment mechanism with a reversed orientation in that the positively charged convex side is actually positioned above the tetrad center. Our structure provides a physiologically relevant basis for the future structure-based rational design of G4-targeted berberine derivatives, and this study demonstrates that it is crucial to validate the ligand-DNA interactions.

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78–7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

Abstract 687: A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design

Abstract Platelet-derived growth factor receptor beta (PDGFR-β) is a cell-surface-receptor tyrosine kinase implicated in platelet-derived growth factor (PDGF) signaling pathways, and overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for cancer therapy. The formation of DNA G-quadruplexes in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter has been found to inhibit PDGFR-β transcriptional activity. We previously determined the major G-quadruplex structure formed in the PDGFR-β promoter using a 22-mer sequence of pu22m1, which adopts a folding pattern containing a unique broken G-strand with the 3' tetrad-guanine coming from a distant site connected by a five nucleotide lateral loop. However, extension of the 3' end flanking segment of pu22m1 results in destabilization of the G-quadruplex, suggesting that this structure may be unstable in vivo. Recently, it has been reported that a unique type of intramolecular G-vacancy-bearing G-quadruplex (GVBQ) can be formed with three G3 tracts and one G2 tract and has an incomplete G-tetrad with a G-vacancy. A guanine derivative, such as GMP or GTP, can fill the G-vacancy with Hoogsteen hydrogen bonding and complete the G-tetrad, resulting in G-quadruplex stabilization. The unique broken-strand structure of PDGFR-β pu22m1 suggests a possibility of the formation of a GVBQ structure in this sequence, which can be completed and stabilized by guanine and guanine derivatives. To test this hypothesis, we examined the formation and stability of G-quadruplex formed in pu22m1 variants and the potential of guanine derivatives to fill in the incomplete G-tetrad and stabilize the overall G-quadruplex, using nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and DMS footprinting experiments. Our results showed that a GVBQ could form in in the PDGFR-β promoter sequence and that dGMP could insert and complete the incomplete G-tetrad to induce the formation of a stable and well-defined dGMP-fill-in-GVBQ in potassium solution. In addition, we discovered two natural plant alkaloids, berberine and chelerythrine, which could stabilize this dGMP-fill-in-GVBQ, with an increase in melting temperature by more than 10 °C. Therefore, it appears that dGMP could interact and stabilize the broken-stranded PDGFR-β promoter G-quadruplex, which may play a role in PDGFR-β transcriptional regulation. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Furthermore, our results suggest that this unique dGMP-fill-in G-quadruplex in the PDGFR-β promoter may serve as a novel anticancer drug target for modulating PDGFR-β gene regulation. Citation Format: Kaibo Wang, Clement Lin, Guanhui Wu, Danzhou Yang. A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 687.

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

ALS and FTD linked GGGGCC-repeat containing DNA oligonucleotide folds into two distinct G-quadruplexes

BackgroundThe most common genetic cause of neurological disorders ALS and FTD is a largely increased number of GGGGCC repeats in C9orf72 gene. Non-canonical structures including G-quadruplexes adopted by expanded repeats are hypothesized to be crucial in pathogenesis.Recently, we have shown that structural polymorphism of oligonucleotide d(G4C2)3G4 is reduced by dG to 8Br-dG substitution. High-resolution structure of one of the two major G-quadruplexes adopts antiparallel topology comprising of four G-quartets. Herein, we describe the topology of the second major G-quadruplex structure and influence of folding conditions on relative populations of the two folds. MethodsInfluence of folding conditions was explored by 1H 1D NMR. Determination of topology was achieved by 2D NMR complemented with PAGE and CD. UV melting experiment was used to explore thermal stability of structures. ResultsTwo structures adopted by oligonucleotide d(G4C2)3GGBrGG denoted AQU and NAN coexist in solution and ratio of their populations is determined by pH and rate of cooling when folding from thermally denatured state in the presence of K+ ions. ConclusionsAQU is kinetically favored and forms by folding at low pH, while NAN is favored thermodynamically and at neutral pH. AQU and NAN share similar antiparallel topology with four G-quartets and three edgewise loops, however they exhibit distinct structural and dynamic properties. General significanceNovel G-quadruplex topology adds insight into diverse polymorphism of DNA sequences comprising potentially pathological GGGGCC repeat. Relative populations of the two structures and their dependence on folding conditions contribute to understanding of factors that govern G-quadruplex folding. This article is part of a Special Issue entitled "Gquadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Abstract 4853: BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels

Abstract The c-MYC proto-oncogene is one of the most deregulated genes in human cancers. Transcriptional repression of c-MYC is considered as one of the attractive strategies in targeting c-MYC. Previous studies revealed that the c-MYC promoter nuclease hypersensitive element (NHE) III1 region, which regulates 80-95% of total c-MYC transcription, can form DNA G-quadruplex (G4) and that stabilization of the c-MYC G4 could repress c-MYC gene expression. We have previously determined the molecular structure of the major G-quadruplex formed in the c-MYC promoter NHE (MycG4), which is an intramolecular parallel-stranded structure. BMVC, a carbazole derivative, was designed as a fluorescent probe for recognizing telomeric G4. However, we found that BMVC binds to MycG4 with much greater affinity and specificity. Cellular studies showed that BMVC was enriched in the nucleus at 48 hr after treatment, leading to markedly reduced c-MYC expression levels. Promoter-driven luciferase assays revealed that BMVC could enhance the inhibitory effects of MycG4, suggesting BMVC can repress c-MYC expression in vivo through stabilization of MycG4. To understand the molecular interactions between BMVC and MycG4, we have determined the molecular structure of the complexes of BMVC with MycG4 by NMR. BMVC appears to bind the 5’-end of the MycG4 with very high affinity, as shown by the slow-exchange binding on the NMR timescale. The structure of the 5’-end complex of BMVC and MycG4 shows that BMVC is paired to the flanking adenine with specific hydrogen bonding interactions, while the BMVC-adenine plane stacks nicely on the 5’-end G-tetrad of MycG4. Compared to the previous MycG4 complex with a quindoline compound, the tighter and more specific binding of BMVC appears to be imparted by the novel arched shape of BMVC and specific pairing recognition of BMVC with the flanking bases of MycG4. BMVC can also bind to the 3’-end of MycG4 with lower affinity to form a second complex. However, the 3’-end complex shows much more dynamic conformation and less specific interactions. Our results indicated that the cellular targets of BMVC include not only telomeric G4, but also the c-MYC promoter G4. Because of the inherent fluorescence and novel binding mode of BMVC, our study provides useful information for future design of improved carbazole-based anticancer agents or specific cellular fluorescent probes, as well as insights into specific recognition of MycG4 by small molecules. Citation Format: Wenting Liu, Guanhui Wu, Clement Lin, Buket Onel, Ding Chen, Ta-Chau Chang, Danzhou Yang. BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4853.

Abstract 3090: Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules

Abstract Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3’ capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3’ capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3’ capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs. Citation Format: Clement Lin, Prashansa Agrawal, Yuwei Chen, Salil Kalarn, Nanjie Deng, Laurence Hurley, Danzhou Yang. Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3090.

The major G-quadruplex formed in the human BCL-2 proximal promoter adopts a parallel structure with a 13-nt loop in K+ solution.

The human BCL-2 gene contains a 39-bp GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of BCL-2 gene expression. Inhibition of BCL-2 expression can decrease cellular proliferation and enhance the efficacy of chemotherapy. Here we report the major G-quadruplex formed in the Pu39 G-rich strand in this BCL-2 promoter region. The 1245G4 quadruplex adopts a parallel structure with one 13-nt and two 1-nt chain-reversal loops. The 1245G4 quadruplex involves four nonsuccessive G-runs, I, II, IV, V, unlike the previously reported bcl2 MidG4 quadruplex formed on the central four G-runs. The parallel 1245G4 quadruplex with the 13-nt loop, unexpectedly, appears to be more stable than the mixed parallel/antiparallel MidG4. Parallel-stranded structures with two 1-nt loops and one variable-length middle loop are found to be prevalent in the promoter G-quadruplexes; the variable middle loop is suggested to determine the specific overall structure and potential ligand recognition site. A limit of 7 nt in loop length is used in all quadruplex-predicting software. Thus, the formation and high stability of the 1245G4 quadruplex with a 13-nt loop is significant. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter is intriguing, suggesting a novel mechanism for gene transcriptional regulation and ligand modulation.

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

The Major G-Quadruplex Formed in the Human Platelet-Derived Growth Factor Receptor β Promoter Adopts a Novel Broken-Strand Structure in K+ Solution

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) has been associated with cancers and vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped G(i)NG(j) motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-β promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression.

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

Thermodynamic Stability and Folding Kinetics of the Major G-Quadruplex and Its Loop Isomers Formed in the Nuclease Hypersensitive Element in the Human c-Myc Promoter: Effect of Loops and Flanking Segments on the Stability of Parallel-Stranded Intramolecular G-Quadruplexes

Overexpression of the c-Myc proto-oncogene is associated with a broad spectrum of human cancers. Nuclease hypersensitivity element III(1) (NHE III(1)) of the c-Myc promoter can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-Myc transcriptional silencing. The major G-quadruplex formed in c-Myc NHE III(1) is a mixture of four loop isomers, which have all been shown to be biologically relevant to c-Myc transcriptional control. In this study, we performed a thorough thermodynamic and kinetic study of the four c-Myc loop isomers in a K(+) solution. The four loop isomers all form parallel-stranded G-quadruplexes with short loop lengths. While the parallel-stranded G-quadruplex has been known to favor short loop lengths, our results show that the difference in thermodynamic and kinetic properties of the four loop isomers, and hence between the parallel G-quadruplexes with similar loop lengths, is more significant than previously recognized. At 20 mM K(+), the average difference in the T(m) values between the most stable loop isomer 14/23 and the least stable loop isomer 11/20 is more than 10 °C. In addition, the capping structures formed by the extended flanking segments are shown to contribute to a stabilization of 2-3 °C in T(m) for the c-Myc promoter G-quadruplex. Understanding the intrinsic thermodynamic stability and kinetic properties of the c-Myc G-quadruplex loop isomers can aid in our understanding of their biological roles and drug targeting.

Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region.

BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. 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. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G3NG3 sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design.