Why to target G-quadruplexes using peptides: Next-generation G4-interacting ligands.

Guanine-rich oligonucleotides existing in both DNA and RNA are able to fold into four-stranded DNA secondary structures via Hoogsteen type hydrogen-bonding, where four guanines self-assemble into a square planar arrangement, which, when stacked upon each other, results in the formation of higher-order structures called G-quadruplexes. Their distribution is not random; they are more frequently present at telomeres, proto-oncogenic promoters, introns, 5'- and 3'-untranslated regions, stem cell markers, ribosome binding sites and so forth and are associated with various biological functions, all of which play a pivotal role in various incurable diseases like cancer and cellular ageing. Several studies have suggested that G-quadruplexes could not regulate biological processes by themselves; instead, various proteins take part in this regulation and can be important therapeutic targets. There are certain limitations in using whole G4-protein for therapeutics purpose because of its high manufacturing cost, laborious structure prediction, dynamic nature, unavailability for oral administration due to its degradation in the gut and inefficient penetration to reach the target site because of the large size. Hence, biologically active peptides can be the potential candidates for therapeutic intervention instead of the whole G4-protein complex. In this review, we aimed to clarify the biological roles of G4s, how we can identify them throughout the genome via bioinformatics, the proteins interacting with G4s and how G4-interacting peptide molecules may be the potential next-generation ligands for targeting the G4 motifs located in biologically important regions.

Magnesium and molecular crowding of the cosolutes stabilize the i-motif structure at physiological pH.

Most of the important genomic regions, especially the G,C rich gene promoters, consist of sequences with potential to form G,C-tetraplexes on both the DNA strands. In this study, we used three C-rich oligonucleotides (11Py, 21Py, and HTPy), of which 11Py and 21Py are located at various transcriptional regulatory elements of the human genome while HTPy sequence is a C-rich strand of human telomere sequence. These C-rich oligonucleotides formed i-motif structures, verified by Circular Dichroism (CD), UV absorption melting experiments, and native gel electrophoresis. The CD spectra revealed that 11Py and 21Py form i-motif structures at acidic pH values of 4.5 and 5.7 in the presence of 100 mM NaCl but remain unstructured at pH 7.0. However, 21Py can form stable i-motif structure even at neutral pH in presence of 1 mM MgCl2 . UV-thermal melting studies showed stabilization of 21Py i-motif at pH 5.7 in the presence of Na+ or K+ with increasing concentration of MgCl2 or CaCl2 from 1 to 10 mM. Significant shift in the CD peak of HTPy sequence was observed as the positive peak from 286 nm shifted to 276 nm while the negative peak from 265 to 254 nm. Further, inevitable necessity of 1 mM Mg2+ to form i-motif structure at neutral pH was observed. Under similar ionic conditions and neutral pH, all the three C-rich sequences were able to form stable i-motif structures (11Py, 21Py) or altered i-motif/homoduplex structures (HTPy) in the presence of MgCl2 and cell mimicking molecular crowding conditions of 40 wt% PEG 200. It is concluded that presence of Mg2+ ions and molecular crowding agents induce and stabilize i-motif structures at physiological solution environment.

Structural analysis of peptide identified from the 2KRR domain of the nucleolin protein with a c-Myc G4 structure using biophysical and biochemical methods.

For the first time, the c-Myc G4 structure is reported to be stabilized by binding of the peptide (derived from the 2KRR domain of the nucleolin protein called the Nu peptide) in the loop region of the G-quadruplex structure by stacking interactions. CD results showed the formation of parallel G4 structure in the presence of 100 mM Na+ or 100 mM K+ with the appearance of two isodichroic points at 229 nm, 254 nm and 252 nm in the presence of 100 mM Na+ or 100 mM K+, respectively. In addition, in UV thermal and CD melting studies, we observed drastic changes with an increase in the hyperchromicity at a DNA : peptide ratio of 1 : 50. On titrating the Nu peptide with c-Myc G4, we calculated the value of binding constant (K a) by plotting fluorescence intensity and DNA concentration as 0.1369 ± 0.008 µM and 0.1277 ± 0.073 µM in Na+ and K+, respectively, which confirms the strong association of Nu peptide with c-Myc G4. The Nu peptide showed preferential cytotoxicity against MDA-MB-231 cells with IC50 values of 5.020 µM and 5.501 µM after 72 and 96 hours. This approach suggests a novel strategy to target G4 structure using natural key peptide segments derived from G4 stabilizing protein.

Identification of G4 motifs of various stem cell markers and their biophysical and biochemical characterization.

Regulatory regions in the human genome, enriched in guanine-rich DNA sequences have a remarkable enrichment of G-rich sequences having a tendency to fold into G-quadruplex structures. To identify the G-quadruplex forming motifs in regulatory regions of stem cell markers, gene sequences of various stem cell markers were downloaded and analyzed to see the abundance of G-rich sequences. We observed the enrichment of G-rich sequences in stem cell markers (CD13, CD19, CD24 and CD38) which could possibly play a critical role in its regulation. We used Circular Dichroism (CD), UV-Thermal denaturation (UV-Tm) and polyacrylamide gel electrophoresis (PAGE) to demonstrate the formation of a G-quadruplex by G-rich sequences present in these stem cell markers. We observed that these G-rich sequences containing minimum consecutive G3 stretch separated by loop length ranging from one to three bases long adopt G-quadruplexes with different molecularity involving two-strands, three-strand and four-strand with parallel and antiparallel conformation. Interestingly, we proposed the formation of three-stranded G-quadruplex by CD13 in 100 mM Na+, CD19 in 100 mM K+, 100 mM K+ with 40 wt% PEG 200, and CD38 in 100 mM K+ + 40 wt% PEG 200. The formation of such diverse G-quadruplex structures in the regulatory regions leaves the fair possibility of recognition by regulatory factors to modulate the gene expression. First time, this study may give insight into the structural polymorphism of G4 forming motifs in different stem cell markers to design the best suitable ligand and to target them for therapeutic development.Communicated by Ramaswamy H. Sarma.

Short designed peptide unfolding human telomeric G-quadruplex: mimicking the helicase function.

Human telomeric DNA can fold into G-quadruplex structures involving the interaction of four guanine bases in a square planar arrangement. The highly distinctive nature of quadruplex topologies suggests that they can act as novel therapeutic targets. In this study, we provide the evidence of human telomeric G4 destabilization in dilute and cell-mimicking molecular crowing conditions upon peptide binding. We have used three human telomeric sequences of different lengths. CD data showed that these sequences folded into anti-parallel G-quadruplex and CD intensity decreased significantly on increasing the peptide concentration. UV-thermal melting results showed significant decrease in hypochromicity due to formation of G4-peptide complex at 295 nm. Fluorescence data showed the quenching on titrating the peptide with human telomere G4. Electrophoretic mobility shift assay confirmed the unfolding of G4 structure. Cell viability was significantly reduced in the presence of QW5 peptide with IC50 values as 8.78 µM and 7.72 µM after 72 and 96 hours of incubation respectively. These results confirmed that QW5 peptide has an ability to bind and unfold to human telomeric G-quadruplex and hence might be the key modulator for targeting diseases having over-representation of G4 motifs and their destabilization will be helpful in increasing the efficiency of DNA replication, transcription or duplex reannealing.Communicated by Ramaswamy H. Sarma.

Significant destabilization of human telomeric G-quadruplex upon peptide binding: dramatic effect of flanking bases.

Human telomere is composed of highly repeated hexanucleotide sequence TTAGGG and a 3' single-stranded DNA tail. Many telomere G4 topologies characterized at atomic level by X-ray crystallography and NMR studies. Until now, various small ligands developed to interact with G-quadruplex mainly to stabilize the structure and least is known for its destabilization. In this study, we provide the first evidence of human telomeric G4 destabilization upon peptide binding in dilute and cell-mimicking molecular crowing conditions due to the changes in flanking bases of human telomeric sequences. Hence, our findings will open the new ways to target diseases related with increasing the efficiency of DNA replication, transcription or duplex reannealing.Communicated by Ramaswamy H. Sarma.

Recognition and unfolding of human telomeric G-quadruplex by short peptide binding identified from the HRDC domain of BLM helicase.

Research in recent decades has revealed that the guanine (G)-quadruplex secondary structure in DNA modulates a variety of cellular events that are mostly related to serious diseases. Systems capable of regulating DNA G-quadruplex structures would therefore be useful for the modulation of various cellular events to produce biological effects. A high specificity for recognition of telomeric G-quadruplex has been observed for BLM helicase. We identified peptides from the HRDC domain of BLM using a molecular docking approach with various available solutions and crystal structures of human telomeres and recently created a peptide library. Herein, we tested one peptide (BLM HRDC peptide) from the library and examined its interaction with human telomeric variant-1 (HTPu-var-1) to understand the basis of G4-protein interactions. Our circular dichroism (CD) data showed that HTPu-var-1 folded into an anti-parallel G-quadruplex, and the CD intensity significantly decreased upon increasing the peptide concentration. There was a significant decrease in hypochromicity due to the formation of G-quadruplex-peptide complex at 295 nm, which indicated the unfolding of structure due to the decrease in stacking interactions. The fluorescence data showed quenching upon titrating the peptide with HTPu-var-1-G4. Electrophoretic mobility shift assay confirmed the unfolding of the G4 structure. Cell viability was significantly reduced in the presence of the BLM peptide, with IC50 values of 10.71 µM and 11.83 µM after 72 and 96 hours, respectively. These results confirmed that the selected peptide has the ability to bind to human telomeric G-quadruplex and unfold it. This is the first report in which a peptide was identified from the HRDC domain of the BLM G4-binding protein for the exploration of the G4-binding motif, which suggests a novel strategy to target G4 using natural key peptide segments.

Significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant (K b) values of the order of 0.05 ± 0.2 µM, 0.12 ± 0.1 µM and 0.05 ± 0.3 µM for complexes in K+ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC50 values of 11.10 µM and 6.44 µM after 72 and 96 hours' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

Sole and stable RNA duplexes of G-rich sequences located in the 5'-untranslated region of protooncogenes.

Guanine- (G-) rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. It is widely held that the formation of a G-quadruplex in RNA is more feasible than in DNA because of the lack of a complementary strand in mRNA. Here, we analyzed sequences of 5'-untranslated regions of protooncogenes and surprisingly found that these regions showed an enrichment of not only guanine (G) but also cytosine (C) nucleotides. Since neighboring cytosine- (C-) rich regions can affect the formation and stability of a G-quadruplex structure, we further investigated the properties of DNA and RNA structures of G-rich and GC-rich regions. We selected typical GC-rich RNA sequences from protooncogenes and corresponding DNA sequences and investigated their structures. It was found that the GC-rich RNA sequences formed stable A-form duplexes as their major structure independent of the surrounding conditions, including the presence of different cations (Na(+), K(+), or Li(+)) or molecular crowding with 40 wt % poly(ethylene glycol) with an average molecular mass of 200 Da although there are a few exceptions in which only a combination of K(+) and molecular crowding induced a G-quadruplex structure of an extremely G-rich RNA sequence. In contrast, structural polymorphisms involving duplexes, G-quadruplexes, and i-motifs were observed for GC-rich DNA sequences depending on the surrounding factors. These results demonstrate the considerable structural and functional differences in GC-rich sequences of the genome (DNA) and transcriptosome (mRNA) with respect to the nucleic acid backbone. Moreover, it was suggested that structural study for a G-rich RNA sequence should be carried out under cell-mimicking condition where K(+) and crowding cosolutes exist.

Monomorphic RNA G-quadruplex and polymorphic DNA G-quadruplex structures responding to cellular environmental factors.

We systematically and quantitatively investigated the structure and thermodynamics of G-quadruplexes of RNAs and corresponding DNAs of the same sequences under molecular crowding conditions that mimic the high osmotic stress induced by the numerous molecules inside of living cells. Structural analyses demonstrated that various telomere RNA sequences folded into parallel-stranded G-quadruplexes in a manner independent of the surrounding conditions with different cations under both dilute and molecular crowding conditions. In contrast, DNA G-quadruplexes showed structural polymorphism. Moreover, we demonstrated that the G-quadruplexes of the RNA sequences were more stable than those of the same DNA sequences. These results show that a single and robust RNA G-quadruplex structure can exist in a manner independent of the sequence and surrounding conditions. To confirm this, we studied a guanine-rich sequence located in the 5'-untranslated region of human bcl-2 mRNA that is thought to play a role in translation. The results revealed a stable parallel G-quadruplex that formed under all conditions tested. For example, a bcl-RNA G-quadruplex in the presence of 5 mM KCl [free energy change at 25 degrees C (DeltaG degrees (25)) of -5.42 kcal/mol] was more stable than its corresponding DNA G-quadruplex (DeltaG degrees (25) = -2.31 kcal/mol). Our results further indicated that water molecules binding to the 2'-OH group of RNA G-quadruplexes play a critical role in their formation and stability.

Metal sensitive and DNA concentration dependent structural rearrangement of short oligonucleotide into large suprastructures.

Formation of higher order structures, such as G-quadruplexes and G-quadruplex based large suprastructures into long G-wires and liquid crystals is promising elements for use in healthcare for drug delivery as they are mechanically and thermally stable. In this study, we studied the structures of short 11-mer oligonucleotide 5'-G2AG5AG2-3'(11Pu) which is observed in 3'-UTR region of c-jun protooncogene. We used circular dichroism, UV-thermal melting, native gel electrophoresis and atomic force microscopy to determine the structure of 11Pu. CD results showed that 11Pu formed a mixed G-quadruplex in the presence of Na+ with and without Mg2+, while it formed a parallel G-quadruplex in the presence of 100 mM K+ with or without Mg2+. Cation selectivity in inducing the formation of large superstructures was observed in the presence of 100 mM K+ with 10 mM Mg2+. On the contrary, 10 mM Ca2+ did not induce the suprastructures. It was further demonstrated that Mg2+ at low concentration induced a parallel G-quadruplex of 11Pu, whereas at 10 mM Mg2+ induced a large suprastructure. AFM Images showed that 11Pu formed a G-wire, a liquid crystals and a crystalline lattice depending on the concentration of 11Pu and Mg2+. These insights may be employed to design G quadruplex-based nanowires for targeted drug delivery as well as interesting candidates for molecular nanowires. Communicated by Ramaswamy H. Sarma.

Selective recognition of human telomeric G-quadruplex with designed peptide via hydrogen bonding followed by base stacking interactions.

We described a novel synthetic peptide in which a glutamine residue binds through hydrogen bonding to a guanine-base and a trytophan residue intercalates with K+ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over its complementary c-rich strand and a double-stranded DNA and its complementary C-rich strand. This peptide offers great potential for cancer treatment by inhibiting the telomere extension by telomerase.

Structural polymorphism exhibited by a homopurine.homopyrimidine sequence found at the right end of human c-jun protooncogene.

Homopurine.homopyrimidine (Pu.Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu.Py DNA segment within human c-jun protooncogene 3'-region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na+/K+ with Mg2+ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na+ in acidic as well as neutral pH facilitated the duplex formation, while K+ favored the formation of self-associated structures. In Na+ and Mg2+, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.

HEPNet: A Knowledge Base Model of Human Energy Pool Network for Predicting the Energy Availability Status of an Individual.

HEPNet is an electronic representation of metabolic reactions occurring within human cellular organization focusing on inflow and outflow of the energy currency ATP, GTP and other energy associated moieties. The backbone of HEPNet consists of primary bio-molecules such as carbohydrates, proteins and fats which ultimately constitute the chief source for the synthesis and obliteration of energy currencies in a cell. A series of biochemical pathways and reactions constituting the catabolism and anabolism of various metabolites are portrayed through cellular compartmentalization. The depicted pathways function synchronously toward an overarching goal of producing ATP and other energy associated moieties to bring into play a variety of cellular functions. HEPNet is manually curated with raw data from experiments and is also connected to KEGG and Reactome databases. This model has been validated by simulating it with physiological states like fasting, starvation, exercise and disease conditions like glycaemia, uremia and dihydrolipoamide dehydrogenase deficiency (DLDD). The results clearly indicate that ATP is the master regulator under different metabolic conditions and physiological states. The results also highlight that energy currencies play a minor role. However, the moiety creatine phosphate has a unique character, since it is a ready-made source of phosphoryl groups for the rapid synthesis of ATP from ADP. HEPNet provides a framework for further expanding the network diverse age groups of both the sexes, followed by the understanding of energetics in more complex metabolic pathways that are related to human disorders.

Structural and Functional Characterization of RecG Helicase under Dilute and Molecular Crowding Conditions.

In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro. We present evidence of a unique protein conformational change in the RecG helicase from an α-helix to a ß-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, the α-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na(+) and Mg(2+). Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that the α-helix and ß-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

Conformational flexibility influences degree of hydration of nucleic acid hybrids.

Four nucleic acid duplexes-DNA/RNA hybrid, RNA/DNA hybrid, RNA duplex, and DNA duplex-were studied under molecular crowding conditions of osmolytes. Destabilization of duplexes (ΔΔG°(25)) indicated that the ΔΔG°(25) values of hybrids were intermediate between those of DNA and RNA duplexes. In the presence of polyethylene glycol 200, the ΔΔG°(25) values were estimated to be +3.0, +3.5, +3.5, and +4.1 kcal mol(-1) for the DNA duplex, DNA/RNA hybrid, RNA/DNA hybrid, and RNA duplex, respectively. Differences in the number of water molecules taken up (-Δn(w)) upon duplex formations between 0 and 37 °C (Δ(-Δn(w))) were estimated to be 44.8 and 59.7 per duplex structure for the DNA/RNA and RNA/DNA hybrids, respectively. While the Δ(-Δn(w)) value for the DNA/RNA hybrid was intermediate between those of the DNA (26.1) and RNA (59.2) duplexes, the value for RNA/DNA hybrid was close to that of RNA duplex. These differences in the thermodynamic parameters and hydration are probably a consequence of the enhanced global flexibility of the RNA/DNA hybrid structure relative to the DNA/RNA hybrid structure observed in molecular dynamics simulations. This molecular crowding study provides information not only on hydration but also on the flexibility of the conformation of nucleic acid duplexes.

Thermodynamic stability of Hoogsteen and Watson-Crick base pairs in the presence of histone H3-mimicking peptide.

We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.

Structural polymorphism at LCR and its role in beta-globin gene regulation.

Information on the secondary structures and conformational manifestations of eukaryotic DNA and their biological significance with reference to gene regulation and expression is limited. The human beta-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I-hypersensitive sites (HSs). Since these HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. Mutations and SNPs may stabilize or destabilize the local secondary structures, affecting the gene expression by changes in the protein-DNA recognition patterns. Various palindromic or quasi-palindromic segments within LCR, could cause structural polymorphism and geometrical switching of DNA. This emphasizes the importance of understanding of the sequence-dependent variations of the DNA structure. Such structural motifs might act as regulatory elements. The local conformational variability of a DNA segment or action of a DNA specific protein is key to create and maintain active chromatin domains and affect transcription of various tissue specific beta-globin genes. We, summarize here the current status of beta-globin LCR structure and function. Further structural studies at molecular level and functional genomics might solve the regulatory puzzles that control the beta-globin gene locus.

Possibility of an antiparallel (tetramer) quadruplex exhibited by the double repeat of the human telomere.

Under physiological concentrations of Na+ and K+, human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K+, whereas Na+ facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K+ and Na+, respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands.