Spectroscopic,biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5′-G₃(T₂AG₃)₃-3′ (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV–vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30)?×?10⁵ M^?1). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV–vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π–π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.     

Dual mode of binding of anti cancer drug epirubicin to G-quadruplex [d-(TTAGGGT)]4 containing human telomeric DNA sequence induces thermal stabilization

Epirubicin exerts its anti cancer action by blocking DNA/RNA synthesis and inhibition of topoisomerase-II enzyme. Recent reports on its influence on telomere maintenance, suggest interaction with G-quadruplex DNA leading to multiple strategies of action. The binding of epirubicin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]₄ comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy. The epirubicin binds as monomer to G-quadruplex DNA with affinity, K_b1 = 3.8 × 10⁶ M⁻¹ and K_b2 = 2.7 × 10⁶ M⁻¹, at two independent sites externally. The specific interactions induce thermal stabilization of DNA by 13.2–26.3 °C, which is likely to come in the way of telomere association with telomerase enzyme and contribute to epirubicin-induced apoptosis in cancer cell lines. The findings pave the way for drug designing in view of the possibility of altering substituent groups on anthracyclines to enhance efficacy using alternate mechanism of its interaction with G4 DNA, causing interference in telomere maintenance pathway by inducing telomere dysfunction.     

NMR based structural studies decipher stacking of the alkaloid coralyne to terminal guanines at two different sites in parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4

BackgroundTelomere elongation by telomerase gets inhibited by G-quadruplex DNA found in its guanine rich region. Stabilization of G-quadruplex DNA upon ligand binding has evolved as a promising strategy to target cancer cells in which telomerase is over expressed.MethodsInteraction of anti-leukemic alkaloid, coralyne, to tetrameric parallel [d(TTGGGGT)]₄ (Ttel7), [d(TTAGGGT)]₄ (Htel7) and monomeric anti-parallel [dGGGG(TTGGGG)₃] (Ttel22) G-quadruplex DNA has been studied using Circular Dichroism (CD) spectroscopy. Titrations of coralyne with Ttel7 and Htel7 were monitored by ¹H and ³¹P NMR spectroscopy. Solution structure of coralyne-Ttel7 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by absorption, CD and ¹H NMR.Results and conclusionsBinding of coralyne to Ttel7/Htel7 induces negative CD band at 315/300 nm. A significant upfield shift in all GNH, downfield shift in T2/T7 base protons and upfield shift (1.8 ppm) in coralyne protons indicates stacking interactions. ³¹P chemical shifts and NOE contacts of G3, G6, T2, T7 protons with methoxy protons reveal proximity of coralyne to T2pG3 and G6pT7 sites. Solution structure reveals stacking of coralyne at G6pT7 and T2pG3 steps with two methoxy groups of coralyne located in the grooves along with formation of a hydrogen bond. Binding stabilizes Ttel7/Htel7 by ~ 25–35 °C in 2:1 coralyne-Ttel7/Htel7 complex.General significanceThe present study is the first report on solution structure of coralyne-Ttel7 complex showing stacking of coralyne with terminal guanine tetrads leading to significant thermal stabilization, which may be responsible for telomerase inhibition.     

Studies on Non-Covalent Interaction of Coumarin Attached Pyrimidine and 1-Methyl Indole 1,2,3 Triazole Analogues with Intermolecular Telomeric G-Quadruplex DNA Using ESI-MS and Spectroscopy

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T₂AG₃) and d(T₂AG₃)₂ sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV–Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T₂AG₃) and d(T₂AG₃)₂ G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.     

A dihydroindolizino indole derivative selectively stabilizes G-quadruplex DNA and down-regulates c-MYC expression in human cancer cells

Background

Telomeric and NHE III1, a c-MYC promoter region is abundant in guanine content and readily form G-quadruplex structures. Small molecules that stabilize G-quadruplex DNA were shown to reduce oncoprotein expression, initiate apoptosis and they may function as anticancer molecules.

Methods

Electrospray ionization mass spectrometry, spectroscopy, isothermal titration calorimetry, Taq DNA polymerase stop assay, real time PCR and luciferase reporter assay. Cell migration assay to find out the effect of derivatives on normal as well as cancer cell proliferation.

Results

Among three different dihydroindolizino indole derivatives, 4-cyanophenyl group attached derivative has shown maximum affinity, selective interaction and higher stability towards G-quadruplex DNA over dsDNA. Further, as a potential G-quadruplex DNA stabilizer, 4-cyanophenyl linked dihydroindolizino indole derivative was found to be more efficient in inhibiting in vitro DNA synthesis, c-MYC expression and cancer cell proliferation among human cancer cells.

Conclusion

The present study reveals that dihydroindolizino indole derivative having 4-cyanophenyl group has potential to stabilize G-quadruplex DNA and exhibit anticancer activity.

General significance

These studies are useful in the identification and synthesis of lead derivatives that will selectively stabilize G-quadruplex DNA and function as anticancer agents.     

Drug recognition and stabilisation of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat

The NMR structure of the parallel-stranded DNA quadruplex d(TTAGGGT)(4), containing the human telomeric repeat, has been determined in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl]acridinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor of telomerase at submicromolar levels (IC(50) value of 0.33(+/-0.13)microM), exhibiting a wide differential between telomerase inhibition and acute cellular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic potency through interaction with, and stabilisation of, four-stranded G-quadruplex structures. RHPS4 forms a dynamic interaction with d(TTAGGGT)(4), as evident from 1H and 19F linewidths, with fast exchange between binding sites induced at 318 K. Perturbations to DNA chemical shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify the 5'-ApG and 5'-GpT steps as the principle intercalation sites; a structural model has been refined using NOE-restrained molecular dynamics. The central G-tetrad core remains intact, with drug molecules stacking at the ends of the G-quadruplex. The partial positive charge on position 13-N of the acridine ring appears to act as a "pseudo" potassium ion and is positioned above the centre of the G-tetrad in the region of high negative charge density. In both ApG and GpT intercalation sites, the drug is seen to converge to the same orientation in which the pi-system of the drug overlaps primarily with two bases of each G-tetrad. The drug is held in place by stacking interactions with the G-tetrads; however, there is some evidence for a more dynamic, weakly stabilised A-tetrad that stacks partially on top of the drug at the 5'-end of the sequence. Together, the interactions of RHPS4 increase the t(m) of the quadruplex by approximately 20 degrees C. There is no evidence for drug intercalation within the G-quadruplex; however, the structural model strongly supports end-stacking interactions with the terminal G-tetrads.     

Role of G-quadruplex located at 5ʹ end of mRNAs

BackgroundSecondary structures in 5′ UTR of mRNAs play a critical role in regulating protein synthesis. Though studies have indicated the role of secondary structure G-quadruplex in translational regulation, position-specific effect of G-quadruplex in naturally occurring mRNAs is still not understood. As a pre-initiation complex recognises 5′ cap of the mRNA and scans along the untranslated region (UTR) before initiating translation, the presence of G-quadruplex in 5′ region may have a significant contribution in regulating translation. Here, we investigate the role of G-quadruplex located at the 5′ end of an mRNA.MethodsBiophysical characterisation of putative G-quadruplexes was performed using UV and CD spectroscopy. Functional implication of G-quadruplex in the context of their location was assessed in cellulo using qRT-PCR and dual luciferase assay system.ResultsPG4 sequences in 5′ UTR of AKT interacting protein (AKTIP), cathepsin B (CTSB) and forkhead box E3 (FOXE3) mRNAs form G-quadruplex whereas it is unable to form G-quadruplex in apolipoprotein A-I binding protein (APOA1BP). Our results demonstrated diverse roles of G-quadruplex located at 5′ end of mRNAs. Though G-quadruplex in AKTIP and CTSB mRNA act as inhibitory modules, it activates translation in FOXE3 mRNA.ConclusionsOur works suggests that G-quadruplex present at the 5′ terminal of an mRNA behaves differently in a different gene context. It can activate or inhibit gene expression.General significanceThis study demonstrated that it is difficult to predict the role of G-quadruplex on the basis of its position in 5′ UTR. The neighbouring nucleotide sequence, the intracellular milieu and the interacting partners might render diverse functions to this secondary structure.     

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)₁/DNA complex (K₁ = 3.7 × 10⁶) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)₁/DNA complex, (K₁ = 7.0 × 10⁴). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)₃/DNA and (TMPyP4)₄/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.     

Comparison of interactions of 5′-derivatives of deoxyoctathymidylate with human DNA polymerize α and HIV reverse transcriptase

K_m and V_max values for d(pT₈) and its derivatives containing various 5′-end groups were estimated in the reaction of DNA polymerization α catalyzed by DNA polymerase α and HIV-RT. The effect of 5′-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT₈ is 200-fold higher than that of d(pT₈). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase α the influence of modifiers on primer affinity is much weaker. The effect of 5′-end residues on the V_max values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.     

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)₂ that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)₂ associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.     

DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P₁ promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K⁺] and 130 mM [K⁺]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

Effect of Cationic Comb-Type Copolymer on Quadruplex Folding of Human Telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA) _n in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA) ₃ GGG) and d((GGGTTA)₇GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA) ₃ GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

The interaction of nemorubicin metabolite PNU-159682 with DNA fragments d(CGTACG)2, d(CGATCG)2 and d(CGCGCG)2 shows a strong but reversible binding to G:C base pairs

The antitumor anthracycline nemorubicin is converted by human liver microsomes to a major metabolite, PNU-159682 (PNU), which was found to be much more potent than its parent drug toward cultured tumor cells and in vivo tumor models. The mechanism of action of nemorubicin appears different from other anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of nemorubicin. The interaction of PNU with the oligonucleotides d(CGTACG)₂, d(CGATCG)₂ and d(CGCGCG)₂ was studied with a combined use of ¹H and ³¹P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC–tandem mass spectra, also combined with endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of intercalators. After digestion with the enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both proton and phosphorus NOESY spectra of PNU bound to d(CGTACG)₂ and to d(CGATCG)₂. The dissociation rate constants (k_off) of the slow step of the intercalation process, measured by ³¹P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for doxorubicin and nemorubicin, leading to a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in solution from restrained MD calculations. The conformation of PNU bound to the oligonucleotides was also derived from the coupling constant values.     

Molecular modeling and biophysical analysis of the c-MYC NHE-III<Subscript>1</Subscript> silencer element

G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III₁ (nuclease hypersensitivity element III₁) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine–cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A₂C₄)₄] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions. Figure Molecular models of the average structure of the final 20 ps of the molecular dynamics simulation of the c-MYC NHE-III₁ (nuclease hypersensitivity element III₁) “silencer” element. The G-Quadruplex structure is at the top-center, and the i-Motif is at the bottom-center of each picture. a “Rotation #1” of the G-Quadruplex, with the T15 loop at the top and rear and the G19/A20 loop at the top and front of the picture. b “Rotation #2” of the G-Quadruplex, with the T15 loop at the top and front of the image, and the G19/A20 loop at the front and adjacent to the G-Quadruplex/i-Motif interface     

Effects of deficient of the Hoogsteen base-pairs on the G-quadruplex stabilization and binding mode of a cationic porphyrin

BackgroundIn stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined.MethodsThe thermal stability of the thrombin-binding DNA aptamer 5′G₁G₂TTG₅G₆TG₈TG₁₀G₁₁TTG₁₄G₁₅ G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured.ResultsThe transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G₁ base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability.ConclusionsReplacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex.General significanceThe Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin.