ARTICLE

Recognition of an important G-quadruplex in the HIV-1promoter with natural small moleculesWeixuan Wang, Yang Sui, Lulu Zhang, Wei Tan, Xiangwei He, and Xiangming Xie

Abstract: Targeting a G-quadruplex with chemical small molecules is a useful strategy for gene therapy for disease. Theguanine-rich sequence d(5=-TG1G2CCTG3G4G5CG6G7G8ACTG9G10G11-3=) in the HIV-1 promoter can form a G-quadruplex structure.In this study, circular dichroism was performed to study the conformation and thermal stability of the HIV-1 G-quadruplexbefore and after adding small molecules. A DMS footprinting assay was used to identify which guanosine can be integrated intothe G-quadruplex structure. Electrospray ionization mass spectrometry was used to evaluate the binding affinities of the smallmolecules with the G-quadruplex. Our results showed that G1, G2, G3, G4, G7, G8, G9, and G10 of the above oligonucleotidesformed a two G-tetrad antiparallel G-quadrulex, and nitidine chloride was found to have the highest binding affinity toward theHIV-1 G-quadruplex among the eight studied small molecules. The Tm value of the G-quadruplex was enhanced from 56.6 to63.2 °C when fourfold nitidine chloride was added. This is potentially a novel approach for anti-HIV-1 drug development.

Key words: G-quadruplex, binding affinity, ESI mass spectrometry, DMS footprinting, circular dichroism.

Résumé : L’utilisation de petites molécules ciblant le G-quadruplexe constitue une stratégie utile pour le traitement de maladies parla thérapie génique. Dans la région du promoteur du HIV-1, la séquence d(5=-TG1G2CCTG3G4G5CG6G7G8ACTG9G10G11-3=), riche enguanine, peut former une structure a quatre brins (G-quadruplexe). Dans le cadre des présents travaux, nous avons utilisé le di-chroïsme circulaire pour étudier la conformation et la stabilité thermique du G-quadruplexe du HIV-1 avant et après l’ajout de petitesmolécules. Une analyse d’empreinte au DMS a permis de déterminer quelles unités de guanosine peuvent s’intégrer dans la structuredu G-quadruplexe. Nous avons utilisé la spectroscopie de masse a ionisation par électronébulisateur pour évaluer l’affinité de liaisondes petites molécules avec le G-quadruplexe. Nos résultats montrent que les guanosines G1, G2, G3, G4, G7, G8, G9 et G10 desoligonucléotides ci-dessus forment un G-quadruplexe antiparallèle a deux G-tétrades. En outre, parmi les huit petites molécules al’étude, le chlorure de nitidine a révélé la plus forte affinité de liaison a l’égard du G-quadruplex du HIV-1. La Tm du G-quadruplexe estpassée de 56,6 a 63,2 °C lorsque nous avons quadruplé la quantité de chlorure de nitidine ajoutée. Cette stratégie pourrait constituerune nouvelle approche pour le développement de médicaments contre le HIV-1. [Traduit par la Rédaction]

Mots-clés : G-quadruplexe, affinité de liaison, spectroscopie de masse a ionisation par électronébulisateur, empreinte au DMS,dichroïsme circulaire.

IntroductionGuanine-rich DNA sequences can fold into a noncanonical high-

order DNA structure known as a G-quadruplex structure (G4s).1–3

The interest in G-quaduplexes has increased gradually over time,with thousands of reports and reviews published on several as-pects of G-quaduplexes, including the biophysical and chemicalcharacteristics as well as biological function in a few mammaliansas well as bacteria species.4–8 Likewise, the presence of putativeG4 sequences has been reported in various viruses genomes, suchas human immunodeficiency virus (HIV-1),9 Epstein–Barr virus(EBV),10 and papillomavirus (HPV).11 In parallel, DNA aptamersthat form G4s have been described as inhibitors and diagnostictools to detect viruses (hepatitis A virus (HAV)),12 EBV,10 severeacute respiratory syndrome virus (SARS),13 and simian virus 40(SV40)14]. G4s are likely present in several significant genomic re-gions and may be a key component in important cellular pro-cesses.15 Targeting these G4s could potentially serve as a basis fornovel antiviral therapies. Hence, as described for cellular tar-gets,16 small ligands that can stabilize the G4 structure may com-pose a potential new class of therapeutic agents to fight viralinfections.17–20

HIV-1 is the main cause of acquired immunodeficiency syn-drome (AIDS), which was first identified in the Western world in1981.21,22 Since then, AIDS has developed into a worldwide pan-demic of disastrous proportions. Considerable progress has beenmade in treating HIV-infected patients using highly active antiret-roviral therapy (HAART) involving multidrug combinations. How-ever, the increasing incidence of drug-resistant viruses along withdrug toxicity among treated people calls for continuous effortsof developing anti-HIV-1 drugs. Targeting genes is considered asan effective therapeutic method for AIDS. The HIV-1 promotercontains a G-rich sequence 50 nucleotides upstream from the

Received 7 May 2015. Accepted 3 October 2015.

W. Wang, Y. Sui, X. He, and X. Xie. College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China.L. Zhang and W. Tan. Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.Corresponding authors: Xiangwei He (e-mail: hexiangwei@bjfu.edu.cn) and Xiangming Xie (e-mail: xxm1005@126.com).

Fig. 1. Schematic diagram of the G-rich sequence in the HIV-1promoter.

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Can. J. Chem. 94: 60–65 (2016) dx.doi.org/10.1139/cjc-2015-0215 Published at www.nrcresearchpress.com/cjc on 19 October 2015.

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transcription-starting site (TSS). This sequence overlaps the so-called minimum promoter composed of three SP1 and two NF-kBbinding sites, which are crucial for the initiation of transcription.23

In this paper, the G-rich sequence d(TGGCCTGGGCGGGACTGGG)located from –76 to –57 in the HIV-1 promoter was chosen as thetarget (Fig. 1). Electrospray ionization mass spectrometry (ESI-MS),circular dichroism (CD) spectrometry, and DMS footprinting wereused to investigate the formation and recognition of the G-quadruplex.The results demonstrated that d(TGGCCTGGGCGGGACTGGG) canform a two G-tetrad antiparallel G-quadrulex. Furthermore, a nat-ural product from Chinese herb, nitidine chloride, was found tohave the highest binding affinity towards the HIV-1 G-quadruplexamong eight natural small molecules (nitidine chloride, jatrorrhi-zine, benzophenanthridine derivative, tetrahydropalmatine, tod-dalolactone, costisine, piperine, and astraglalin named as P1–P8 inFig. 2). The CD melting experiment indicated that P1 can enhancethe stability of the HIV-1 G-quadruplex, and this structure is po-tentially a novel target for anti-HIV-1 drug development.

Materials and methods

MaterialsSingle-strand oligonucleotides d(TGGCCTGGGCGGGACTGGG) (S)

were purchased from the Sangon Company (Shanghai, China)with HPLC purification. The obtained oligonucleotides were dis-solved in deionized water and the concentration was determinedwith a Nanodrop 2000 spectrophotometer (Thermo Scientific) andthe stock DNA solution was diluted to 750 �mol/L. The smallmolecules used in this study were provided by Professor Gu Yuanof Peking University.

ESI-MSAll ESI-MS experiments were carried out on a Finnigan LCQ

Deca XP Plus ion-trap mass spectrometer (Thermo Finnigan, SanJose, CA). The instrument was used in the negative ion mode, withthe capillary voltage set to 2.7 kV. The capillary temperature wasset to 120 °C. The sheath gas flow rate was 25 arb. For theG-quadruplex experiments, the strand concentration was 5 �mol/Land the buffer contained 50 mmol/L NH4OAc and 30% CH3OH (thepH value was 7.0). The samples were injected at a flow rate of2 �L/min.

CDThe CD spectra were obtained using a JASCO J-815 spectropola-

rimeter equipped with a Peltier junction temperature-controlledcell holder. CD experiments were carried out on samples at aconcentration of 5 �mol/L DNA (30 mmol/L Tris–HCl and 50 mmol/LKCl, pH = 7.4). For the melting experiments, the small molecules

Fig. 2. Structures of the studied natural product molecules P1–P8.

Fig. 3. ESI-MS spectra of 5 �mol/L DNA in 50 mmol/L NH4OAc buffer.

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were added to the 10 �mol/L oligonucleotides to make the finalconcentration ratio of small molecules to DNA 4:1. Melting exper-iments were measured at 293 nm and the Tm values were calcu-lated from the normalized CD melting curves according to themethods described previously.24

DMS footprintingThe DMS footprinting assay was performed as previously de-

scribed.25 The FAM-labeled oligodeoxygenucleotide was dilutedwith 60 mmol/L Tris–HCl buffer (pH = 7.4) to 0.1 �mol/L whilecontaining 0 or 150 mmol/L KCl; afterward, preannealing at 95 °Cwas performed for 10 min before slowly cooling to 4 °C. The an-nealed samples were treated with 10% DMS for 5 min before beingquenched and extracted with a Tris–phenol–chloroform solution(pH = 8.0). The aqueous phase was precipitated using ethanol at–80 °C. The dry precipitate was dissolved in 10% piperidine andincubated at 90 °C for 30 min followed by precipitation. Thetreated sample was resolved using 20% denatured PAGE gel at1500 V for 4 h in a 4 °C cold room before being imaged with a GEHealthcare Typhoon 9400 gel scanner.

Results and discussions

G-quadruplex formationThe potential G-quadruplex formation in the HIV-1 promoter

was evaluated by ESI-MS, CD spectrometry, and DMS footprinting.The ESI-MS spectra of d(TGGCCTGGGCGGGACTGGG) (S) (calcu-lated molecular weight = 5941.87) in NH4OAc solution are shownin Fig. 3. The ESI-MS spectra showed that the base peak (100%) andthe main peak (80%) were two complex ions of DNA and oneammonium ion at m/z 1190.2 ([S + NH4

+ – 6H+]5– abbreviated to[G]5–) and one at m/z 1487.7 ([S + NH4

+ – 5H+]4– abbreviated to [G]4–),respectively. These were characteristic of the formation of theG-quadruplex structure.26 Since ammonium ions were known tosit between each G-tetrad layer to stabilize the G-quadruplexstructure, it was reasonable that there were two G-tetrad layers inthe G-quadruplex that possessed one ammonium ion. This resultwas confirmed by CD spectroscopy. The CD spectrum of the S inwater (Fig. 4A) showed a positive peak at 252 nm, which was theunfolded state, while in the KCl and NH4OAc solution, it showed anegative peak at 264 nm and a positive peak at 293 nm, which was

characteristic of the antiparallel G-quadruplex. Thus, the resultsfrom both ESI-MS and CD spectra indicated the formation of a twoG-tetrad antiparallel G-quadrulex.

To identify which guanosine can participate in the formation of theG-quadruplex structure, the DMS footprinting assay was performed.The 11 guanosines (5=-TG1G2CCTG3G4G5CG6G7G8ACTG9G10G11-3=) arepresent in the sequence. When the G participated in G-quadrulexformation, the difference of band brightness between in waterand KCl buffer was significant. Figure 4B shows that the bands ofG1, G2, G3, G4, G7, G8, G9, and G10 were weaker than those of G5,G6, and G11. Thus, the DMS footprinting experiment demon-strated that G1, G2, G3, G4, G7, G8, G9, and G10 were integratedinto the G-quadrulex structure.

Noncovalent interaction between small molecules and theG-quadruplex

In this study, eight natural product molecules were selected toexamine the recognition affinities to the HIV-1 G-quadruplex byESI-MS. The mass spectra of the HIV-1 G-quadruplex and the smallmolecules (Fig. 2) in a molar ratio of 1:4 as examples are shownin Fig. 5. In the ESI-MS spectra, [G]5– and [G]4– denoted theG-quadrulex ions with –5 and –4 charges, respectively, and [G + nP]5–

and [G + nP]4– denoted the 1:n complex ions of the G-quadrulex (G)and small molecule (P) with –5 and –4 charges, respectively. ForP1, the intensity of the complex ion [G + P1]4– at m/z 1575.2 was100% (base peak) and the intensity of [G + P1]5– at m/z 1259.7 wasnearly 72%. Moreover, the peaks of the G-quadruplex DNA for P1([G]5– m/z 1190.3 and [G]4– m/z 1487.7) were only 40% and 47%, re-spectively, while the peaks of [G]4– for P2–P8 were the base peak.For P2 and P3, the complex ions with one small molecule and twosmall molecules were relatively strong, but for P6–P8, the peaks of[G + 2P]4– and [G + 2P]5– cannot be detected. These results indicatedthat P1 had the highest affinity in binding with the G-quadruplex.

To evaluate the binding affinities of small molecules (P1–P8) tothe G-quadruplex, a parameter, IRa is introduced. IR[G + P], IR[G +2P], and IR[G + 3P] denote the ability of the G-quadruplex to bindwith one to three small molecules. IRa is a ratio of the total inten-sity of the complex ion to the total intensity of the G-quadruplexion and denotes the relative binding affinity of the small mole-cules to the G-quadruplex:

Fig. 4. (A) CD spectra of DNA in water, 100 mmol/L NH4OAc, and 100 mmol/L KCl buffer; (B) DMS footprinting assay of 3=-labeled DNAincubating in 30 mmol/L Tris–HCl buffer and 150 mmol/L KCl.

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Fig. 5. ESI-MS spectra of 5 �mol/L G-quadruplex DNA (G) with 20 �mol/L small molecules (P): (A) P1, (B) P2, (C) P3, (D) P4, (E) P5, (F) P6, (G) P7,and (H) P8.

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IR[G � nP] ��Ir(G � nP)

�Ir(G)�n � 1, 2, or 3�

IRa ��Ir(G � P) � �Ir(G � 2P) � �Ir(G � 3P)

�Ir(G)

where �Ir(G) are the total intensities of the G-quadruplexes,�Ir(G + nP) are the total intensities of the 1:1, 1:2, and 1:3 complexions, respectively, and P represents the small molecule.

All of the IRa values were calculated according to the formula andare shown in Table 1. Comparing the IRa in Table 1, nitidine chloride(P1) had the best binding affinity with an IRa value of 2.86. jatrorrhi-zine (P2) and benzophenanthridine derivative (P3) had good affinitywith an IRa value of 1.18 and 1.54, respectively. The IRa values ofP4–P8 were less than 0.8, which indicated that they had lower bind-ing affinity towards the HIV-1 G-quadruplex. Therefore, the relativebinding affinity order is P1 > P3 > P2 > P5 > P4 > P6 > P7 > P8.

P1 enhanced the HIV-1 G-quadruplex thermal stabilityThe thermal stability of the HIV-1 G-quadruplex and the com-

plex was evaluated using CD spectroscopy with the melting exper-iment. The CD melting experiment revealed that the Tm value ofthe G-quadruplex was enhanced from 56.6 to 63.2 °C when four-fold nitidine chloride (P1) was added (Fig. 6); the CD melting curvesof the G-quadruplex with the other molecules are provided in thesupporting information (see Supplementary material section. Ac-cording the to melting experiments, P1 had the highest Tm value

compared with the other seven molecules, which was identicalwith the mass spectrometry method. These results indicated thatP1 can stabilize the HIV-1 G-quadruplex and had the best bindingaffinity.

ConclusionsThis research has demonstrated that the G-rich sequence

d(TGGCCTGGGCGGGACTGGG) in the HIV-1 promoter can form aG-quadruplex as detected by ESI and CD spectroscopy. Further-more, DMS footprinting indicated that G1, G2, G3, G4, G7, G8, G9,and G10 were integrated into the formation of the G-quadruplex.In addition, a natural small molecule, nitidine chloride, wasfound to have the highest binding affinity to the G-quadruplexamong the eight small molecules and it could enhance theG-quadruplex thermal stability. Our study provided importantclues that nitidine chloride may regulate the expression of theHIV-1 promoter and can be a prodrug for anti-HIV-1 treatment.

Supplementary materialSupplementary material is available with the article through

the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjc-2015-0215.

AcknowledgementsWe thank Professor Yuan Gu for providing the laboratory and

small molecules for our experiments. This work was supported bythe Fundamental Research Funds for the Central Universities(BLX2013027) and the National Natural Science Foundation ofChina (31400085).

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Table 1. IR values of small molecules with theG-quadruplex.

IR[G + P] IR[G + 2P] IR[G + 3P] IRa

P1 1.95 0.73 0.18 2.86P2 0.77 0.31 0.10 1.18P3 0.71 0.54 0.28 1.54P4 0.37 0.16 0.07 0.53P5 0.53 0.18 0.00 0.71P6 0.49 0.00 0.00 0.49P7 0.30 0.00 0.00 0.30P8 0.21 0.00 0.00 0.21

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