Interactions between a Bioflavonoid and c-MYC Promoter G

Subscriber access provided by LUNDS UNIV

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Interactions Between a Bioflavonoid and c-MYC Promoter GQuadruplex DNA: Ensemble and Single-Molecule Investigation Sneha Paul, Sk Saddam Hossain, Bala Divya Mallavarapu, and Anunay Samanta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b00335 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Interactions Between a Bioflavonoid and c-MYC Promoter G-Quadruplex DNA: Ensemble and Single-Molecule Investigation Sneha Paul, Sk Saddam Hossain, Bala Divya M. and Anunay Samanta* School of Chemistry, University of Hyderabad, Hyderabad-500046, India

ABSTRACT Small molecules capable of stabilizing the G-Quadruplex structure of the nuclease hypersensitivity element III1 (NHE III1) are useful in controlling the overexpression of the cMYC oncogene. In this study we have probed the interactions of a 22-mer c-MYC promoter quadruplex-forming sequence (Pu22) with a bioflavonoid 3, 4′, 5, 7-tetrahydroxyflavone, commonly known as kaempferol (KF). Ensemble FRET experiments on labelled Pu22 indicate that KF decreases the affinity of the former towards its complimentary strand, suggesting the stabilization of the quadruplex structure of Pu22. Considering that binding dynamics plays an important role in supramolecular interactions, but there is hardly any information on this aspect for quadruplex-flavonoid systems, we have studied the kinetics of KF-Pu22 complexation and de-complexation processes at the single-molecule level employing fluorescence correlation spectroscopy (FCS) technique. The binding dynamics is characterized by a fast relaxation time of 10-50 µs. This leads to a high association rate constant (k+) of 109 M-1s-1 which is close to the pure-diffusion controlled limit. However, it is the low dissociation rate constant (k-) of 104 s-1 that is mainly responsible for the stability of the KF-Pu22 complex. Molecular docking study shows that KF binds near the 3′-end of Pu22 by forming several H-bonds with the bases. These findings suggest that KF is a potential binder of the c-MYC promoter quadruplex DNA and can be useful in anti-cancer therapies.

*Corresponding author, Email: [email protected]

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION DNA has a tendency to adopt secondary structures among which guanine-rich (G-rich) sequences are known to fold into G-Quadruplexes (GQs). Four guanines associate via WatsonCrick and Hoogsteen H-bonding to form a planar quartet, called G-tetrad, and stacking of two or more such G-tetrads gives rise to GQs.1, 2 These structures are stabilized by metal ions such as Na+ and K+.3, 4 Over the last few years, there has been growing evidence of in vivo presence of GQs

5-8

and their role in pivotal cellular processes like telomere maintenance, DNA replication

and gene expression.9-12 One of the potential quadruplex forming candidate is the nuclease hypersensitivity element III1 (NHE III1) in c-MYC promoter consisting of a 27 base-pair long purine (Pu)-rich segment with five consecutive runs of guanine (mycPu27, Figure 1A).13 mycPu27 folds into at least two types of GQ structure with the major one involving the G-tracts II, III, IV, V (mycPu22, Figure 1A).13, 14

mycPu22 exists as a parallel GQ structure in presence of K+.14, 15 The overexpression of c-

MYC oncogene is associated with cell proliferation, which is one of the most common genetic disorders found in several cancers.16-18 NHE III1 controls 80% transcription of the c-MYC genes and its GQ structure acts as a transcription repressor.19, 20 Small molecules and proteins, which can stabilize this GQ structure, can thus down-regulate the transcription of c-MYC genes and help in anti-cancer therapies.21-25 GQ binders are usually planar molecules with heteroaromatic rings that can stack in between the end tetrads and loops or fit into the grooves of quadruplex DNA.26 Various classes of molecules have been documented as potential GQ binders 26-32 among which naturally occurring flavonoids are one of the promising candidates.33-36 The most abundant subclass of flavonoids is the


Page 2 of 31

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


flavonols, which are found in fruits, vegetables and beverages, and known for their therapeutic applications.37-40 One such flavonol is 3, 4′, 5, 7-tetrahydroxyflavone, commonly known as kaempferol (KF, Figure 1C), which is an anti-oxidant capable of preventing cancer cell growth and modulating several key elements involved in apoptosis.41,

42

KF is a planar system

containing two benzene rings (A and B rings) connected by a pyrone ring (ring C) and fulfills the conditions of a prospective quadruplex binder. Further, the hydroxyl groups at the periphery can participate in additional interactions with DNA and stabilize its structure. The above facts motivated us to undertake the present study and probe the interactions between KF and c-MYC promoter GQ both at the ensemble and single molecule level. Considering that the actual c-MYC promoter sequence, mycPu22 has several conformations of the loop region and might complicate the binding event,14 we have used in the present study a slightly modified sequence (Pu22, Figure 1A), where G14 and G23 are replaced with thymines (T) as it adopts a single conformation, whose structure is known (Figure 1B).43 It is worth mentioning here that although flavonoids-GQ interactions have been studied earlier at the ensemble level, to the best of our knowledge, the present work represents the first single molecule study. Moreover, the information gained from the fluorescence correlation spectroscopy (FCS) measurements, such as the rate constants of the complexation dynamics of the KF-Pu22 system, is first of its kind.


The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 31

(A) 1 2

3

4 5

6 7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

mycPu27 5′- T G G G G A G G G T G G G G A G G G T G G G G A A G G -3′ I II III IV V mycPu22 5′- T G A G G G T G G G G A G G G T G G G G A A -3′ Pu22 labPu22 Cstrand

5′- T G A G G G T G G G T A G G G T G G G T A A -3′ 5′-FAM-T G A G G G T G G G T A G G G T G G G T A A-TAMRA-3′ 5′- T T A C C C A C C C T A C C C A C C C T C A -3′

(B) (C) B A

C

Figure 1: (A) Wild-type 27-mer sequence of the NHE III1 element in c-MYC promoter, mycPu27, the 22-mer G-rich sequence that folds into the major GQ conformation, mycPu22 in K+ rich aqueous solutions, Pu22, which is a modified sequence of mycPu22, obtained by replacing G14 and G23 with T (shown in red), the dual-labeled Pu22, labPu22 with fluorescein (FAM)- and tetramethylrhodamine (TAMRA)- based probes at the 5′ and 3′ ends respectively and the complimentary strand of Pu22. (B) Parallel folding topology of Pu22 with three propeller type loops (major conformation of c-MYC GQ in K+ solutions). Yellow, red and blue circles represent guanine, adenine and thymine respectively. (C)


Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure of 3, 4′, 5, 7-tetrahydroxyflavone (kaempferol, KF) with rings A, B and C labeled. EXPERIMENTAL SECTION Materials and sample preparation Kaempferol, KCl, KH2PO4, K2HPO4 and methanol were procured from Sigma Aldrich and used without purification. The unlabeled (Pu22) and dual-labeled (5′ FAM and 3′ TAMRA, labPu22) c-MYC promoter based oligomers and the complimentary strand (Cstrand) of Pu22 were HPLC purified and purchased from Integrated DNA Technologies. For the preparation of G-Quadruplex DNA, the oligomers (labeled/unlabeled) were suspended in a 10 mM phosphate buffer (pH 7.4) containing 60 mM KCl. The solutions were heated at 95oC for 10 minutes and then allowed to cool to room temperature (25oC) slowly for ̴6 hours. The solutions were incubated at 4oC for 24 hours prior to use. The concentrations of Pu22 and Cstrand were estimated using the molar extinction coefficient values of 2,28,700 and 1,79,900 M-1cm-1strand-1 at 260 nm as provided by the manufacturer. The concentration of labPu22 was calculated by measuring the absorbance of FAM moeity attached to it and using a molar extinction coefficient of 4.1 x 104 M-1cm-1 at 496 nm.44 Stock solution of KF was prepared in methanol. In all experiments, the amount of methanol in the aqueous buffered solutions was restricted to 1) or quenched (q1 case is attained at much lower binder concentrations as compared to q

Interactions between a Bioflavonoid and c-MYC Promoter G

Recommend Documents