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Multi-Spectroscopic and Theoretical Exploration on the Comparative Binding Aspects of the Bio-Flavonoid Fisetin with Triple and Double Helical Forms of RNA Sutanwi Bhuiya, Lucy Haque, Rapti Goswami, and Suman Das J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07972 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017
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Multi-Spectroscopic and Theoretical Exploration on the Comparative Binding Aspects of the Bio-Flavonoid Fisetin with Triple and Double Helical Forms of RNA
Sutanwi Bhuiya, Lucy Haque, Rapti Goswami and Suman Das* Department of Chemistry Jadavpur University Raja S. C. Mullick Road, Jadavpur Kolkata 700 032 India
*Corresponding author Tel.: +91 94 3437 3164, +91033 2457 2349 Fax: +91 33 2414 6266 E-mail:
SD:
[email protected] SB:
[email protected] LH:
[email protected] RG:
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ABSTRACT The interactions of RNA triplex (U.A*U) and duplex (A.U) with the naturally occurring flavonoid fisetin (FTN) have been examined at pH 7.0 using various spectroscopic, viscometric and theoretical studies. Experimental observations showed that the ligand binds with both double and triple helical forms of RNA, although the binding affinity is greater for the triplex structure (5.94×106 M-1) compared to the duplex counterpart (1.0×105 M-1). Thermal melting experiments revealed that the Hoogsteen base paired third strand of triplex was stabilized to a greater extent (~14 °C) compared to the Watson-Crick base paired second strand (~4 °C) in presence of FTN. From fluorimetric study, we observed that U.A*U and A.U primarily bind the photo-produced tautomer of FTN in the excited state. Steady-state and time-resolved anisotropy measurements illustrate considerable modulations of the spectroscopic properties of the tautomeric FTN within the RNA environment. Viscometric, fluorescence quenching and thermal melting studies all together support the mode of binding to be intercalation. Theoretical study explains the experimental absorption and emission (dual fluorescence) behaviour of FTN along with the ESIPT process.
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INTRODUCTION In the research field of molecular biology, nucleic acids are considered mostly for their participation as the transporter of information in expressions of their sequences and on the involvement of proteins in terms of their functions. Usually, a neglected area in this field is about the structural and functional exploration of RNA molecules. This is probably because they do not possess ‘structure’ related functional role to take care in contrast to DNA. However, RNA plays a crucial role in ‘central dogma’ of molecular cell system. Various biological functions governed by RNA are determined following the complex structures of RNA stabilized by secondary and tertiary interactions. Tertiary structure of RNA is the threedimensional arrangement of RNA building blocks including helical duplexes, triple-stranded RNA structures and other components that are held together through association jointly expressed as RNA tertiary interactions.1 An RNA triplex, a member of RNA tertiary structures, is a complex structure stabilized by multiple base triples and formed by following sequence specific binding rules.2 RNA duplex can accommodate single strand [known as triplex forming oligonucleotide (TFO)] resulting the formation of a major-groove or a minorgroove triplex through Hoogsteen hydrogen bonds with purine rich strands. The antiparallel arrangement of one purine strand flanked between two pyrimidine strands (Figure 1) is referred to as the ‘pyrimidine·purine*pyrimidine’ triplex motif, which in this context is poly(U).poly(A)*poly(U) triplex [where .(dot) represents the Watson-Crick base pairing and *(asterisk) denotes the Hoogsteen base pairing, herein after U.A*U]. In the year 1957, Felsenfeld and his colleagues first focus the spotlight on the formation of the RNA triple helical nucleic acid by mixing poly(U) and poly(A) in a molar ratio of 2:1.3,4 However, the triplex structures had not been given any biological relevance until the existence of triple helix regions were come into notice in H-DNA structure in natural DNA samples.5 Since then, their implication in several biological functions have given renewed interest by means
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Figure 1. Base pairing scheme in triplex (U.A*U) and duplex (A.U) RNA.
of antisense and antigene strategies, post-transcriptional RNA processing, gene regulation etc.6-8 The natural pyrimidine motif major-groove triplex (U.A*U) structure was first found to form in the human telomerase RNA pseudoknot in absence of Mg2+ ions.9 U.A*U triplex is particularly stable in H-type pseudoknots and plays the driving force in various functions that includes telomerase activity,9 sensing metabolite by riboswitches in mRNA untranslated regions,10 stimulating -1 ribosomal frame shifting in mRNA coding regions11 etc. On that note, Bailey and his group have reviewed a detail insight about the in vivo cellular functions of different triple helical nucleic acid structures.12 It has been found that formation of local triple helix may inhibit the process of transcription of a specific gene.13 In comparison to the Watson-Crick pairing in double helix, the binding of Hoogsten base paired TFO is relatively weak. This results a lowering in the thermodynamic stability of the triplex structure compared to the duplex counterpart which critically limits their cellular application. One means of 4|Page ACS Paragon Plus Environment
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increasing the stabilization of triplex is through the association of small molecules with triple helical nucleic acids where the ligands (small molecule) selectively bind to the triplex strand thereby drawing the equilibrium in the direction of triplex formation (Scheme 1).14
Scheme 1. Equilibrium between free and bound state of RNA triplex.
Thus, small molecules that possess the potential to stabilize the specific sequences of triple helices through binding are of crucial importance. In this regard, intercalators are known for their active participation to stabilize the triple helices15-17 although it cannot be generalized for all intercalating ligands.18 Understanding the association of small molecules with RNA19,20 may thus be an area of importance in the contemporary RNA targeted small molecules interaction studies. Therefore, the development of molecules capable of controlling RNA activity is now being investigated for the progress of medicinal as well as chemicobiological research. Albert Szent-Györgyi, the Nobel laureate from Hungary, discovered flavonoid compounds in 1936.21 Flavonoids or bioflavonoids (flavus – yellow), are the largest group of polyphenolic compounds available from various dietary resources in most plants.22 Flavonoids have been reported by several authors for having anti-inflammatory, antibacterial, anti-viral, anti-allergic, anti-mutagenic, anti-neoplastic, anti-thrombotic and vasodilatory actions.23-25 The said pharmacological effects are governed by the anti-oxidant activity of flavonoids resulting from their ability to scavenge free radicals.26 These low molecular weight substances are of high pharmacological potency and of low cytotoxicity which make them promising alternatives to conventional therapeutic drugs.22 The core structure of flavonoid molecules are the assembly of carbon atoms in two aromatic rings, 5|Page ACS Paragon Plus Environment
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usually denoted by A (benzoyl ring) and B (cinnamoyl ring) rings, which are joined by a “bridge” of three-carbon atoms: C6-C3-C6, thus forming a diphenyl-propane structure with the central unit being a benzo-γ-pyrone (chromone, ring C) moiety (Figure 2A).22 Several hydroxyl groups, oxygen, sugar, or methyl groups are joined to this core structure. On the basis of the oxidation state of the heterocyclic ring, flavonoids are classified into different categories as flavones, flavanonols, flavonols, flavanones or isoflavones etc.22 Fisetin (C15H10O6) (3, 7, 3′, 4′-tetrahydroxyflavone, herein after FTN, Figure 2B) belongs to the flavonol (3-hydroxy flavone) group of the family of polyphenols. It is found in
Figure 2. Chemical structure of (A) Flavonoid skeleton and (B) FTN.
many dietary plant resources such as herbs, fruits, vegetables and specially strawberries, grapes, apples, persimmons, onions and cucumbers at concentrations in the range of 2–160 µg/g.27 It has been reported to act as a neuroprotective agent,28 which suggests FTN as a potential drug for the treatment of memory disorders like Alzheimer’s disease.29 Another report reveals that FTN has been used with chemotherapeutic drug cis-platin for probable combination therapy where it was recognized that FTN could definitely show a possible way to the removal of embryonal carcinoma cells acting as an anticancer agent.30 FTN has been reported to show inhibitory effects against human low-density lipoprotein (LDL) oxidation in
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vitro.31 Thus in the recent past years, FTN is a growing field of research because of its presence in various human foods and its role in antiproliferative,32 apoptotic29 and antioxidant33 activities. It was proposed that the presence of o-dihydroxy group in the B ring, the 3-hydroxy group and 2,3-double bond in the C ring are the fundamental reason for exerting the antioxidant activity of FTN (Figure 2B).34 Malignant melanoma is a fatal humanoid cancer with no effectual cure for the disease but the treatment of FTN with metastatic melanoma cells of human resulted in decreased cell feasibility with G1-phase arrest.35 FTN is a potential inhibitor of protein kinase C (PKC)36 and HIV-1 proteinase37. However, similar to other natural products, FTN has imposed restriction on its usages due to its low water solubility (
