http://nar.oxfordjournals.org/cgi/content/full/33/14/4466

"Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter".

Kangkan Halder and Shantanu Chowdhury*

Institute of Genomics and Integrative Biology, CSIR Mall Road, Delhi 110007, India

*To whom correspondence should be addressed. Tel: +91 11 2766 6157; Fax: +91 11 2766 7471;
Email: shantanuc@igib.res.in

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE IIII) in the c-myc promoter, making the quadruplex a strong anti-cancer target. With respect to this, the competing equilibrium between intramolecular quadruplex folding and bimolecular duplex formation is poorly understood and very few techniques have addressed this problem. We present a method for simultaneously determining the kinetic constants for G-quadruplex folding/unfolding and hybridization in the presence of the complementary strand from a single reaction using an optical biosensor based on surface plasmon resonance (SPR). Using this technique, we demonstrate for the first time that quadruplex formation in the c-myc promoter is favored at low strand concentrations. Our results indicate favorable quadruplex folding (equilibrium folding constant K_F of 2.09 calculated from the kinetic parameters: folding rate constant, k_f = 1.65 x 10 ^-2 s ^-1 and unfolding rate constant, k_u = 7.90 x 10 ^-3 s ^-1) in 150 mM K⁺. The hybridization rate constants detected concurrently gave a bimolecular association constant, k_a = 1.37 x 10 ⁵ M ^-1 s ^-1 and dissociation constant, k_d = 4.94 x 10 ^-5 s ^-1. Interestingly, in the presence of Na ⁺ we observed that G-quadruplex folding was unfavorable (K_F = 0.54). Implication of our results on the c-myc transcription activation model is discussed in light of aberrant c-myc expression observed on destabilization of the G-quadruplex.

Introduction:

Expression of the oncogene c-myc is associated with cellular proliferation and control of differentiation. As a result, loss of regulation resulting in overexpression of c-myc is correlated with a large number of human and animal cancers (1–4). Antisense oligonucleotide mediated transcription silencing has been observed to induce differentiation in myelocytes indicating the role of aberrant c-myc overexpression in differentiation (5,6). Transcription regulation of c-myc is complex and involves multiple promoters, P1 and P2 being prominent among them [for reviews see (4,7)]. The nuclease hypersensitive element (NHE IIII), corresponding to –147 to –117 bases relative to P1 transcription initiation site controls >80% of c-myc transcription and hence is an important anti-cancer target (8–11). It has been observed that the purine-rich anti-sense strand of the NHE adopts a G-quadruplex conformation and it was recently shown that the structure could be a regulatory switch for c-myc (12,13). Based on this and various other observations, postulated models of regulatory control entail a switch between the G-quadruplex and the duplex DNA, which could be central in elucidation of the mechanism of c-myc transcription and design of antisense therapy (8,9,11,14). The orchestration of the structural transitions driving this quadruplex–duplex competition is poorly understood.

The G-quadruplex constitutes a four-strand fold-back structure of stacked guanine-tetrads. These tetrads are coplanar arrangement of four guanines held together by Hoogsteen hydrogen bonds (15,16). Apart from the promoter region of c-myc, sequences that form G-quadruplex in vitro have been found in the telomeres (17) and within the switch regions of immunoglobin heavy-chain genes (18). Interestingly, recent evidence implicates these unusual DNA structures as ‘at risk motifs’ (19) owing to their involvement in genome rearrangements induced by polymerase slippage events in the nematode Caenorhabditis elegans on inactivation of a putative helicase, DOG-1 (20). In a genomic context, formation of G-quadruplex competes with duplex formation and thus the kinetics and thermodynamics of the structural transitions would be the underlying factors determining its functional role.

Determination of the competing rate constants (G-quadruplex folding and hybridization) requires simultaneous determination of the folding/unfolding rates and the duplex formation rates. Nanomotors have been designed based on the folding/unfolding of G-quadruplex motifs, which were demonstrated using FRET (21). The rates of folding/unfolding determined the efficiency of the nanomachine and could be regulated using a duplex trap. A FRET-based study has been used to observe the quadruplex folding constants in the presence of a PNA trap, where the PNA strand concentration was maintained such that hybridization was very fast (22). However, in vivo extrapolations can be made only when the strand concentrations are equimolar and very low. A recent report addresses this problem using human telomeric G-quadruplex hybridization on an optical biosensor based on surface plasmon resonance (SPR) and suggests a possible quadruplex–duplex competition mechanism at low equimolar concentration of the complementary strand (23).

Based on DNase I hypersensitivity, it was reported that the major regulatory element of c-myc exists in a strand-separated form rendering this location as a NHE IIII (8). We hypothesized that the underlying inherent kinetics of duplex formation may play a significant role, in conjunction with other cellular factors, which could be important for the crucial regulatory mechanism. In this study, we used SPR-based biosensor to observe competing hybridization versus G-quadruplex formation in the c-myc regulatory region at physiological conditions. Using an analytical component resolution method described here for the first time, we could not only simultaneously determine the individual rate constants of folding/unfolding (of G-quadruplex) and association/dissociation (of hybridization) but we also separated the two components of the hybridization reaction. One resulting from hybridization with pre-equilibrated unstructured oligonucleotides present on sensor surface and the other owing to hybridization with immobilized molecules unfolding in the presence of the complementary strand (during injection). We observed that both the folded and the unfolded forms have short half-lives of <90 s and our results further indicated that the rate-limiting step changes as a result of complementary strand concentration. At low-strand concentration hybridization is slow and determines the overall rate while with increasing concentration motif transition becomes rate determining. Based on our results we conclude that G-quadruplex may be the predominant state at the low intracellular strand concentrations because duplex formation is kinetically unfavorable.

Kinetic Analysis:

...Please see: http://nar.oxfordjournals.org/cgi/content/full/33/14/4466

Additional References:

1. Hovsepian JA, and Frenster JH, "Sense and antisense during RNA initiation of the DNA transcription bubble". (Sense vs. antisense during initiation of gene transcription).

2. Frenster JH, Allfrey VG, and Mirsky, AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes". (Intranuclear Na ⁺ and K ⁺ effects).

3. Yafe A, Etzioni S, Weisman-Shomer P, and Fry M, "Formation and properties of hairpin and tetraplex structures of guanine-rich regulatory sequences of muscle-specific genes".  (Enhancer or promoter regions of muscle-specific genes).

4. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter".

5. Simonssen T, "G-Quadruplex DNA Structures -Variations on a Theme", Biol. Chem. vol. 382, pp. 621-628 April, 2001). http://www.bcbp.gu.se/simonsson/BC_2001.pdf
 

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).