Poster abstracts

Poster number 120 submitted by Madeline Sherlock

Structural Studies of G-Quadruplexes using their Intrinsic Fluorescence

Madeline E. Sherlock (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA), Chun Kit Kwok (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA), Philip C. Bevilacqua (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA )

Abstract:
The Guanine quadruplex structure (GQS) has been of particular interest in recent years and is important to regulation of replication, transcription and translation. The general nucleotide pattern for a GQS motif is “GwLaGxLbGyLcGz”, where w, x, y, and z are ≥2 nucleotides, and loops (L) a, b, and c have lengths of ≥1 nucleotide. The unique quartet structure and folding properties of GQSs lead to unique spectroscopic features including an inverse UV melting profile at 295 nm, distinct circular dichroism (CD) features, and the recently discovered intrinsic fluorescence in DNA GQSs. We investigated the effect of increasing loop and G-stretch lengths on GQS folding in several RNA systems. We found that longer loops generally have a weaker potassium ion binding affinity (K+1/2), which is likely due to an increase in flexibility. As the length of the G-stretch increases (i.e. G2 GQS to G6 GQS), we observed a general decrease in folding cooperativity (Hill coefficient, ‘n’), leading to a broad ~5-log response range to K+ ions. We demonstrated that the observed decrease in cooperativity is due to intermediates in the GQS folding pathway, showing a clear 3-state transition in G3 and G4 GQS. Lastly, we found that RNA GQSs also exhibit the property of intrinsic fluorescence.1
The intrinsic fluorescence of GQS was further explored in DNA systems with varying loop sequence, loop length, and G-stretch length. Potassium ion titrations were performed and folding was monitored by fluorescence and CD spectroscopy. We found that longer G-stretches (i.e. G3 GQS vs G2 GQS) exhibit stronger fluorescence, even per G-stretch length. Interestingly, we also observed that the GQS with the shortest loops (1 nucleotide) have the strongest fluorescence emission intensity. Looking into the available NMR structures of dG3T and dG2A, we found that the T loops in dG3T are oriented away with respect to the G-quartets, whereas the A loops in dG2A are oriented toward and can interact with the G-quartet. This result is especially interesting as dG2A has a heptad structure, which suggests that A loops (λmax >385 nm) exhibit maximal fluorescence at longer wavelength than T loops (λmax <385 nm) due to extended conjugation. Also, the extruded orientation of the T loops in dG3T likely minimizes fluorescence quenching by loop bases and thus leads to stronger fluorescence.2
Overall, GQS folding can occur in a highly cooperative (molecular switch-like) or uncooperative (rheostat-like) mode, depending on the G-stretch length. Also, the intrinsic fluorescence of GQS is useful for nucleic acid studies and the development of label-free detection methods. These studies serve to provide a deeper understanding of structure and folding properties of GQS folding, and shed light on possible roles of GQS in cells.

References:
1. Kwok, C. K., Sherlock, M. E., and Bevilacqua, P. C. (2013) Angew. Chem. Int. Ed. 52, 683-686.
2. Kwok, C. K., Sherlock, M. E., and Bevilacqua, P. C. (2013) Biochemistry 52, 3019-3021.

Keywords: G-quadruplex, folding cooperativity, fluorescence