A sequence-independent analysis of the loop length dependence of intramolecular RNA G-quadruplex stability and topology

Abstract

G-Quadruplexes are noncanonical nucleic acid secondary structures based on guanine association that are readily adopted by G-rich RNA and DNA sequences. Naturally occurring genomic G-quadruplex-forming sequences have functional roles in biology that are mediated through structure. To appreciate how this is achieved, an understanding of the likelihood of G-quadruplex formation and the structural features of the folded species under a defined set of conditions is informative. We previously systematically investigated the thermodynamic stability and folding topology of DNA G-quadruplexes and found a strong dependence of these properties on loop length and loop arrangement [Bugaut, A., and Balasubramanian, S. (2008) Biochemistry 47, 689-697]. Here we report on a complementary analysis of RNA G-quadruplexes using UV melting and circular dichroism spectroscopy that also serves as a comparison to the equivalent DNA G-quadruplex-forming sequences. We found that the thermodynamic stability of G-quadruplex RNA can be modulated by loop length while the overall structure is largely unaffected. The systematic design of our study also revealed subtle loop length dependencies in RNA G-quadruplex structure.

Figure 1

RNA oligonucleotide library design.

Figure 2

Nondenaturing gel electrophoresis of RNA oligonucleotide libraries (20 μM) and naturally occurring intramolecular RNA G-quadruplexes. Lanes 1–7: L111, L112, TRF2, L113, MT3, L333, and TERRA, respectively.

Figure 3

Fraction folded of RNA oligonucleotide libraries as a function of temperature.

Figure 4

ΔG_VH as a function of total loop length: RNA libraries (◆), naturally occurring RNA G-quadruplex sequences (◇), and DNA libraries (red diamonds). Errors are given as the standard deviation of triplicate experiments.

Figure 5

Overlays of CD spectra generated from RNA oligonucleotide libraries with total loop lengths of (A) 3–5 and (B) 6–9.