A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes

Abstract

G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences.

Figure 1

Arrows indicate 5′ to 3′ polarity. (A) Different arrangements of strand polarity for G-quadruplexes: (a) all strands parallel, (b) three parallel strands and one antiparallel strand, (c) two pairs of adjacent parallel strands, and (d) alternating antiparallel strands. (B) Examples of loop topologies leading to different arrangements of strand polarity.

Figure 2

General design of the quadruplex libraries (Ljkl) used for this study.

Figure 3

Fraction folded as a function of temperature for the 21 DNA quadruplex libraries.

Figure 4

Nondenaturing gel electrophoresis of quadruplex libraries representative of total loop lengths of not more than five nucleotides.

Figure 5

Changes in ΔG_VH as a function of total loop length.

Figure 6

Overlays of the CD spectra generated by (A) quadruplex libraries with a total loop length of not more than five nucleotides and (B) quadruplex libraries with a total loop length of more than five nucleotides.

Figure 7

Arrows indicate 5′ to 3′ polarity. (A) Possible folds for quadruplex-forming sequences with two single-nucleotide loops (red) and a di- or trinucleotide central loop (blue) that (a) can adopt a double-chain-reversal conformation or can fold back, bridging either (b) two adjacent or (c) two diagonally opposite antiparallel strands [even though molecular dynamics simulations have previously shown that thymidine dinucleotide loops might be too short to span the diagonal of the G-tetrad (43)]. (B) Possible folds for quadruplex-forming sequences with two consecutive single-nucleotide loops (red) and trinucleotide loops (blue) that (d) can adopt a double-chain-reversal conformation or (e) can fold back to bridge two adjacent antiparallel strands.