Metal Cations in G-Quadruplex Folding and Stability

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes T-ALL development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5′UTR sequences such as the 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. Hence, the 5′UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Repeats of Gn sequences are detected as single strand overhangs at the ends of eukaryotic chromosomes together with associated binding proteins. Such telomere sequences have been implicated in the replication and maintenance of chromosomal termini. They may also mediate chromosomal organization and association during meiosis and mitosis. We have determined the three-dimensional solution structure of the human telomere sequence, d[AG3(T2AG3)3] in Na(+)-containing solution using a combined NMR, distance geometry and molecular dynamics approach (including relaxation matrix refinement). The sequence, which contains four AG3 repeats, folds intramolecularly into a G-tetraplex stabilized by three stacked G-tetrads which are connected by two lateral loops and a central diagonal loop. Of the four grooves that are formed, one is wide, two are of medium width and one is narrow. The alignment of adjacent G-G-G segments in parallel generates the two grooves of medium width whilst the antiparallel arrangement results in one wide and one narrow groove. Three of the four adenines stack on top of adjacent G-tetrads while the majority of the thymines sample multiple conformations. The availability of the d[AG3(T2AG3)3] solution structure containing four AG3 human telomeric repeats should permit the rational design of ligands that recognize and bind with specificity and affinity to the individual grooves of the G-tetraplex, as well as to either end containing the diagonal and lateral loops. Such ligands could modulate the equilibrium between folded G-tetraplex structures and their unfolded extended counterparts.

We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled oligonucleotides). All sequences were capable of forming stable quadruplexes, with T m above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and T m was found in K+: each added base leads to a 2°C drop in T m or ∼0.3 kcal/mol loss in ΔG°. The trend was less clear in Na+, with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).