DNA guanine quadruplexes: When less is more

Guanine quadruplexes (G4) are nucleic acid secondary structures frequently implicated as important regulators of diverse cellular processes across the genomes and transcriptomes of many organisms. The relationship between the primary sequence and the resulting secondary structure of potential quadruplex-forming sequences (PQS) is still not completely clear, particularly for sequences with more than four G-tracts or longer G-tracts. In such cases, additional often contradictory phenomena may arise, including tract sliding within G-registers associated with loop length and conformation polymorphism. Using a combination of computational, biophysical and biochemical methods, we analyzed a subset of naturally occurring DNA PQS characterized by four equally long G-tracts of increasing length, separated by single non-G nucleotides, allowed to form intramolecular G4s with increasing number of tetrads, the principal determinants of G4 stability. We observed that extension of G-tracts length beyond three G does not lead, within timescales relevant to major nuclear processes on DNA, to the expected increase in G4 stability associated with the formation of additional tetrads. Our data indicate that such PQS become kinetically trapped in three-tetrad intermediates, with the surplus guanines positioned within destabilizing extended loops or overhangs. Even after thermal unfolding these intermediates and providing sufficient time, potential monomolecular four-tetrad parallel G4s are thermodynamically outcompeted by the antiparallel ones. From the perspective of cellular processes, longer G-tracts do not confer higher stability of the resulting G4s; they may, however, enhance the conformational modularity and regulatory potential of the relevant PQS. • A subset of natural DNA PQS sites with (GnT)3Gn central motif was studied for n = 3–5. • At fast timescales, G-tracts extension beyond 3G does not increase the G4 stability. • 4-tetrad ps G4 does not form even with 5-G-tract sequences in our system. • 3-tetrad ps and 4-tetrad aps G4s compete due to kinetic vs thermodynamic preference. • Extended G-tracts facilitate polymerase passage, supporting reduced G4 stability.