The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.

Nucleic acid quadruplexes, based on the guanine quartet, can arise from one or several strands, depending on the sequence. Those consisting of a single strand are folded in one of two principal topologies: antiparallel, in which all or half the guanine stretches are antiparallel to each other, or parallel, in which all guanine stretches are parallel to each other. In the latter, all guanine nucleosides possess the anti conformation about the glycoside bond while in the former, half possess the anti conformation, half the syn conformation. While antiparallel is the more common fold, examples of biologically important, parallel quadruplexes are becoming increasingly common. Thus it is of interest to understand the forces that determine the quadruplex fold. Here we examine the influence of individual nucleoside conformation on the overall folding topology by selective substitution of rG for dG. We can reverse the antiparallel fold of the thrombin binding aptamer (TBA) by this approach. Additionally, this substitution converts a unimolecular quadruplex into a bimolecular one. Similar reverse substitutions in the all-RNA analog of TBA result in a parallel to antiparallel change in topology and alter the strand configuration from bimolecular to unimolecular. Based on the specific substitutions made, we conclude that the strong preference of guanine ribonucleosides for the anti conformation is the driving force for the change in topology. These results demonstrate how conformational properties of guanine nucleosides govern not only the quadruplex folding topology but also impact quadruplex molecularity and provide a means to control these properties.

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Among the many unusual conformations of DNA and RNA, quadruplex structures, based on the guanine quartet, possess several unique properties. These properties, along with the general features of guanine quadruplexes, are described in the context of possible roles for these structures in biological systems. A variety of experimental observations supporting the notion that quadruplexes are important in vivo is presented, including proteins known to specifically bind to quadruplex structures, guanine‐rich DNA, and RNA sequences endowed with the potential for forming quartet‐based structures in telomeres and regulatory regions, such as gene promoters, quadruplexes as DNA aptamer folding motifs arising from in vitro selection experiments, and potential chemotherapeutic, quadruplex‐forming oligonucleotides. Taken together, all of these observations argue cogently not only for the presence of quadruplexes in biological systems but also for their significance in terms of their roles in various biological processes. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 209–227, 2001