1The growing recognition of the important roles of guanine quadruplex structures in regulating gene expression and telomere biology have motivated considerable efforts to design or discover synthetic molecules that can bind to quadruplexes with high affinity and selectivity.1–3
Quadruplex-binding ligands might find applications in chemical biology, where they could report the presence or perturb the biological function of a quadruplex.4,5
Clinical applications of drug-like small molecules targeted to G-quadruplexes are also envisioned.6
Small molecules bind to G-quadruplexes primarily through shape-based recognition. An alternative strategy relies on sequence-based recognition, where complementary C-rich oligonucleotides bind via Watson-Crick base pairing to form heteroduplexes7–10
or homologous G-rich oligonucleotides bind via Hoogsteen-based G-tetrad formation to yield heteroquadruplexes.11–15
Peptide nucleic acid (PNA) probes can bind to G-quadruplexes by either of these mechanisms, leading to stable hybrid structures.
Our recent focus has been on PNA-DNA/RNA heteroquadruplex formation, based on the high affinity (low nanomolar Kd
s) of the PNA for its homologous DNA11,13,14
targets, as well as excellent sequence discrimination for homologous versus complementary targets that can be achieved through PNA backbone modification.14
Short PNAs consisting of two G2
tracts and various nucleobase or abasic loops successfully invade G-quadruplex targets under physiological conditions to form stable heteroquadruplex structures. The heteroquadruplex stoichiometries are typically 2 PNA: 1 DNA or RNA. Hence, the short PNAs disrupt the folded homoquadruplex then hybridize such that each of two PNA strands recognizes two of the four G-tracts that make up the quadruplex target.
The current report describes our first steps toward combining sequence- and shape-based recognition of quadruplexes through the simultaneous application of PNAs and small molecules. In our prior work, we used a covalently conjugated fluorogenic cyanine dye to provide a fluorescence response to PNA binding, as in the “light-up” probes originally reported by Ishiguro and coworkers for DNA17
and subsequently by Svanvik and coworkers for PNA.18,19
Recently, Ladame and coworkers15
combined a PNA G3
tract that could form one edge of a heteroquadruplex with an acridone stacking ligand previously shown to end-stack on G-tetrads.20
Although the examples cited above clearly illustrate that a covalently conjugated dye can interact with a PNA-DNA heteroquadruplex, it was unclear to what extent the dye would interact with the heteroquadruplex if not for the covalent linkage to the PNA terminus. While still composed of G-tetrads found in DNA homoquadruplexes, PNA-DNA heteroquadruplexes offer unique structural characteristics that could be useful for small molecule recognition. For example, the negative charge density in a heteroquadruplex is less than half that of a homoquadruplex, due to the uncharged nature of the backbone and the presence of cationic termini on the PNA. Thus, electrostatic interactions offered by the two types of quadruplex will be quite different. Similarly, the van der Waals surfaces of the quadruplex grooves should vary considerably between homo- and heteroquadruplexes.
Cyanine dyes have found numerous applications in nucleic acid recognition,21,22
with various reports on intercalation, groove binding and end-stacking on duplexes,23
The wide range of absorption/emission wavelengths accessible to the cyanines, the ease with which the net charge on the molecule can be varied through substituents, and the versatility of the dyes, in terms of either having environmentally sensitive or insensitive quantum yields, have been exploited in these diverse studies. In this report, we describe two carbocyanine dyes that bind noncovalently to PNA-DNA heteroquadruplexes. These dyes can exhibit variable fluorescence enhancements upon binding, depending on the loop composition of the PNA strand. These results open the door to using cyanine dyes in combination with G-rich PNAs to form and detect heteroquadruplex structures.