Phasor Diagram Analysis of Telomeric G-Quadruplex Stability and Heterogeneity

Abstract

Telomeres function to protect the end of linear chromosomes from damage and degradation. Human telomeres consists of several kilobases of the tandem repeat d(TTAGGG), which terminate in a 3′-single stranded overhang capable of forming G-Quadruplex structures. Telomeric G-Quadruplexes have potential roles in the maintenance of telomere integrity and the control of telomerase activity, an enzyme responsible for elongation of the telomeric overhang that is over expressed in a wide variety of cancers. Investigation of intramolecular G-Quadruplexes corresponding to the human telomere is complicated by the formation of multiple rapidly interconverting conformations. The multiple telomere G-Quadruplex conformations are identified by unique folding geometries containing distinct connecting loop regions.Time-resolved fluorescent measurements have been conducted to characterize the stability and heterogeneity of the telomeric G-Quadruplex forming sequence d(AGGG(TTAGGG)3). The fluorescent adenine analog, 2-aminopurine, served as an internal fluorescent probe of G-Quadruplex conformation and was used to specifically monitor loop regions. 2-aminopurine demonstrates a complex fluorescent decay upon incorporation into nucleic acid polymers that can be simplified by transformation of time-resolved data to an (S,G) phasor diagram. Phasor diagrams were used to monitor G-Quadruplex formation upon addition of NaCl or KCl and elucidated a unique KCl-dependent transition. The digestion of G-Quadruplex structure by a single-strand specific nuclease and acrylamide quenching are also evaluated through phasor diagrams. This work demonstrates the utility of phasor diagrams for the study of nucleic acid systems and the ability to sensitively monitor complex fluorescent decays that may complicate analysis through nonlinear regression techniques. Supported by awards RO1CA35635 and RO1GM077422 from the NCI and NIGMS.