Guanine Quadruplex Disrupting PNAs: Potential Anticancer Agents

Go back to projects

Guanine Quadruplex Disrupting PNAs: Potential Anticancer Agents

 

 

The guanine (G) quadruplex is a secondary structure formed by G-rich sequences of DNA and RNA. G quadruplexes consist of G tetrads that are stabilized by hydrogen bonding and cation coordination. Quadruplex-forming sequences (QFSs) typically follow the pattern:

 

                                       QFS: G_n-X_m-G_n-X_m-G_n-X_m-G_n

 

where n = 3, X can be any of the nucleobases, and m = 1 or greater. Synthetic oligonucleotides containing this sequence motif fold into G quadruplexes (Figure 1) which can be characterized by optical and NMR spectroscopy and X-ray crystallography.

 

         

 

Figure 1. Top: Structure of a hydrogen-bonded guanine tetrad. Bottom: Folding of a QFS allows formation of G-tetrads and pi stacking to form the final G quadruplex structure.

 

 

QFSs are often found in important regulatory regions of genomic DNA, including telomeres and promoters, as well as in pre-messenger RNAs. It is very difficult to tell if these sequences actually fold into G-quadruplexes in vivo, although G-quadruplex binding proteins have been identified in a number of organisms.

Of great interest from a biomedical perspective are the suspected roles played by QFSs in a number of cancers: (1) Telomeres are replicated in many cancer cells by telomerase, whereas the enzyme is not present in most normal cells. Maintenance of telomere length is essential for avoiding senescence and apoptosis. (2) QFSs in promoter regions regulate transcription of oncogenes. (3) QFSs in pre-mRNA control splicing to form the mature mRNA such as the mRNA that leads to expression of telomerase. QFS-binding agents can be envisioned to impair these key molecular steps in cancer.

In our lab, we synthesize peptide nucleic acid (PNA) oligomers that bind with high affinity to QFSs. In the most obvious approach, the PNA is complementary to the QFS and therefore binds to form a hybrid PNA-DNA or PNA-RNA duplex according to the Watson-Crick rules for base pairing (Figure 2A). In a second, less obvious strategy, the PNA is homologous (i.e. has the same sequence) to the QFS. In these cases, a hybrid PNA-DNA or PNA-RNA quadruplex is formed (Figure 2B). The specific DNA and RNA targets we have studied so far are described in the publications listed at the end of this section.

 

 

 

Figure 2. Top: Hybridization of a complementary PNA results in disruption of the G-quadruplex target and formation of PNA-DNA duplexes. Bottom: Hybridization of a homologous PNA results in disruption of the G-quadruplex target and formation of PNA-DNA quadruplexes.

 

 

With each new QFS target that we choose, the corresponding homologous PNA is prepared in our lab by solid-phase peptide synthesis. Then, optical spectroscopic experiments are performed to characterize the hybrid quadruplexes that form. These experiments provide information on the stoichiometry and structure of the hybrid. Affinities are typically high, with binding occurring readily even at low nanomolar PNA concentrations under physiological conditions. Our current efforts are focused on QFS targets that are present in the promoter regions of the MYC oncogene and the VEGF gene, telomeric DNA and pre-mRNA for the hTERT gene. Ultimately, our goal is to test QFS-targeted PNAs in cell culture, animals and eventually humans.

 

Funding and Collaboration

 

This work is currently funded by the National Institutes of Health. A major requirement for these biological experiments is that the PNAs be able to cross cell membranes in order to reach their intracellular targets. Our collaboration with Prof. Danith Ly of the CMU Chemistry Department will help us to accomplish this goal. Prof. Ly’s group specializes in synthesizing modified PNAs that exhibit efficient cellular uptake. In addition, we collaborate with Prof. W. David Wilson of the Department of Chemistry at Georgia State University to perform surface plasmon resonance and calorimetry experiments to better understand the thermodynamics and kinetics of hybrid quadruplex formation.

 

Personnel

 

Subhadeep Roy (5th Year graduate student)

Connor Murphy (2nd Year graduate student)

Halimatu Mohammed (1st Year graduate student)

Dr. Gloria Silva (Visiting Assistant Professor)

Henry Huang (Senior chemistry major)

 

 

Publications

 

1. Marin, V. L.; Armitage, B. A. “Hybridization of Complementary and Homologous Peptide Nucleic Acid Oligomers to a Guanine Quadruplex-Forming RNA” Biochemistry 2006, 45, 1745-1754.

2. Marin, V. L.; Armitage, B. A. “RNA Guanine Quadruplex Invasion by Complementary and Homologous PNA Probes” J. Am. Chem. Soc. 2005, 127, 8032-8033.

3. Datta, B.; Schmitt, C.; Armitage, B. A. “Formation of a PNA₂-DNA₂ Hybrid Quadruplex” J. Am. Chem. Soc. 2003, 125, 4111-4118.

4. Datta, B.; Armitage, B. A. "Hybridization of PNA to Structured DNA Targets: Quadruplex Invasion and the Overhang Effect" J. Am. Chem. Soc. 2001, 123, 9612-9619.

 

The Armitage Group