Small molecule/nucleic acid interactions play sophisticated regulatory roles in cellular functions. Aberrant interactions have been implicated in diseases, and nucleic acids are targets of small molecule therapeutics. Spermine, a polyamine, binds specifically to RNA tertiary structures to regulate cell growth and protein expression. Polyamines levels are elevated in neoplastic cells and polyamine biosynthesis is a target of chemotherapeutic agents, but how polyamines function is largely unknown. Pentamidine, an FDA approved drug for treating fungal and protozoan infections also has antitumor activity and was recently identified as a lead compound for treatment of Myotonic Dystrophy, is presumed to act by binding nucleic acids but the molecular basis of its activity is not known. To better understand the interactions of these small molecules with DNA and RNA, we will develop a new technique to map their nucleic acid binding sites to nucleotide precision. The proposed protocol will employ analogs of the molecule of interest containing alkynyl or biotinyl moieties that will allow the molecule to be selectively purified from cellular debris with affinity chromatography. The analogs will be crosslinked to DNA (RNA) using ultraviolet light, and the small molecule/nucleic acid adduct will be purified using the engineered molecular affinity. The proposed protocol is an adaptation of techniques routinely used to identify the nucleic acid binding sites of proteins (e.g. Crosslinking and Immunoprecipitation: CLIP) in the cell. Literature experiments indicate that spermine and pentamidine will crosslink with nucleic acids and preliminary mass spectrometric data indicates that pentamidine will crosslink with a short hairpin RNA. The sequence information we obtain will allow us to determine which genes and regulatory elements are most affected by polyamines and which DNA(RNA) sequences pentamidine binds to exert its cellular functions. This information will define the role of polyamines in normal and neoplastic cell growth and a deeper understanding of polyamine function will be valuable in identifying new therapeutic targets and designing chemotherapeutic agents. An understanding of the molecular mechanism of pentamidine's function will guide second-generation analogue synthesis for more specific, less toxic drugs. This approach, small molecule affinity purification and sequencing (SMAP-seq), may find application as a general protocol for investigations of small molecule-nucleic acid interactions.
The technology we are developing will be a valuable tool for learning about the molecular basis of many diseases and how drug therapies work. By focusing on pentamidine and polyamines we will gain information that will help us to identify new drug targets and make new, less toxic and more specific drugs for treating fungal and protozoan infections, as well as drugs for the treatment of cancer and myotonic dystrophy.
