The Formation Of G-quadruplexes In Double-stranded DNA And Dissecting The Structure Of G-quadruplexes By Footprinting

Nucleic acid can adopt a four-stranded DNA structures known as G-quadruplex. Large numbers of guanine-rich sequences with potential to form G-quadruplexes have been identified in genomes of various organisms. It is now believed that G-quadruplexes play important role in regulating gene expression and thus constitute valuable therapeutic targets against cancer and other diseases. In addition to Telomere 3'overhang, G-quadruplex-forming sequences are constrained at both ends by long DNA duplex with a complementary strand in close proximity to compete for duplex formation. G-quadruplex/duplex competition in long double-stranded DNA has rarely been studied. In this work, we prepared long dsDNA from human genome carrying G-quadruplex-forming sequences with flanking duplex at both sides and studied G-quadruplex formation under both dilute and molecular crowding conditions during the process of in vitro transcription and heat denaturation/renaturation. Our data revealed that molecular crowding creates an essential environment for stable G-quadruplex to form in dsDNA. Using DMS footprinting and gel electrophoresis, we prove the formation of G-quadruplex. Our results also showed that the heat denaturation/renaturation treatment followed by gel electrophoresis could provide a simple method to quantitatively access the ability of G-quadruplex formation in long double-stranded DNA. The effect of K+ and PEG concentration was investigated and we found that stable G-quadruplexes could only form under the crowding condition with PEG at concentrations near the physiological concentration of biomass in living cells. This observation reveals a physicalbasis for the formation of stable G-quadruplexes in genome and supports its presence under the in vivo molecular crowding condition.The wide spread of G-quadruplex-forming sequences in genomic DNA and their role in regulating gene expression has made G-quadruplex structures attractive therapeutic targets against a variety of diseases. Information on the structure of G-quadruplexes is crucial for understanding their physiological roles and designing effective drugs against them. Resolving the structures of G-quadruplexes in double-stranded DNA is critic for searching and designing drugs.Using the quantitatively method to measure the ability of G-quadruplex formation in long double-stranded DNA as previous, we find a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc). This ligand have especially good shape complementarity with the quartet plane and interact with G-quadruplex via stacking externally to the terminal G-quartets. Furthermore, under light irradiation, phthalocyanines cleave DNA via generation of singlet oxygen. Because of these properties, we developed a photocleavage footprinting technique to determine the folding orientation of each individual G-tract in intramolecular G-quadruplex formed in both single- and double-stranded nucleic acids. Based on the differential photocleavage induced by a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc) to the guanines between the two terminal G-quartets in a G-quadruplex, this method identifies the guanines hosted in each terminal G-quartets to reveal G-tract orientation. The method is extremely intuitive, straightforward, and requires little expertise. Besides, it also detects G-quadruplex formation in long single- and double-stranded nucleic acids.