摘要
A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclero- sis （ALS） and frontotemporal dementia （FTD）. We examined G-quadruplex forming ability of GGGGCC- repeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K~ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5＇- and 3＇-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[（GaC2）3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance （NMR） spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when sub- stituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG sub- stitution at position 21 and fine tuning of folding conditions facilitate folding of d[（G4C2）3GG^BrGG] into （mostly） a single G-quadruplex, and thus enable determi- nation of its high-resolution structure by high-field NMR.
A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclero- sis （ALS） and frontotemporal dementia （FTD）. We examined G-quadruplex forming ability of GGGGCC- repeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K~ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5＇- and 3＇-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[（GaC2）3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance （NMR） spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when sub- stituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG sub- stitution at position 21 and fine tuning of folding conditions facilitate folding of d[（G4C2）3GG^BrGG] into （mostly） a single G-quadruplex, and thus enable determi- nation of its high-resolution structure by high-field NMR.
