NMR Solution Structures Of G-quadruplexes Formed By Short Fragments Of Telomeric DNA Sequence

G-quadruplex(GQ)is a four-stranded helical structure of guanine-rich sequence widespread in organisms,and plays an important role in many biologically relevant processes in vivo.As a fundamental structural element,GQ has found broad applications in the fields of medical diagnosis,biosensor,and nanotechnology.The orientation of any two adjacent strands within a GQ can be either parallel or anti-parallel with respect to each other.However,tetrameric GQs assembled by the tetramerization of single-repeat fragments of telomeric DNA almost exclusively adopt the all-parallel strand orientations rarely with an exception,which greatly limits its application.In this work,NMR structural studies have been performed on a variety of short fragments with different lengths of telomeric DNA.We found for the first time that d(GGGTTA)and d(GTTAGG)enable the formation of novel GQs.These discoveries multiply the diversity of GQ and thus perform potential applications.The DNA fragment of d(GGGTTA)folds into a(2+2)alternating antiparallel tetrameric GQ of three-tetrads in K+solution,and the same structural topology is also preserved either in Na+or NH4+solutions.Within this four-stranded GQ,two diagonally-positioned strands are symmetric and have the same strand orientation;whereas two adjacent strands are asymmetric and have the opposite strand orientation.The glycosidic bond angles(GBAs)of consecutive guanines along each G-tract of two adjacent asymmetric strands are 5'-syn-anti-anti-3' and 5'-syn-syn-anti-3 ',respectively.In contrast,all GBAs of consecutive guanine residues in a conventionally parallel tetrameric GQ adopts anti conformations exclusively.In addition,we found that a spontaneous transformation from this tetrameric antiparallel GQ structure into another octamer staggered higher-order structure gradually took place over time.This structural transformation can be regulated by the concentration of DNA and K+ions,and the addition of molecular crowding reagent PEG-200.The other DNA sequence d(GTTAGG),is self-trimerized into a novel asymmetric trimolecular GQ(tri-GQ)with two-stacked G-tetrads preferentially in Na+solution,tolerant to an equal amount of K+cation.Usually,the conventional G-columns which are composed of the sugar-phosphate backbone of contiguous G-tract support stacked G-tetrads between each layer.Remarkably in this tri-GQ,except three successive G-columns with the GBA arrangement of 5'-syn-anti-3',there is the fourth broken G-column which is composed of two scattered anti 5'-G residues,head to head in spatial arrangement,from two differently folded strands of d(GTTAGG).Meanwhile,the overhung 5'-G residue with a syn GBA of the third d(GTTAGG)strand protrudes out of GQ core.Notably,this tri-GQ is featured with two width-irregular grooves adjacent to the broken G-column,in which the width combinations are variable along a single groove.This unique dimension of the width-variable grooves is expected to provide a distinctive target platform for potential GQ-specific binders.In addition,NMR EXSY experiments reveal that the tri-GQ of d(GTTAGG)is not just simply a static structure but rather a dynamic assembly.Fast strand exchanges spontaneously occur on a timescale of seconds at 17?,between folded tri-GQ and unfolded single-strand of d(GTTAGG)that both species coexist in dynamic equilibrium.This is the first solution structure of a well-organized GQ actually assembled dynamically.