The Investigation Of Telomere DNA Topology Using AFM-based Single Molecule Force Spectroscopy

Telomeres are eukaryotic linear chromosomal protection ends.In mammals,telomeric DNA is composed of thousands of(TTAGGG)n/(CCCTAA)n repeats,and the G-rich strand forms a short(?50–500 nt)single-stranded overhang on the 3'end,which can invade into upstream duplex telomeric DNA resulting in a telomere loop(T-loop).At the same time,both G-rich and C-rich strands can form unusual higher-order DNA structures(G-quadruplex and i-motif),so telomere DNA has a more complex topology in different solutions compared with normal double-stranded DNA(dsDNA).And the function of telomeres is closely related to their structural specificity.Telomere shortening is one of the factors that causes cell senescence.Telomeric DNA is also critical for genomic integrity and plays an important role in cancer.Due to the difficulty in synthesizing repeated sequences and the complexity of long tandem higher-order structures,people do not fully understand the topological structure of double-stranded or single-stranded long-chain telomeric DNA.A deeper understanding of telomere topology close to its actual length will be helpful to provide new ideas for designing anticancer drugs.Here,long double-stranded telomere sequences and long G-rich single-stranded sequences are synthesized by EM-PCR,TA cloning and rolling circle amplification.Single molecule force spectroscopy(SMFS)based on atomic force microscopy(AFM),circular dichroism(CD),UV and gel electrophoresis were used to study the structure properties of telomere DNA.The main findings are as follows:1)We investigated the folding/unfolding behavior of human telomeric sequence with the length over 10 knt through CD spectroscopy and AFM-based single molecule force spectroscopy.A new type of intermediate with two or four guanines at 5'and 3'ends of every G-quadruplex forming sequence missing from the tetraplex was captured in the long G-rich DNA,which is named as pre-G-quadruplex(pre-GQ).According to the number of guanine involved in folding,pre-GQ is divided into two types.In comparison to single G-quadruplex,the tandem pre-G-quadruplexes exhibited a 10 pN lower unfolding force suggesting the destabilization due to their incompletely folded state.By performing the repeatedly stretching and relaxing manipulation,we inferred that the folding of long telomere G-strand may undergo a two-step pathway which involves the fast transition from unstructured ssDNA to G-hairpin state(in seconds)and the slow folding of free guanines into the vacant sites in tetraplex to form type 1,type 2 pre-GQ and complete GQ,which could be accelerated in the presence of 40%PEG.This study sheds light on the structure and dynamic folding mechanism of long-chain tandem G-quadruplexes.2)The factors affecting the topological structure of long-chain double-stranded telomeric DNA were investigated by SMFS.To do that,we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA.The CD results show that the long(700 bp)telomeric dsDNA can even keep its double helix structure in the presence K~+in acidic aqueous solution at room temperature,which is different from shorter(e.g.,21 bp)telomeric sequence.Using mechanical manipulation assays based on single molecule atomic force microscopy and some traditional methods,we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation higher-order structures(G-quadruplexes or i-motifs).Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structure(G-quadruplex and i-motif)at the same time(e.g.,in the presence of 100 mM KCl under pH 4.7),the higher-order structure(s)can remain after the external force is removed.In the presence of monovalent K~+,single wall carbon nanotubes(SWCNTs),acidic conditions,or short G-rich fragments(?30 nt)can switch the transition from dsDNA to higher-order structures.Our results provide a new way to regulate the topology of telomeric DNA.The results deepen our understanding of the relevant biological functions of telomeres since mechanical force exists in many biological processes,such as DNA replication and transcription.In addition,this method can be used to study other long-chain dsDNA with tandem repeat sequences.3)Single molecule force spectroscopy was used to explore the interaction between G-quadruplex and TRF2.As an important member of telomere binding proteins(shelterin),TRF2 plays an important role in the formation of T-loop and the stabilization of telomere structure.Firstly,we demonstrated by AFM imaging that TRF2 protein can specifically bind to the G-quadruplex.Then using single molecule force spectroscopy,we found that when the TRF2 protein binds to the G-quadruplex,the unfolding force of G-quadruplex is decreased.And through the calculation of the hysteresis area between the stretching curve and the relaxation curve by the Jarzynski model,the work equivalent to the change in free energy of a single G-quadruplex unfolding before and after adding TRF2 can be obtained.These results prove that the binding of TRF2 to G-quadruplex will reduce the stability of G-quadruplex.And it may be one of the reasons that why TRF2 is beneficial for stabilizing the T-loop structure.