Cytocine Rich DNA Strand Forming I-motif Nonconvalent Complex Studied With Electrospray Ionization Mass Spectrometry

Human telomere and centromere and animal telomeres are rich of cytosine (C)oligodeoxynucleotide DNA sequences. The C-rich olignucleotide sequences can forma four-stranded structure by the intercalation of two parallel duplexes containinghemiprotonated C·CH+in acidic environment, which is called i-motif. Based onnumbers of the C stretches in DNA strand, the four-stranded i-motif structures can beformed by the unimolecular folding, folded bimolecular or tetramolecular interactions.As telomeric C-rich oligonucleotides can form i-motif structures in vitro, and it canselectively bind with some proteins. It has been proposed that they may play a role inbiological process. The property of i-motif formation derived by protons can beapplied in nano-device. The common methods used for studying i-motif are NMR,X-ray diffraction, circular dichroism, UV-thermal melting, and so on. Electrosprayionization mass spectrometry (ESI-MS) has been widely used for the analysis ofnon-convalent complexes formed by variety of DNA strands due to ‘soft ionization’technique. In this work we use ESI-MS to investigate the formation of thetetramolecular i-motif structure by measuring the molecular mass of thetetromolecular i-motif in the gas phase.Firstly, the ESI-MS instrumental parameters and solution conditions for theformation of the tetramolecular complexes are optimized using DNA strandd(TCCCCC); the optimal pH is consistant with the reports studied by the othertechniques. Under the same conditions, the effect of cytocine length on the i-motifformation were investigated, results show that the tetromolecular [dCn]4was detectedwhen n>3.Secondly, in order to find the effect of additional bases and base numbers addedonto5′or3′end on the formation of the tetramolecules, a series of DNA strands withsequence d(XnCCCCYm) were designed, where X, Y=thymine, adenine or guanine, and n, m comprised between0and2. In our conclusion, i-motif formation is favoredby sequences with additional base residues located at5′end. In addition, the basespecies can make a dramatic influence on the formation of the tetramoleculesfollowing this order: T> A> G. The influence of the base number on the tetramerformation varied with base species.Thirdly，with interest to the unknown peak which appearred in the mass spectrum,correlative experiments were performed. The neutral loss of furfuryl alcohol and baseloss from the short DNA strands were observed in the mass spectra of during ESIprocess. In addition, the purine base hydrolysis occurred in annealing process in waterbath.Futhermore, CD spectroscopic method is used to study the tetromolecules in thesame solution conditions as they were studied by MS and consistant results areobtained comfirming that MS method is fast and effective for the study of the i-motif.