| Being a carrier of genetic information,nucleic acids participate in guiding the operation of life function and play important role in all biological,biochemical and medical sciences.The variety of composition,sequence and conformation result in the complexity of nucleic acids structure,and the chemical properties and structures determine the biological function of nucleic acids.The rapid growth of human genome projects as well as the novel therapeutics based on antisense oligonucleotides has stimulated the search for development of fast and reliable methods for the separation and analysis of nucleic acids.However,the large size and extraordinary conformational flexibility of nucleic acids provide key challenges to the separation.By virtue of its high resolving capability,short cycle times,and full automation capability,high performance liquid chromatography(HPLC)becomes one of the common separation methods,and is widely used in the separation and analysis of nucleic acids.The structure characteristics of nucleic acids can be reflected in chromatographic behavior,otherwise the chromatographic behavior can reveal the structure information of nucleic acids.In this paper,reversed-phase liquid chromatography(RPLC)being selected as the main separation technique,the influence of base composition,base sequence,secondary structure,stationary phase,mobile phase and etc.on retention behavior of nucleic acids was investigated.The relationship between nucleic acids structure-retention behavior,and the interaction mechanism between nucleic acids and RPLC stationary phase both were explored.The exiting state and structure behavior of nucleic acids in solution were studied further.The major contents of this paper are described as follows:(1)The retention behaviors of oligonucleotides and double-stranded deoxyribonucleic acids were investigated on silica-based C18 stationary phase by ion-pair reversed phase liquid chromatography(IP-RPLC).It is found that the retention of oligonucleotides was influenced by base composition and base sequence other than size,and oligonucleotides prone to form self-dimer have weaker retention than those not prone to form self-dimers but with same base composition.However,homo-oligonucleotides are suitable for the size-dependent separation as the special case of oligonucleotide.For double-stranded deoxyribonucleic acids,the retention is also influenced by base composition and base sequence,as well as size.This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase.In addition,no specific influence of guanine and cytosine contents was confirmed on retention of double-stranded deoxyribonucleic acids.Notably,the space effect resulted from the stereo-structure of nucleic acids is also the influence factor for their retention behavior in IP-RPLC.(2)Monolithic octadecylsilane column and particle-packed octadecylsilane column were used to investigate and compare the retention behaviors of oligonucleotides under different column temperature by using IP-RPLC.It is found that,on monolithic column,hairpin oligonucleotides always have weaker retention than corresponding random coil oligonucleotides with same base composition,whereas,the retention orders of hairpins and corresponding random coils on particle-packed columns have no rule to follow.In addition,there is a relative poor linear correlation between retention factor(k)of oligonucleotides and reciprocal of temperature(1/T)on particle-packed column compared with monolithic column,especially for hairpins.Moreover,the smaller particle size of particle-packed columns,the poorer correlation between k and 1/T.Results also revealed that the overall retention order on particle-packed column with small particle(3 ?m)differed greatly from that on monolithic column,however,the retention order on 10 ?m particle-packed column was approaching to that on monolithic column.From the above,we infer that oligonucleotides can keep their natural conformations when passing through monolithic column,attributed to the special pore structures of the monolith.However,the conformations of oligonucleotides are suppressed or even be destroyed when passing through particle-packed columns due to the narrow and tortuous channels among the stacked stationary phase particles,leading to more complex and unequable retention behaviors difficult to sum up.Therefore,monolithic column exhibits better retention regularity for oligonucleotides with secondary structure especially for hairpins than particle-packed column.It is noteworthy that the separation based on monolith opens an intriguing prospect in accurately elucidating the IP-RPLC retention behaviors of oligonucleotides.(3)The retention behaviors of oligonucleotides were investigated under mobile phase containing mixed ion-pair reagents of triethylamine/propylamine-acetate(TEA/PA-AA)and compared with that under classical mobile phase containing triethylamine acetate(TEAA)as ion-pair reagent by IP-RPLC.It is found that the retention of oligonucleotides under TEA/PA-AA system was always weaker than that under TEAA system when the concentration of ion-pair reagents was kept the same.In addition,retention time for most oligonucleotides became longer and longer as the increasing concentration of ion-pair reagents(20-120 mM).As the special case,the retention time of(dC)n(n>10)was relatively unchanged.It should be that the retention of(dC)n increases fast with the enhanced concentration of ion-pair reagents,and the maximum retention is achieved under relatively low concentration of ion-pair reagents.Additionally,short chain(dT)n and hetero-oligonucleotides both had better resolution under TEA/PA-AA system than TEAA system.Nevertheless,(dA)n and(dC)n both had better resolution under TEAA system.What's more,the dominated separation mechanism of oligonucleotides under the two systems both were ion-pair model,deduced from the relationship between k of oligonucleotides and total ion-pair reagents concentration(CP).In general,TEA/PA-AA especially under moderate concentration is superior to TEAA in the separation of hetero-oligonucleotides having same chain length and similar structure.Ion-pair reagent with low concentration improves the compatibility with fowwowing electrospray ionization-mass spectrometer(ESI-MS),which benefits the qualitative analysis of oligonucleotides.(4)The polymorphism of three sequences d[(G3T)3G3],d[(G3C)3G3]and d[(G3T)4]annealed in six different solutions were investigated by means of RPLC,liquid chromatography-mass spectrometry(LC-MS),fluorescence spectroscopy,circular dichroism spectroscopy,together with native-polyacrylamide gel electrophoresis.Different G-quadruplex(G4)conformations of these three sequences can be separated clearly by RPLC,and further characterized by on-line LC-MS analysis.It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G3T)3G3]and d[(G3C)3G3]under the annealing conditions.However,flanking loop impeded d[(G3T)4]to form higher-order structures than dimer.In addition,the nature and concentration of cation,as well as the annealing solution component,all have decent influence on the stability and relative ratios of various G4 building blocks.Based on the above findings,RPLC and LC-MS combined with spectroscopy and electrophoresis can be used as a facile and powerful tool for quick separation and identification of G4s in solutions,and for effective assessment of DNA sequences and annealing environments on G4 polymorphism.The established protocol provides a novel strategy for evaluating G4 polymorphism,which will facilitate elucidation of quadruplexes structure and their biophysical function.The research towards nucleic acids retention behavior in this present work benefits to the selection of chromatographic separation systems in order to purify,characterize and quantitate nucleic acids,also can reveal the existing state of nucleic acid in solution,which will help the study of nucleic acids structure and their biophysical properties. |
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