The Novel Post-SELEX Optimization, Characterization And Sensing Methods Toward Minimized Aptamer

Oligonucleotide aptamer(aptamer)refers to a short nucleotide(ssDNA or RNA)that is screened and generated by the Systemically Evolution of Ligands by EXponential Enrichment(SELEX)technique.The aptamer can fold into a unique three-dimensional structure,which is complementary to the target molecule to form high affinity and high specificity,that has been called “chemical antibody”.The molecular recognition pattern between aptamer and target molecule is very similar to that of antibodies,but the specificity and affinity are even higher.Aptamer rivals antibody in several aspects,such as good stability,prepared by automated chemical synthesis,no immunogenicity,and non-toxic.In addition,aptamer itself is an oligonucleotide,it owns the features of fast and reversible denaturing and renaturation,easy functional modification and labeling,and as an excellent nano-device,etc.Aptamers are a class of important “star molecules”.With the rapid development of new SELEX screening techniques,high throughput sequencing,biochip assay,and other sequencing/bioactivity evaluation techniques,the number of aptamers is increasing fast.The target molecules varied from ions,small molecules,transcription factors,proteins(enzymes),to cells,tissues,bacteria,viruses,etc.Aptamers have great potential in the analysis,sensing,clinical diagnosis and treatment.However,to date,only a handful of aptamers have entered the clinical trial phase,and only one aptamer drug has been approved by the US Food and Drug Administration,that is Pegaptanib,for the clinical treatment of age-related macular degeneration.There are few aptameric candidates for commercial clinical diagnostic use.It is not a good match with the trend of prosperous development of aptamers.The main technical bottlenecks are the secondary or tertiary structures of aptamers are not so clear,the interaction mechanism with drug target is unknown,and the bioavailability of therapeutic aptamers should be improved and so on.From the molecular interaction level,most aptamers have structural flexibility and diversity.When recognizing the target molecules,they mostly form “adaptive” conformation with high specificity and high affinity.Most precise structural models of the aptamer-target molecules have not yet been obtained.Most understandings on recognition patterns are partial and empirical.In addition,in the full-length sequence of the aptamer screened by SELEX,only sequence segment has binding affinity.There is an urgent need to develop effective methods to guide the structural truncation and modification,and to find the aptamer with higher affinity and more specificity without reductant sequence,so as to improve tolerance,versatility,and stability in aptamer recognition researches.This makes SELEX optimization(post-SELEX)a highlighted research area.In the Post-SEELX approaches,the aptamer sequence was more randomly tailored or site-directed mutated according to the empirical analysis of the secondary structure and free energy of the nucleic acid(?G).The main drawback is that the random way is inefficient and difficult.There is no systematic theoretical guidance and a better solution.Toward fundamental issues occurred in post-SELEX,the main content of this thesis is an in-depth study,which is divided into five chapters.The first chapter is the introduction(literature review).We have summarized the characteristics,the application prospect and the main problems met in the researches on the aptamer.We have gleaned and presented the comments on the optimization of the aptamers in post-SELEX,the corresponding methods on interaction evaluation and optimization,the aptamer structure characterization techniques,and the aptamers as analytical and sensing modules.Finally,the basis of the thesis and the main research contents is aroused.The second chapter is the construction of a stepping combinatorial library,and method development of affinity evaluation by surface plasmon resonance(SPR).In this chapter,based on the concept of combination chemistry,aptamer 807-39 nt for recombinant human erythropoietin-?(EPO-?)was used as the model molecule,we have invented and designed four clusters of stepping sequence,constitute a new multidirectional,stepping small library of aptamer analogues.The library contains four clusters,such as left contraction,right contraction,inner contraction,and out extension clusters of a total of 35 sequences.We have also developed a label-free SPR method,to rapidly and efficiently screen and evaluate the above-mentioned aptamer analogues.The minimized aptamer-In27 was picked out,which has a sole loop,without original stem portion from the initial aptamer 39 nt but retains the whole binding affinity.It confirms the rationality and high efficiency of the construction of this small stepping library.The third chapter is the investigation on the degenerated sequences and binding sites of minimized aptamer In27.In this chapter,we used a method of site-directed mutagenesis,combined with domain division,to screen and obtain the key base of great effect on the binding in the In27 sequence.Considering that In27 still retains the sequence of consecutive guanine nucleotides in the aptamer 39 nt,which has a good accordance with the general principles of G-quadruplex(GQ)formation,the base of In27 was divided into D1~D5 domains by interval-GG(G)-skeleton on the basis of CD characterization.Six clusters of site mutations were constructed,named as D1,D2,D3&4,D5,GQ and degenerated sequences in a total of 32 sequences.We used the SPR screening technique to derive the degenerated sequence of In27,and to clarify the key bases on the aptamer In27.Further,we used affinity and competitive analysis to define the binding site of a variety of recognition molecules,such as the aptamer 39 nt and In27,the EPO receptor(EPOR),the monoclonal antibody of EPO,and the wheat germ agglutinin(WGA)on the EPO-? protein.The above research has laid the foundation for the investigations on the structural characteristics of the aptamer,and the aptameric sensing method development.The fourth chapter is an investigation on the characteristics of the active tertiary structure of In27 with a variety of spectroscopic techniques.The structure of the aptamer is diverse,and its “adaptive” dominant conformation is more susceptible to the influence and regulation of the microenvironment including pH,cation type,stabilizing/protecting agent,and even interface.It is the very reason that the interaction between aptamer and target molecule is urgent to be clarified.In this chapter,we have developed SPR,CD and fluorescence probe techniques to illustrate the relationship between In27 activity and conformation.We found that the presence and concentration of cationic Na+ were the key factors that affected the specific affinity recognition of In27.Cation Na+ induced aptamer In27 form a parallel/antiparallel mixed GQ conformation.Two GQ-specific fluorescent probes,thysmenonine T(ThT)and Nmethylporphyrin IX(NMM)can be used as a pair of GQ probes for this active hybrid conformation of In27.The addition of EPO-? can compete with ThT and NMM,so the “light switch-on/off” sensing methods can be conveniently developed by using both GQ fluorescent probes.The above investigations provide an effective combination of techniques for elucidating the active conformation of a novel aptamer,and provide a class of modules with clear interaction features for analytical,sensing,and diagnostic applications.The fifth chapter is a label-free sensing method development on aptamer.Biolayer Interferometry(BLI)is a new surface sensing technique,which is characterized by label-free,sensitive,fast and diverse modes.In this chapter,we immobilized the biotinylated aptamer 39 nt or In27 on the streptavidin(SA)sensor chip,and have optimized the orientation,concentration,and elimination of non-specific effects,and regeneration conditions,etc.We have further used WGA as a module of signal amplification,based on that WGA and aptamer have different binding sites on EPO-?,to build a sensitive platform for the EPO-? protein detection in solution.We at last compared the SPR and BLI methods,and explained the possible reasons.This research broadens the applicability of the aptamers as a sensing module.In conclusion,our thesis focused on the construction of an innovative small multidirectional stepping combinatorial library of aptamer analogues.We have high efficiently screened and optimized the minimized aptamer-In27.We then have built a degenerated sequence on the basis of clarifying the contribution of key base.We clarified and simplified the key microenvironmental factors influencing the active structure of the aptamer by a composition of spectroscopic techniques.The characteristics of tertiary structure of the In27 induced by Na+ were elucidated.Finally,a label-free,highly sensitive sensing method for EPO-? protein was developed.It provides a kind of suitable aptamer for the analysis,sensing,diagnosis and other applications.It also provides an effective method for optimizing the structure of the aptamer and elucidating the structural characteristics of the aptamer and the target protein.