Screening Of Affinity Nucleic Acids For Human TGF-β RⅡfrom SsDNA Random Library By SELEX

Background:Systematic evolution of ligands by exponential enrichment (SELEX)is such a process that permits isolation of oligonucleotides which can bind target molecular with high affinity and specificity from synthetic random library of ssDNA or RNA. The essence of SELEX is artificial evolution in tube. The selected oligonucleotides are called aptamer. Diversity inthree-dimensionalstructures of oligonucleotides in the library is the basis of interaction between oligonucleotides and the target molecules. Compared with antibodies, nucleic acid aptamers possess some advantages such as wider range of target molecules, easily modified, chemical synthesis. Aptamers have found applications in the fields of basic research, clinical diagnosis and drug development.TGF-β family members play an important role in fibrotic diseases. TGF-βs have three types of receptors: TGF-βRⅠ, TGF-βRⅡ, TGF-βRⅢ. TGF-βRⅢ transfers TGF-βs in stroma to TGF-βRⅡ and enhances their affinity. Both TGF-βRⅠand TGF-βRⅡ belong to the Ser/Thr kinase receptor family. TGF-βRⅡ can combine TGF-βs freely while TGF-βRⅠ can only recognize TGF-βRⅡ–ligand complex. TGF-βRⅠactivated by TGF-βRⅡ–ligand complex induces signal cascades such as dislocation of Smads into nucleus and brings a series biological effect. Therefore, TGF-βRⅡ is a good target to block the activity of TGF-βs. Antagonists against TGF-βRⅡ could be of therapeutic value potentially. Aptamers for TGF-βRⅡ have not been reported so far. This study was designed to screen enriched nucleic acid library affinity for human TGF-(RⅡ from ssDNA random library by SELEX.Methods and materials:1. Constructing initial random libraryAn 108 nt ssDNA template containing 60 random nucleotides flanked by invariant primers was designed. It was generated from solid-phase synthesis and completely deprotected and gel purified. 2. SELEX procedure1) The first selection round: Synthetic random ssDNA library (1.2×1015 individual sequences) was incubated with TGF-βRⅡ. Nucleic acid-target complexes were captured in nitrocellulose membrane. The bound nucleic acids were extracted from membrane and amplified by PCR method with a 5( end biotinylated antisense primer. The PCR product was purified on a denaturing polyacrylamide gel, then mixed with Streptavidin-Agarose. The non-biotinylated strand was released by 0.15N NaOH and recovered for the next round selection. 2) The selection rounds 2～4: TGF-βRⅡ was absorbed on the solid matrix Phenyl-Agarose, then incubated with enriched library obtained from the previous round. The bound nucleic acids were obtained from Phenyl-Agarose and amplified by PCR method with a 5( end biotinylated antisense primer. The separation of desired ssDNA was completely the same as that applied in the first selection round. The negativeselection aganist solid matrix Phenyl-Agarose was introduced from theselection round 3 on.3) The selection rounds 5～8: Compared with the selection rounds 2～4, two adjustments were made : (1) In negative selection procedure, enriched library was incubated with solid matrix Phenyl-Agarose absorbed BSA in advance. (2) In selection procedure, yeast tRNA was added into the selecting buffer. The stringency was increased as the selection proceeded.3. Evaluating affinity of enriched library for TGF-βRⅡ1) Nitrocellulose membrane-binding assay:Both random library and enriched library were end radiolabeled with 32P and purified, then incubated with TGF-βRⅡ and BSA in binding buffer. The reaction mixture was applied to the rinsed nitrocellulose membrane and filtered through the membrane. The membranes were dried. The radioactivity remained on the membrane was determined in a scintillation counter as CPM. The pure ssDNA was used as control. The affinity of ssDNA to target molecule was calculated as a percentage by the following formula:(The radioactivity of nucleic acid-protein group remained on the membrane – The radioactivity of nucleic acid control group remained on the membrane) / The radioactivity of...