Screening and Identification of Nucleic Acid Aptamers Specifically Binding to Human Lipocalin 6 / 中国生物化学与分子生物学报

It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science. Nucleic acid aptamers targeting sperm cell-surface proteins can be used for the separation and purification of sperms from mixed stain samples. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and is associated with sperm maturation. Using the systematic evolution of ligands by the exponential enrichment (SELEX) technique, magnetic bead-bound hLCN6 was used as the target molecule to screen for aptamers with high affinity and specificity to hLCN6 from a random single-stranded DNA (ssDNA) library. Through 15 rounds of positive selection and 3 rounds of negative selection, 24 clones were selected and subjected to sequence analysis. Subsequently, 4 candidate aptamers were selected and further examined for their binding affinity and specificity by enzyme-linked oligonucleotide adsorption (ELONA) and cell binding assays. One aptamer (H2) against hLCN6 with a high affinity and specificity was isolated and investigated by dot blotting and immunofluorescence staining. The result revealed that the candidate aptamer H2 with a dissociation constant of (3. 21 ± 0. 75) nmol/ L was able to recognize and specifically bind to hLCN6. The aptamer H2 also showed high affinity and specificity to human sperms in vitro, which establishes the foundation for the separation of sperm cells from mixed stain based on nucleic acid-protein interactions and provides a new scheme.

Selection of DNA aptamers to cervical intraepithelial neoplasia by SELEX / 生物工程学报

An in vitro synthesized random ssDNA library was subjected to 12 rounds of selection against anti-screening cells and sieving cells by SELEX. Normal and inflammatory cervical exfoliation cells were selected as anti-screening cells, and the cervical exfoliation cells of low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2, CIN3) and cervical carcinoma were selected as sieving cells during the screening process. Then, the highly specific aptamer CIN-Ap4 was established by the analysis of the specificity, affinity and cell immunofluorescence, which can be used as biomarker for Cervical Intraepithelial Neoplasia. Prime Premier 5.0 was applied to design a random ssDNA library. According to the fixed sequence at both ends of the library, a pair of primers were designed and synthesized. At the same time, the optimal annealing temperature, cycle times and primer concentration ratio of PCR procedure were selected. The results under the optimal condition are shown as follows. In the 50 μL reaction system, the optimum reaction conditions of symmetry PCR are as follows: annealing temperature is 49.5 ℃, number of cycles is 15. The optimal reaction conditions of indirect asymmetric PCR are as follows: the primer concentration ratio is 80:1, and the number of cycles is 35. The experiment proves that the oligonucleotide library is constructed successfully, and the highly specific dsDNA and ssDNA can be obtained under optimal PCR conditions with good repeatability, which establishes the foundation for the further exploration and experimentation.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen / 中国病毒学·英文版

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial 115 mer library of ～1.1×1015 random-sequence RNA molecules using the SELEX procedure.The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells.This is the first reported RNA aptamer which could bind to a HBV specific antigen.This newly isolated aptamer could be modified to deliver imaging,diagnostic,and therapeutic agents targeted at HBV-infected cells.

Developing aptamers into tumor diagnostics and therapeutics / 中国肿瘤生物治疗杂志

Aptamers are small single-stranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent non-conventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.