设计合成了一种长臂发夹型核酸探针,结合核酸外切酶Ⅲ水解反应建立了一种免标记荧光信号放大高灵敏检测DNA的新方法。当不存在靶DNA时, SYBR Green Ⅰ荧光染料能够嵌入发夹型探针的茎部而发出很强的荧光,而当存在靶DNA并与发夹型探针杂交后,核酸外切酶Ⅲ从杂交产物的3’端开始水解发夹型探针,释放出靶DNA,并触发下一个酶水解反应,同时SYBR Green Ⅰ染料也随发夹型探针水解而释放,导致荧光信号降低,从而实现了对DNA的免标记荧光信号放大高灵敏检测。该方法的检出限低至320 fmol/L,比传统双标的分子信标的方法降低了4~5个数量级,且该方法还具有免标记、简单、快速的特点。
A pH-sensitive controlled release system was proposed in this work, which consists of mesoporous silica nanoparticles（MSNs） functionalized on the pore outlets with poly（4-vinylphenybronic acid-co-2-（dimethylamino）ethyl acrylate） [P（VPBA-DMAEA）]. Four kinds of P（VPBA-DMAEA）-gated MSNs were synthesized and applied for the p H-sensitive controlled release. The results showed that P（VPBADMAEA） can work as a p H-sensitive nanovalve. The release behavior of the hybrid nanoparticles could be adjusted by changing the mole ratio of VPBA and DMAEA. With the increasing of the mole ratio of VPBA,the leakage of the entrapped molecules in the pores of MSNs could be decreased at neutral and alkaline conditions. By altering the p H of buffer from 4.0 to 8.0, the valve could be switched ‘‘on＇＇ and ‘‘off＇＇reversibly. In addition, cells viability results indicated that these P（VPBA-DMAEA）-gated MSNs had good biocompatibility. We believe that these MSNs based p H-sensitive controlled release system will provide a promising nanodevice for sited release of drug delivery.
Yu-Jie ChangXi-Zhen LiuQing ZhaoXiao-Hai YangKe-Min WangQing WangMin LinMeng Yang
Prostate cancer is the most common malignancy in men lack of efficient early diagnosis and therapeutics,calling for effective molecular probes.Herein,we performed cell-based systematic evolution of ligands by exponential enrichment（cell-SELEX） to obtain specific recognition of human prostate cancer cells PC-3M.Four aptamers were successfully obtained that can bind to target cells with high affinity and specificity.A 51-nt truncated sequence named Xq-2-C1 was identified after further elaborative analysis on the secondary structure.More importantly,the achieved aptamer Xq-2-C1 not only demonstrated excellent specific to target cells,but also revealed specific recognition to clinical prostate cancer tissue.The tissue imaging results showed that Xq-2-C1 had better recognition ratio for clinical prostate cancer tissue samples（85%） compared to the random sequence（9%）.These results demonstrate that these newly generated aptamers would furnish potential applications in the early diagnosis and clinical treatment of prostate cancer.
DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.
Daiqi LiGuoyan LuChunyang LeiZhen WangLijun LiZhou NieYan HuangShouzhuo Yao
