全新靶向小分子核酸适配体特异性检测肺癌患者血清标志物的应用研究

目的以纳米磁珠为筛选介质,利用富集配体系统进化技术(SELEX)从肺癌患者血清中筛选得到高特异性、强亲和力的肺癌血清核酸适配体,并进一步构建基于核酸适配体的肺癌血清标志物检测方法。方法以羧基官能团修饰的磁性纳米颗粒为载体、肺癌患者血清为筛选正向靶标、正常人血清为负向靶标,利用SELEX技术从含有77个碱基的随机ssDNA文库中筛选出与肺癌血清特异性结合的核酸适配体,流式细胞术监测筛选进程;当筛选达到饱和时对富集文库进行克隆测序并鉴定克隆的靶标结合特异性。选择性能最优的核酸适配体用于肺癌患者血清标志物的检测,通过50例肺癌患者血清和50例正常人血清鉴定该方法的特异性与敏感度。结果SELEX筛选进行到第9轮时,富集达到最大化。对筛选文库克隆测序,发现seq24和seq85的结合特异性更优,与正常人血清和白蛋白几乎不结合。核酸适配体seq24的Kd值为98.56 nmol/L,seq85的Kd值为128.69 nmol/L;将构建的基于核酸适配体seq24的肺癌患者血清检测方法与目前临床应用的方法比较,两者结果一致,阳性检出率为100%;此外,所构建的方法可区分早期和晚期肺癌患者。结论核酸适配体seq24能以高特异性和亲和力识别肺癌患者血清,以其为基础的全新检测体系可为肺癌的早期诊断提供新方法。     

肺癌肿瘤标志物神经元特异性烯醇化酶抗原适配子的筛选及其应用

目的提出一种基于适配子快速检测肺癌肿瘤标志物的方法。方法利用指数级富集配体的系统进化技术筛选其高特异性适配子,通过流式细胞术检测亲和力,最终选出一条高亲和力、强特异性的适配子。枸橼酸钠还原法制备纳米金颗粒。建立基于免标记纳米金和适配子的分析方法,并对其方法进行考察。结果通过 6 轮筛选得到三条 NSE 的高特异性、强亲和力的适配子,其中,a 适配子特异性最高。同时利用筛选得到的肺癌肿瘤标志物 a 适配子和纳米金颗粒建立一种快速检测 NSE 抗原的方法。结论该方法具有较好的准确度,为肺癌的早期检测提供了一种新的手段。     

肝癌血清特异标志物Dickkopf-1核酸适体的筛选及其鉴定

目的筛选和鉴定肝癌血清特异标志物Dickkopf-1核酸适体(Apt)。方法采用配体指数级富集系统进化(SELEX)技术,以DKK1为靶蛋白,羧基化琼脂磁珠为筛选介质,从随机ssDNA文库中筛选出一组能够特异性与其结合的核酸适体。利用生物信息学方法对核酸适体进行序列分析和二级结构预测,表面等离子体共振仪分析测定核酸适体的亲和力。结果经过6轮消减SELEX筛选,次级ssDNA文库与DKK1靶标蛋白的亲和力趋向稳定,将第 6 轮筛选产物经PCR扩增进行高通量测序。表面等离子体共振仪检测结果表明,筛选得到的DKK1核酸适体与DKK1的结合解离常数均在纳摩尔级水平,Apt-5的K_d值最小,亲和力最高,Apt-26和Apt-28核酸适体亲和力相对较弱。二级结构预测分析表明,茎环和茎凸环结构为主要的结构形式。圆二色光谱分析结果显示,3个候选核酸适体（Apt-5, Apt-26, Apt-28）能特异形成G-四链体结构识别DKK1靶标蛋白。结论获得了与DKK1靶标蛋白特异性结合的核酸适体,为后续核酸适体的应用研究以及 DKK1蛋白功能的研究奠定了基础。     

RhD抗体ssDNA适配体的筛选与鉴定

目的获得具有拮抗RhD抗体能力的特异性核酸适配体。方法利用SELEX技术,从体外合成的82 nt随机单链DNA文库中,筛选出与RhD抗体特异结合的核酸适配体。采用荧光标记法检测核酸适配体与RhD抗体结合的亲和力及特异性,并对中和RhD抗体的剂量效应关系进行分析。结果通过筛选得到的4个核酸适配体解离常数达到nmol/L水平,其中3号核酸适配体特异性最强,其次为1号,而2号与4号无特异性。1号与3号核酸适配体联合使用,浓度分别为50 pmol/L时,可完全中和RhD抗体。结论利用随机单链寡核苷酸文库成功获得与RhD抗体特异性结合的核酸适配体,所获得的核酸适配体具有拮抗RhD抗体的能力。     

癌胚抗原ssDNA核酸适配子的体外筛选及鉴定

目的通过SELEX技术筛选获得癌胚抗原(CEA)特异性核酸适配子,为建立CEA适配子检测方法和肿瘤靶向治疗奠定实验基础。方法构建长度为88bp的随机ssDNA文库,体外筛选人血清CEA特异性适配子,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测特异性适配子结合蛋白。结果经过优化体外筛选条件,经12轮筛选,获得特异性核酸适配子,经SDS-PAGE证实了其识别并特异性结合的物质为人血清CEA。结论经体外12轮筛选,证实了所获得的CEA适配子可特异性地与人血清CEA蛋白结合,为后续建立CEA特异性检测平台提供基础,也为靶向治疗药物的研制提供一定的思路。     

核酸适配子的研究进展

核酸适配体（Aptamer ,又称核酸适体、适配子）是采用模拟自然进化过程的人工筛选技术-指数富集配体系统进化技术（SELEX）筛选得到的具有识别功能的新型核酸分子。其本质是由一段单链核酸（20～60碱基）通过折叠形成特定的三维结构,能与靶分子高亲和力、高特异性结合。1990年,T uerk等从人工合成的随机单链寡核苷酸文库中筛选出了分别能与噬菌体T4DNA聚合酶和有机染料高特异性、高亲和力结合的单链寡核苷酸配体［1］,筛选出的配体称核酸适配子（aptamer）,该词源于拉丁语aptus ,即to fit ,“适合”的意思。     

针对prominin-1的C-末端肽的适配子筛选和初步鉴定

目的:采用SELEX技术筛选针对人prominin-1的C-末端18肽(C18)的适配子并进行初步鉴定.方法:采用微孔板法进行SELEX筛选.用合成的KLH-C18作为筛选靶标,进行7轮筛选后进行克隆测序.选取适配子C6进行结合ELISA检测,同时进行与转染prominin-1质粒的U87细胞的免疫组化;利用链亲和素磁珠来进行适配子C6的免疫共沉淀.结果:经过7轮SELEX筛选得到具有特定二级结构的适配子.适配子C6可结合C18肽和表达人prominin-1的U87细胞.Pull down实验证明了C6可识别天然状态的prominin-1蛋白.结论:利用“靶标切换”的SELEX技术,初步获得能与人prominin-1的C-末端18肽及其天然蛋白结合的适配子,为肿瘤的诊疗奠定实验基础.     

Cell-SELEX技术筛选急性早幼粒细胞白血病NB4细胞适配体

目的用以细胞为靶标的指数富集配体的系统进化(Cell-SELEX)技术获得与急性早幼粒细胞白血病(APL)细胞株NB4高亲和力、高特异性的适配体。方法以NB4细胞为靶标,用Cell-SELEX技术从随机单链DNA(ssDNA)文库中筛选出一组适配体,将筛选得到的适配体群进行克隆、测序及结合力测定,用流式细胞仪和荧光显微镜对结合率最高的适配体进行亲和力和特异性分析。结果第1,4,7,10,13,16,19轮筛选获得的次级文库与NB4细胞的结合率分别为(1.6%,3.8%,6.3%,11.4%,15.4%,19.9%,16.7%);第16轮筛选的21个阳性克隆菌测序结果表明,存在有3种高度富集的适配体,分别为CX1序列2个,CX5序列3个,CX9序列16个;流式细胞仪检测3种序列的结合率分别为9.7%,12.6%,17.2%;CX9的解离常数(Kd)为16.2 nmol/L;荧光显微镜下发现,CX9与NB4细胞结合的荧光强度高于K562细胞。结论用Cell-SELEX技术成功筛选到针对NB4细胞的适配体CX9,为荧光标记适配体快速鉴定APL提供实验依据。     

核酸适配子在生物医学领域中的应用

1990年Tuerk和Ellingto报道了一种在体外应用随机单链寡核苷酸文库筛选、扩增特定配体的技术,此种技术可以得到能与非核酸靶分子具有高亲和力、高特异性结合的寡核苷酸序列,此技术称为指数富集配基的系统进化技术（systematic evolution of ligands by exponential enrichment,SELEX）,筛选出的寡核苷酸序列称为核酸适配子（aptamer）。     

急性髓系白血病CD33~+/CD34~+细胞表面核酸适配体的筛选及结构分析

目前,对急性白血病细胞表面特异性分子标记物所知甚少,因而白血病的诊断尚缺乏白血病细胞的特异性方法。为此,本研究采用细胞-指数富集配体系统进化(cell-systematic evolution of ligands by exponentialenrichment,cSELEX)技术,筛选与急性髓系白血病(acute myeloblastic leukemia,AML)M2型(AML-M2)患者CD33+/CD34+细胞结合的核酸适配体(aptamer),为寻找AML-M2白血病细胞表面特异性分子标记物提供基础。首先,通过免疫磁珠方法分选出AML-M2患者骨髓CD33+/CD34+细胞并将其作为靶细胞,正常人CD33+/CD34+细胞为反筛选细胞,采用cSELEX技术,从单链DNA(single strand deoxyribonucleic acid,ssDNA)文库中筛选与AML-M2CD33+/CD34+细胞结合的适配体。随后,通过克隆和测序分析各适配体的结构。结果显示,经过13轮次的反复筛选,ssDNA文库的适配体与AML-M2患者CD33+/CD34+细胞的富集度从0.7%增加到52.9%,至第13轮时趋于稳定。对所获得的30个适配体序列分析表明,大多数适配体含有CCCCT、CTCTC和CTCAC保守序列中的一种。二级结构分析显示,30个适配体中含有3种不同类型的模拟二级结构。结论 :本研究成功筛选到AML-M2型白血病CD33+/CD34+细胞的适配体,这为进一步寻找AML-M2白血病细胞表面特异性分子标记物以及AML-M2型白血病的分子诊断创造了基础。     

Selection and preliminary application of a single stranded DNA aptamer targeting colorectal cancer serum

Colorectal cancer is one of the common causes of malignant tumors in recent years, thus the discovery of potential compounds that detect the occurrence of colorectal cancer by efficient approaches is necessary. In this study, the method of systematic evolution of ligands by exponential enrichment (SELEX) was used for recognizing serum from colorectal cancer patients by a single-stranded DNA library of aptamers assisted by single-walled carbon nanotubes (SWCNTs) to remove single-stranded DNA with low affinity. Ten rounds of selection were applied using colorectal cancer serum as a target with the serum of healthy individuals as a control. As the result, we have successfully identified four candidate aptamers after high-throughput genome sequencing analysis, comparison analysis and secondary structure prediction. Among them, aptamer Seq-2 exhibited the highest affinity and the strongest selectivity with an equilibrium dissociation constant (K_d) of 11.31 ± 3.25 nM and a C_t difference value of 4.25 ± 0.38 between the colorectal cancer group and the healthy group. Moreover, with fifty negative control serum samples, the positive detection rate of fifty positive serum samples tested by aptamer Seq-2 was over 90%. In particular, aptamer Seq-2 can strongly bind the colorectal cancer serum, less strongly bind the non-colon cancer serum and hardly bind the healthy serum. Therefore, aptamer Seq-2 presents enormous potential for exploring as a tumor diagnostic kit and detecting unknown tumor markers in serum to reflect colorectal cancer.

Aptamer Seq-2 with high affinity and selectivity was screened against colorectal cancer serum directly for clinical application.     

DNA aptamers from whole-serum SELEX as new diagnostic agents against gastric cancer

Gastric cancer is still among the leading causes of cancer deaths worldwide. Despite the improvements in diagnostic methods, the status of early detection has not been achieved so far. Early diagnosis of gastric cancer may significantly improve the cure rate of patients. Therefore, a new diagnostic method is needed. In this study, subtractive SELEX was performed to screen gastric cancer serum-specific DNA aptamers by using gastric cancer serum and normal serum as the target and negative serum, respectively. Four highly specific aptamers generated for gastric cancer serum, Seq-3, Seq-6, Seq-19 and Seq-54, were developed using whole-serum subtractive SELEX technology with K_d of 128 ± 26.3 nM, 149 ± 23.6 nM, 232 ± 44.2 nM, 202 ± 25.6 nM, respectively. These generated aptamers showed higher specificities toward their target serum by differentiating normal serum but closely related other cancer serums. The selected four high affinity DNA aptamers were further applied to the development based on qPCR method for the early detection of gastric cancer. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer binding proteins. Among these potential biomarkers, APOA1, APOA4, PARD3, Importin subunit alpha-1 showed a relatively high score probability. Therefore, these four ssDNA aptamers generated in our study could be a promising molecular probe for gastric cancer diagnosis.

Gastric cancer is still among the leading causes of cancer deaths worldwide.     

筛选环孢霉素A适体的SELEX技术的建立

摘　要：目的:运用指数富集的配体系统进化(SELEX)技术, 筛选并鉴定环孢霉素A(CsA)特异性的适体.方法:合成一个全长78个核苷酸中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,通过 SELEX方法,即以磁珠作为筛选介质,利用生物素-链亲和素-辣根过氧化物酶系统检测每轮ssDNA文库与CsA的亲和力,筛选针对CsA的适体,将最后一轮筛选产物克隆测序并运用相关软件进行一级结构和二级结构分析.结果:经过10轮的筛选,ssDNA文库与CsA的亲和力呈上升趋势,随机挑选的19 个克隆适体根据一级结构的同源性可分为5个家族,二级结构预测以茎环(发夹)为主.结论:我们通过改良SELEX方法筛选得到了CsA特异性的适体.     

Identification of a novel DNA aptamer that selectively targets lung cancer serum

Lung cancer is the leading cause of cancer-related deaths worldwide. Early diagnosis and treatment is critical to improving the 5 year survival rate of lung cancer. The identification of new options for early-stage diagnosis and therapy of lung cancer still represents a crucial challenge. Therefore, a new diagnostic method is urgently needed. In this study, we used a new modified SELEX, called serum-SELEX, to isolate aptamers that can specifically bind lung cancer serum, without any prior knowledge of their target. Among the obtained candidate aptamer sequences, Ap-LC-19 was identified as the optimal aptamer probe with the lowest dissociation constant (K_d) value of 15 ± 8.6 nM and higher affinity assessed by qPCR. Furthermore, this molecule could be a suitable aptamer for lung cancer serum and could be used as a recognition element in aptamer-based biosensors for efficient early diagnosis of lung cancer or as an innovative tool for targeted therapy. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer targeted proteins. CLEC3B could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.

Lung cancer is the leading cause of cancer-related deaths worldwide.     

自组装核酸适配子探针在深部真菌感染可视化检测中的应用

目的:建立一种自组装核酸适配子探针技术以实现深部真菌感染中外周血(1,3)-β-D-葡聚糖的可视化检测。方法:用碱酶解法提取白念珠菌(1,3)-β-D-葡聚糖并使用葡聚糖酶体外消化处理,获得的水溶性葡聚糖作为靶标,利用指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)从单链DNA文库中筛选能够特异性识别并结合葡聚糖的DNA适配子;对筛选出的适配子进行结构设计,使其能够形成含血晶素(hemin)的G四联体结构,从而具有拟过氧化物酶特性,进一步催化TMB底物显色,肉眼判断颜色变化进行结果判读;对149例临床血清标本分别用建立的方法和G试验检测(1,3)-β-D-葡聚糖,评价检测效能。结果:成功提取白念珠菌(1,3)-β-D-葡聚糖并使用葡聚糖酶消化处理后经SDS-PAGE分离、糖原染色发现,其水溶性的(1,3)-β-D-葡聚糖主要分布在相对分子质量<1700、约4600和10000~15000区域。其中相对分子量小于10000的(1,3)-β-D-葡聚糖比例较高,将其作为适配子筛选靶标。经过8轮正向筛选和4轮负向筛选,将最后一轮ssDNA文库扩增为dsDNA并克隆至pUC19质粒,对单个阳性克隆扩增所得适配子进行相对结合力比较,获得6个结合力较高的适配子(命名为A1—A6)。竞争试验结果表明6个适配子能够识别4个不同位点。将高结合力适配子对不同浓度(1,3)-β-D-葡聚糖进行亲和力检测,肉眼可辨检出限为3.125 pg/mL,线性范围为1.6 pg/mL^400 pg/mL。对149例血清(1,3)-β-D-葡聚糖检测结果显示,适配子自组装显色系统检测性能优于G试验(χ^2=4.373,P=0.0365)。结论:成功自建可视化核酸适配子探针,实现外周血(1,3)-β-D-葡聚糖检测,且有望开发深部真菌床旁检测试剂盒。     

In vitro selection of DNA aptamers for the development of chemiluminescence aptasensor for neuron-specific enolase (NSE) detection

Neuron-specific enolase (NSE) is one of the most commonly used serum tumor biomarker in clinical practice for small cell lung cancer screening, early diagnosis, evaluation of therapeutic efficacy and prognosis. In this study, we obtained DNA aptamers with great affinity and selectivity to NSE via subtractive SELEX approach. After 10 rounds, three candidate aptamers were successfully selected and identified. Their affinities were measured by surface plasmon resonance. Apt-5 aptamer with high binding affinity and good specificity were obtained, which had the dissociation constant (K_D) values of 12.26 nM. In addition, electrophoretic mobility shift assay (EMSA) experiment also further indicated that the Apt-5 had a highly specific affinity to NSE without binding to HSA. The circular dichroism (CD) analysis revealed that the three aptamers formed stable B-form, stem-loop conformations. The selected aptamers were used to construct a chemiluminescence (CL) aptasensor biosensing platform to detect NSE from actual serum samples. Experimental results confirmed that the CL immunosensing platform had good sensitivity with detection limits of 1–100 ng mL⁻¹. The results demonstrated that our obtained the Apt-5 aptsensor was highly specific in the detection of NSE in serum samples. The detection limit was 0.1 ng mL⁻¹, which was lower than the 0.25 ng mL⁻¹ limit of the ELISA used at the hospital. Moreover, the aptasensor can contribute to better detection of small cell lung cancer (SCLC).

Neuron-specific enolase (NSE) is one of the most commonly used serum tumor biomarker in clinical practice for small cell lung cancer screening, early diagnosis, evaluation of therapeutic efficacy and prognosis.