CN117487813A - 特异性识别阿奇霉素的单链dna适配体序列及其应用 - Google Patents
特异性识别阿奇霉素的单链dna适配体序列及其应用 Download PDFInfo
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Abstract
本发明公开了特异性识别阿奇霉素的单链DNA(ssDNA)适配体及其应用。通过磁珠‑SELEX技术,将双链DNA文库固定至磁珠,加入阿奇霉素竞争置换有亲和力的序列的方法,经过数轮正筛选和反筛选,根据高通量测序富集数和序列同源性比对,挑选Apt3进行亲和力和特异性验证,并对最佳适配体Apt3进行截短优化和单碱基突变优化。最后得到一条长度为27个核苷酸的适配体Apt3‑27T,该适配体对阿奇霉素的亲和力较原始适配体有所提高,并且能有效区分阿奇霉素和其结构类似物红霉素,对阿奇霉素有高度特异性,因此被选作最优阿奇霉素适配体。本发明为阿奇霉素检测提供了性能优良的识别元件和检测方法。
Description
技术领域
本发明涉及特异性识别阿奇霉素的单链DNA适配体序列及其应用,属于生物检测技术领域。
背景技术
随着科学技术的不断进步,抗生素污染已成为水安全领域中一个重要的问题。抗生素是一类包括天然、半合成和合成的抗菌化合物,被广泛应用于预防和治疗人类和动物的感染性疾病。尽管抗生素得到广泛使用,但废水处理设施对抗生素的去除效果有限,导致这些物质持续地排放入河流和海洋环境中。这种情况导致细菌逐渐对这些抗生素产生耐药性,从而对生态系统和人类福祉造成威胁。
阿奇霉素(Azithromycin)是一种大环内酯类抗生素,具有抑制细菌蛋白质合成、减少生物膜形成和群体感的有效作用。由于阿奇霉素具有广泛的组织通透性和较长的半衰期,因此在上呼吸道感染、中耳感染、性传播感染等多种感染中被广泛应用。需要注意的是,阿奇霉素也是废水中浓度最高的抗生素之一,因此监测环境中是否存在阿奇霉素并采取积极措施防止污染至关重要。常用于检测阿奇霉素的方法包括色谱技术,尤其是高效液相色谱(HPLC)和液相色谱-串联质谱(LC-MS/MS)。此外,还有电化学技术和量子点技术等其他检测方法可用于阿奇霉素的检测。然而,这些方法通常需要大量时间、昂贵设备和专业人员,虽然灵敏度和精确度较高。因此,迫切需要开发一种快速、经济、高效的阿奇霉素检测方法。
指数富集的配体系统进化技术(systematic evolution of ligands byexponential enrichment,简称为SELEX)是一种从随机寡核苷酸文库中体外筛选能与各种靶标物质特异性结合的寡聚核苷酸片段的分子生物学技术。靶标物质可以是蛋白质、细胞、小分子、金属离子、核酸或者药物,筛选得到的寡聚核苷酸片段被称为适配体。SELEX技术的基本原理是构建人工合成的随机寡核苷酸文库,将其与靶标物质孵育,保留与靶标物质相互作用的序列,经多轮扩增及筛选,即可获得高亲和力和特异性的核酸适配体。核酸适配体具有亲和力高、特异性和稳定性强、成本低廉、易于修饰等优点。然而,目前尚未有关于阿奇霉素适配体的相关报道。
发明内容
为解决上述问题,本发明提供了一系列特异性识别阿奇霉素的ssDNA适配体,通过磁珠SELEX技术,借助磁性分离获得与靶分子特异性结合的高亲和力寡核苷酸序列,经过12轮筛选和相应的反筛选后,进行高通量测序获得适配体序列,并通过序列截短突变优化获得最优适配体序列。这些适配体是阿奇霉素的新型识别元件,具有稳定性良好、灵敏度高、成本低、易制备、易修饰和标记的高特异性的优势,并能应用于多种检测方法的构建。
本发明的第一个目的是提供一种特异性识别阿奇霉素的核酸适配体,所述核酸适配体选自以下任一种:
(1)SEQ ID NO.1所示的核苷酸序列;
(2)SEQ ID NO.1所示的核苷酸序列优化后的序列之一。
进一步地,SEQ ID NO.1所示的核苷酸序列优化后的序列如SEQ ID NO.2-6所示。
进一步地,所述核酸适配体的5′端或3′端修饰有功能基团或分子。
进一步地,所述功能基团或分子用于提高稳定性或用于提供检测信号。
进一步地,所述功能基团或分子选自同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基、巯基中的至少一种。
本发明的第二个目的是提供上述核酸适配体在检测阿奇霉素中的应用。
本发明的第三个目的是提供一种用于检测阿奇霉素的产品,该产品中含有上述任一核酸适配体。当然,本领域技术人员可以知晓,所述产品的形式包括但不限于组合物、试剂盒、试纸、芯片、传感器等任何可以实现检测的形式。
进一步地,所述产品为荧光传感器。本发明进一步构建了一种基于适配体的比例荧光生物传感器,实现了对阿奇霉素的检测。该传感器利用氧化石墨烯淬灭荧光基团的特性,并引入硫黄素(ThT)。当适配体和ThT共同孵育时,它们之间的结合会导致荧光增强。然而,在存在阿奇霉素的情况下,阿奇霉素与适配体竞争性地结合,导致ThT从适配体表面脱离,荧光强度随之降低。氧化石墨烯被用于吸附未与阿奇霉素结合的适配体并淬灭其荧光基团。通过测量荧光强度的变化,可以实现对阿奇霉素的定量分析。当不存在阿奇霉素时,适配体将被氧化石墨烯吸附,无法进行后续步骤。这种传感器将阿奇霉素的浓度检测转化为荧光强度的测定,具有较高的灵敏度和特异性。
进一步地,所述荧光传感器中包括:上述核酸适配体中的一种或多种;还包括荧光基团和荧光淬灭剂。
进一步地,所述荧光基团包括但不限于6-羧基荧光素、CY3、六氯-6-甲基荧光素、6-羧基四甲基罗丹明、ROX、CY5、异硫氰酸荧光素、5-羧基荧光素、染料硫代黄素T等。
进一步地,所述荧光淬灭剂包括但不限于氧化石墨烯、金纳米颗粒、二氧化锰纳米片、4-(4’-二甲基氨基偶氮苯基)苯甲酸、二甲氨基偶氮苯甲酰等。
本发明的第四个目的是提供上述产品在检测阿奇霉素中的应用。
本发明的有益效果:
本发明采用磁珠-SELEX技术将寡核苷酸文库固定至磁珠上,通过竞争置换和扩增富集,结合反复严格的反筛选得到与阿奇霉素高特异结合的ssDNA适配体,为阿奇霉素的检测提供了稳定性良好、灵敏度高、成本低、易制备、易修饰和标记的高特异性检测识别元件和可能的检测方法。
附图说明
图1为磁珠-SELEX筛选阿奇霉素特异性适配体原理图。
图2为测定适配体序列Apt3和Apt3-39的Kd值拟合曲线图和特异性图。
图3为测定适配体序列Apt3-27、Apt3-27A、Apt3-27T、Apt3-27C的Kd值拟合曲线图和特异性图。
图4为荧光适配体生物传感器法检测阿奇霉素的标准曲线图。
图5为荧光适配体生物传感器的特异性验证。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
本发明所使用的缓冲液材料信息:
PBS缓冲液:137mmol·L-1NaCl,2.7mmol·L-1KCl,pH 7.4;
结合缓冲液:137mmol·L-1NaCl,8mmol·L-1Na2HPO4,2.5mmol·L-1KCl,1.5mmol·L-1KH2PO4,1mmol·L-1CaCl2,0.5mmol·L-1MgCl2·6H2O,pH 7.4
本发明涉及的筛选方案如下:
(a)构建初始ssDNA随机文库:5′-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3′,其中N代表碱基A,T,C,G中任一个;
正向引物1:5′-FAM-AGCAGCACAGAGGTCAGATG-3′;
正向引物2:5′-AGCAGCACAGAGGTCAGATG-3′;
反向引物1:5′-Biotin-TTCACGGTAGCACGCATAGG-3′;
反向引物2:5′-TTCACGGTAGCACGCATAGG-3′;
(b)使步骤(a)中的正向引物1与反向引物1对ssDNA文库按照以下条件进行PCR扩增:ssDNA8μL,正向引物8μL(μmol·L-1),反向引物8μL(μmol·L-1), DNA聚合酶,去离子水26μL;工作温度循环为95℃15s,95℃15s,55℃15s,72℃15s,扩增循环数为15;
(c)链霉亲和素磁珠:粒径200nm,浓度为10mg·mL-1,用PBS缓冲液清洗3-7次;
(d)使步骤(c)的链霉亲和素磁珠与步骤(b)中扩增的DNA在适宜的条件下结合,适宜的条件包括室温20-30℃,结合时间1-3h;
(e)使步骤(d)中的混合物用磁力架进行分离后,去上清,再用结合缓冲液清洗3-7次,然后与阿奇霉素溶液混合,在室温20-30℃孵育1-3h;
(f)使步骤(e)中的混合物用磁力架进行分离后,取上清,收集上清中与阿奇霉素结合的ssDNA序列;
(g)重复步骤(b)~(f)4-8次;
(h)引入反筛选,使步骤(d)中的混合物用磁力架进行分离后,去上清,再用结合缓冲液清洗3-7次。然后与红霉素、链霉素、青霉素、氯霉素溶液混合,在室温20-30℃孵育1-3h后,磁性分离去上清。随后加入阿奇霉素溶液,在室温20-30℃孵育1-3h,磁性分离后收集上清中与阿奇霉素结合的ssDNA序列。重复反筛2-6次;
(i)重复步骤(b)~(f)1-5次;
(j)收集经以上步骤得到的阿奇霉素和ssDNA的混合液,用正向引物2和反向引物2进行PCR扩增,随后进行高通量测序。
本发明利用磁珠-SELEX技术,在双链DNA分子中反义链的5’端修饰生物素,利用链霉亲和素和生物素之间的相互作用,将双链DNA分子修饰在磁珠表面,随后再与靶标分子孵育,与靶分子亲和力高的ssDNA序列被竞争性的结合下来从而游离在体系中,通过磁性分离,将ssDNA分离出并作为下一轮筛选的次级文库,经过多轮筛选后,最终保留下来的ssDNA序列与阿奇霉素具有较高的亲和力。同时,在筛选的最后几轮中引入反筛选手段,以红霉素、链霉素、青霉素、氯霉素等干扰物为反筛选目标,从富集的对阿奇霉素有亲和力的序列中除去能同时结合红霉素、链霉素、青霉素、氯霉素的序列,大大提高了适配体的特异性,最终获得了具有高亲和力、高特异性的阿奇霉素适配体。并进一步根据二级结构和分子对接模拟结果对适配体截短优化和单碱基突变优化,最终得到最优适配体Apt3-27T。本发明为阿奇霉素检测提供了稳定性良好、亲和力高、易制备、易修饰和标记的高特异性适配体序列。
实施例1:随机ssDNA文库及其引物的构建
(a)构建长度80个碱基的随机ssDNA文库
5′-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3′,其中N代表碱基A,T,C,G中任一个。
(b)合成正向引物:
正向引物1:5′-FAM-AGCAGCACAGAGGTCAGATG-3′;
正向引物2:5′-AGCAGCACAGAGGTCAGATG-3′;
(c)合成反向引物:
反向引物1:5′-Biotin-TTCACGGTAGCACGCATAGG-3′;
反向引物2:5′-TTCACGGTAGCACGCATAGG-3′。
实施例2:核酸适配体的体外筛选
为筛选出与阿奇霉素有高亲和力和高特异性的ssDNA适配体,共进行了12轮核酸适配体的筛选,1-6轮为正筛,7-9轮引入反筛,第10轮再次开始正筛直至上清荧光稳定。
(a)100μL的PCR扩增体系如表1所示。
表1
正向引物 | 10μmol·L-1 | 8μL |
反向引物 | 10μmol·L-1 | 8μL |
模板DNA | 8μL | |
2×PrimeSTARMax Premix | 50μL | |
ddH2O | 补充至100μL体系 |
(b)文库固定:采用正向引物1反向引物1扩增ssDNA文库。PCR循环条件为95℃初始变性5min,然后进行4个循环:95℃变性15s,55℃退火15s,72℃延伸15s,72℃延伸5min。将95μL PCR产物与105μL结合缓冲液混合。将混合物加入含有0.5mg链霉亲和素磁珠的管中,并在25℃下连续振荡孵育1小时。通过生物素和链霉亲和素之间的相互作用,双链DNA(dsDNA)固定在磁珠表面。最后,使用结合缓冲液洗涤混合物3次,以分离未结合的寡核苷酸。
(c)体外筛选:在整个筛选过程中,包括三轮反向筛选和多轮正向筛选。每轮开始时,向含有固定化文库的磁珠中添加190μL结合缓冲液和10μL 10mM阿奇霉素,然后在25℃孵育1小时。接着进行磁分离,收集上清液作为下一轮ssDNA子文库,并在激发波长为495nm、发射波长为520nm下测量荧光强度。随后,收集每轮上清液进行PCR扩增,PCR循环条件为95℃初始变性5min,然后进行15个循环:95℃变性15s,55℃退火15s,72℃延伸15s,72℃延伸5min。重复上述固定化的dsDNA文库和筛选过程,直到正向筛选的荧光强度稳定。最后,通过添加红霉素、链霉素、青霉素和氯霉素进行三轮反向筛选,以增强最终适配体筛选的特异性。在反筛后,继续进行正向筛选,直至上清的荧光强度再次稳定。
实施例3:筛选得到的ssDNA克隆、测序
ssDNA克隆测序:经过终轮筛选得到的ssDNA,用正向引物2和反向引物2进行PCR扩增,扩增产物送上海生工进行高通量测序。
实施例4:用荧光法测定候选适配体序列的解离常数Kd值
对实施例3中的高通量测序结果进行分析处理,挑选出Apt3,截短序列Apt3-39、Apt3-27,突变序列Apt3-27A、Apt3-27T、Apt3-27C进行解离常数(Kd)测定。将有荧光基团6-羧基荧光素(FAM)修饰的不同浓度的适配体序列加入到结合缓冲液中,并用结合缓冲液补充体积至190μL,95℃加热10min,然后冰浴10min,然后在室温下稳定10min。再将10μL10mmol·L-1阿奇霉素溶液加入到溶液中,轻微震荡,室温摇动反应1后,再加入氧化石墨烯,室温条件下孵育30min,将混合液以15000rpm离心10min,取上清。最后将全部上清液加入到96孔板中用酶标仪测其荧光强度。
由方程:y=Bmax×free ssDNA/(Kd+free ssDNA),可对每一条适配体序列的Kd值进行分析。方程中y表示核酸适配体结合的阿奇霉素占总的阿奇霉素的比例,即饱和度;Bmax表示最大结合位点的数目,free ssDNA表示未与阿奇霉素结合的游离的ssDNA浓度。拟合曲线如图2和图3所示,测得Apt3、Apt3-39、Apt3-27及Apt3-27A、Apt3-27T、Apt3-27C的Kd值分别为235.07±27.48nmol·L-1、225.66±23.77nmol·L-1、218.93±21.46nmol·L-1、349.96±42.38nmol·L-1、215.84±24.75nmol·L-1和247.19±24.96nmol·L-1,均具有较高的亲和力,其中截断突变后的Apt3-27T与阿奇霉素具有最高的亲和力。
所述Apt3或Apt3-39、Apt3-27、Apt3-27A、Apt3-27T、Apt3-27C的序列分别为:
Apt3:
5′-AGCAGCACAGAGGTCAGATGGTTCCGTCCTGGGGCTGTCGGAGTGTTTAGCGTCTCGTCGCCTATGCGTGCTACCGTGAA-3′(SEQ ID NO.1)
Apt3-39:
5′-AGCAGCACAGAGGTCAGATGGTTCCGTCCTGGGGCTGTC-3′(SEQ ID NO.2)
Apt3-27:5′-AGCAGCACAGAGCGTCCTGGGGCTGTC-3′(SEQ ID NO.3)
Apt3-27A:5′-AGCAGCACAGAGCGTCCTGAGGCTGTC-3′(SEQ ID NO.4)
Apt3-27T:5′-AGCAGCACAGAGCGTCCTGTGGCTGTC-3′(SEQ ID NO.5)
Apt3-27C:5′-AGCAGCACAGAGCGTCCTGCGGCTGTC-3′(SEQ ID NO.6)
实施例5:荧光法验证适配体序列的特异性
向荧光基团6-羧基荧光素(FAM)修饰的相同浓度的适配体中加入200μL结合缓冲液,95℃加热10min,然后冰浴10min,然后在室温下稳定10min。再加入10μL 10mmol·L-1的阿奇霉素、红霉素、链霉素、青霉素和氯霉素溶液,轻微震荡,室温反应1h后,再加入氧化石墨烯,室温条件下孵30min,将混合液以15000rpm离心10min,取上清。最后将全部上清液加入到96孔板中用酶标仪测其荧光强度。比较荧光比值(F-F0)/F0,其中F0代表对照组的荧光读数,F代表实验组的荧光读数。
实施例6:构建荧光适配体生物传感器检测阿奇霉素标准品
由于Apt3-27T的G含量高达33.3%,引入ThT作为一个元素来构建检测阿奇霉素的GO传感器。在适配体和ThT共孵育时,结合发生,导致荧光增强。然而,在阿奇霉素存在的情况下,阿奇霉素与适配体竞争性结合导致ThT从适配体表面脱离,导致荧光强度下降。此外,GO被用来吸附未结合的适配体并猝灭其荧光。通过测量荧光强度的变化,可以实现对阿奇霉素的定量分析,并且具有较高的灵敏度和选择性。将有荧光基团6-羧基荧光素(FAM)修饰的适配体先在95℃加热10min,然后冰浴10min,然后在室温下稳定10min。将50μL 2μM的适配体溶液和10μL 400μM的ThT溶液混合,用结合缓冲液稀释至190μL。再加入10μL不同浓度的阿奇霉素,使其终浓度分别为25、50、100、200、400、600、1000、2000、5000nM,然后在25℃振荡孵育30min,以促进靶标与适配子的完全结合。随后加入20μL GO,在25℃振荡孵育30min。样品孵育后,以15000rpm离心10min,取出上清液,在激发波长425nm/发射波长495nm和激发波长495nm/发射波长520nm下测量荧光强度。得到的图以相对荧光比值(F/F)/(F0/F0)-1为纵坐标,以系统中阿奇霉素的终浓度为横坐标,确定合适的线性响应范围,其中F、F0代表实验组和对照组的FAM荧光读数,f、f0代表实验组和对照组的ThT荧光读数(图4)。
实施例7:荧光适配体生物传感器的特异性验证
为了验证荧光适配体生物传感器的特异性,采用其他抗生素包括红霉素、链霉素、青霉素和氯霉素作为对照组,实验方法参照实施例6。如图5所示,阿奇霉素存在时的相对荧光强度明显高于其他对照组。因此,构建的荧光适配体传感器对阿奇霉素具有较高的特异性。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种特异性识别阿奇霉素的核酸适配体,其特征在于,所述核酸适配体选自以下任一种:
(1)SEQ ID NO.1所示的核苷酸序列;
(2)SEQ ID NO.1所示的核苷酸序列优化后的序列之一。
2.根据权利要求1所述的核酸适配体,其特征在于:SEQ ID NO.1所示的核苷酸序列优化后的序列如SEQ ID NO.2-6所示。
3.根据权利要求1所述的核酸适配体,其特征在于:所述核酸适配体的5′端或3′端修饰有功能基团或分子。
4.根据权利要求3所述的核酸适配体,其特征在于:所述功能基团或分子用于提高稳定性或用于提供检测信号。
5.根据权利要求3所述的核酸适配体,其特征在于:所述功能基团或分子选自同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基、巯基中的至少一种。
6.权利要求1-5任一项所述的核酸适配体在检测阿奇霉素中的应用。
7.一种用于检测阿奇霉素的产品,其特征在于:含有权利要求1-5任一项所述的核酸适配体。
8.根据权利要求7所述的产品,其特征在于:所述产品为荧光传感器。
9.根据权利要求8所述的产品,其特征在于,所述荧光传感器中包括:所述核酸适配体中的一种或多种;还包括荧光基团和荧光淬灭剂。
10.权利要求7-9任一项所述的产品在检测阿奇霉素中的应用。
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