CN116732096A - 不引入额外片段的rna体外成环方法 - Google Patents
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Abstract
本发明提出一种核酸载体。其中,所述核酸载体相对于SEQ ID NO:1所示的核苷酸序列,具有下列突变的至少之一:缺失第134~194位核苷酸之间的任一或多个核苷酸;或第132位核苷酸发生突变。所述核酸载体可以在维持PIE系统较高外显子成环效率的同时,有效缩短外显子序列长度,增加用目的基因片段替代外显子片段的可能性,降低目的基因序列设计与成环位点选择的盲目性与试错成本,实现不引入额外片段的Ana PIE成环系统。
Description
技术领域
本发明涉及生物技术领域,具体地,本发明涉及一种不引入额外片段的RNA体外成环方法,更具体地,本发明涉及一种核酸载体、构建核酸载体的方法、使使得目的基因成环的方法、核酸药物或疫苗、药物组合物及其用途。
背景技术
对mRNA表达载体的工程化改造在生产工艺和递送技术上的突破而在近年间备受关注。并且在传染病、肿瘤等应用场景的探索有疗效好、安全性高、成本低、周期短等方面的优势使其成为科研领域的热点。然而,线性RNA载体进入体内后会受到mRNA相关代谢通路的调控,也会激活一系列天然免疫反应,从而限制了线性RNA载体在体内长期稳定发挥功能。未经修饰与递送载体包裹的线性RNA在体内的半衰期只有数小时。这使得线性RNA载体在面对一些需要更宽的有效剂量窗口的应用场景只能采取增大给药剂量或增加给药频次的方式,而这增加了其可行性与可接受性方面的挑战。虽然通过特定核苷酸修饰、使用LNP(脂质纳米颗粒)包裹递送可以有效降低线性RNA载体在体内的免疫原性并提升其在体内的稳定性,但仍无法完全改善线性RNA载体在蛋白替代等应用中面临的困境。
环状结构RNA在原核与真核天然系统中均被发现,其特性与功能不同于广泛存在的线性结构RNA,其形成机制、生理作用与代谢过程也是一直以来的研究热点。目前的研究表明:环状RNA的天然形成机制往往依赖于与体系中相关盐离子相耦合的特定的RNA二/三级结构,可能还会涉及相关辅助蛋白参与;环状RNA可以作为核酸适配体或miRNA海绵参与体内信号通路调控,部分环状RNA也可作为模板翻译蛋白质;环状RNA在体内不会直接受到基于RNA外切的线性RNA代谢通路调控,而是通过某些特定具有RNA内切活性的蛋白酶破坏环状结构后再进入线性RNA代谢途径。环状RNA作为工程载体更适合于在机体中的稳定存在与目的蛋白的长效表达。
I型内含子在mRNA剪接成熟的过程中会在剪接位点形成复杂的三级结构,介导自身成环,其中部分内含子的成环过程可以不依赖其它蛋白辅助而在体外完成,具有工程潜力。此前的研究中,研究者保留I型内含子剪接位点附近的内含子与外显子序列,并将5'与3'内含子前后颠倒,构建了PIE系统以实现前后内含子间的目的基因片段成环。后续的研究中,研究者优化了成环条件,并添加了同源臂序列用于辅助成环。成环后虽然可以通过纯化工艺将作为反应副产物的内含子片段分离,但一小段原剪接位点附近的外显子序列仍会留在所形成的环上,这不仅制约了目的基因片段设计,后续研究中也发现其作为外源片段可能具有一定的免疫原性。
针对上述问题,研究者发现如来源于T4 td基因的I型内含子在两侧剪接位点与附近的外显子片段可能形成一小段配对,仅保留这一小段配对就可以有效实现体外成环,而工程上可以对目的基因片段进行合理设计,使其与这一小段配对的外显子片段重叠,从而通过对目的基因序列与成环位点的设计代替了PIE系统原本的外显子片段,实现不引入额外片段的PIE成环系统。
然而,这种策略并不适用于来源于其它物种其它基因的所有I型内含子。以目前工程上广泛采用的来源于鱼腥藻的另一种I型内含子Ana为例,它在一侧与外显子片段有3bp配对,而另一侧与外显子片段无配对关系。虽然此前的研究中对Ana PIE系统的工程优化已经实现了近90%的外显子成环效率,但Ana PIE系统无法直接使用前述策略实现不引入额外片段的PIE成环系统。
因此,亟需开发一种不引入额外片段而保留较高外显子成环效率的Ana PIE成环系统的实现方法。
发明内容
本发明旨在至少一定程度上解决现有技术中存在的技术问题至少之一。
在本发明的第一方面,本发明提出了一种核酸载体。根据本发明的实施例,相对于SEQ ID NO:1所示的核苷酸序列,具有下列突变的至少之一:缺失第134~194位核苷酸之间的任一或多个核苷酸;或第132位核苷酸发生突变。
根据本发明的实施例,SEQ ID NO:1为天然鱼腥藻I类内含子Ana PIE序列,其中,第132~182位为上游外显子,第183~197位为下游外显子。
需要说明的是,所述“第134~194位核苷酸之间”包括第134位、第135位~第193位和第194位核苷酸。
根据本发明的实施例,所述核酸载体可以在维持PIE系统较高外显子成环效率的同时,有效缩短外显子序列长度,增加用目的基因片段替代外显子片段的可能性,降低目的基因序列设计与成环位点选择的盲目性与试错成本,实现不引入额外片段的Ana PIE成环系统。
根据本发明的实施例,上述核酸载体进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述核酸载体具有下列突变的至少之一:
c.缺失第160~182位核苷酸;
d.缺失第156~189位核苷酸;
e.缺失第141~189位核苷酸;
f.缺失第136~194位核苷酸;
h.缺失第135~194位核苷酸;
i.缺失第134~194位核苷酸;
o.第132位核苷酸发生突变。
根据本发明的实施例,第132位核苷酸突变为G。
根据本发明的实施例,进一步包括:
s.在第182位后与第183位前插入第一配对茎结构,所述第一配对茎结构匹配长度不小于19bp,错配率不超过10%;优选的,所述第一配对茎结构具有CGCCGGAAACGCAAUAGCCGCGGCUAUUAUGCGUUACCGGCG所示的核苷酸序列;或
t.在第182位后与第183位前插入第二配对茎结构,所述第二配对茎结构匹配长度不小于11bp,错配率不超过10%;优选的,所述第二配对茎结构具有CGCCGGAAACGCGCGUUACCGGCG所示的核苷酸序列;或
u.在第182位后与第183位前插入第三配对茎结构,所述第三配对茎结构匹配长度不小于6bp,错配率不超过10%;优选的,所述第三配对茎结构具有CGCCGGCCGGCG所示的核苷酸序列;或
v.在第182位后与第183位前插入第四配对茎结构,所述第四配对茎结构的匹配长度不小于5bp,错配率不超过10%;优选的,第四配对茎结构具有CGCCGCGGCG所示的核苷酸序列;或
w.在第182位后与第183位前插入第五配对茎结构,所述第五配对茎结构的匹配长度不小于4bp;优选的,所述第五配对茎结构具有CGCCGGCG所示的核苷酸序列;或
x.在第182位后与第183位前插入第六配对茎结构,所述第六配对茎结构的匹配长度不小于3bp;优选的,所述第六配对茎结构具有CGCGCG所示的核苷酸序列;或
y.在第182位后与第183位前插入第七配对茎结构,所述第七配对茎结构的匹配长度不小于2bp;优选的,所述第七配对茎结构具有CGCG所示的核苷酸序列;或
z.在第182位后与第183位前插入第八配对茎结构,所述第八配对茎结构的匹配长度不小于1bp;优选的,所述第八配对茎结构具有CG所示的核苷酸序列。
根据本发明的实施例,所述核酸载体进一步具有上述突变或处理时,所述核酸载体的外显子成环效率进一步提高。
根据本发明的实施例,进一步包括:a.在第1位核苷酸前插入体外转录模板序列,且在第312位核苷酸后插入体外转录模板序列;
任选地,所述体外转录模板序列具有SEQ ID NO:2所示的核苷酸序列;
任选地,所述体外转录模板序列具有SEQ ID NO:3所示的核苷酸序列。
进而有利于待插入或携带目的基因的表达。
根据本发明的实施例,进一步包括:b.在第182位后与第183位前插入克隆载体序列。进而待待插入或携带目的基因可克隆载体序列替换,实现目的基因插入所述核酸载体。
根据本发明的实施例,进一步包括在配对茎结构的中间位置插入克隆载体序列,所述中间位置两边的配对茎结构的核苷酸长度差异不超过2nt。
根据本发明的实施例,进一步包括在第一配对茎结构的CGCCGGAAACGCAAUAGCCG核苷酸序列后,CGGCUAUUAUGCGUUACCGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第二配对茎结构的CGCCGGAAACGC核苷酸序列后,GCGUUACCGGCG前插入克隆载体序列;或
进一步包括在所述第三配对茎结构的CGCCGG核苷酸序列后,CCGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第四配对茎结构的CGCCG核苷酸序列后,CGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第五配对茎结构的CGCC核苷酸序列后,GGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第六配对茎结构的CGC核苷酸序列后,GCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第七配对茎结构的CG核苷酸序列后,CG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第八配对茎结构的C核苷酸序列后,G核苷酸序列前插入克隆载体序列。
根据本发明的实施例,所述克隆载体序列具有SEQ ID NO:4所示的核苷酸序列。
AACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCAAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAA(SEQ ID NO:1);
GGGACACCCUCGACCGUCGAUUGUCCACUGGUC(SEQ ID NO:2);
GACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:3);
AAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAA(SEQ ID NO:4)。
根据本发明的实施例,相对于SEQ ID NO:1所示的核苷酸序列,具有表1中成环能力为“+”的任一的突变类型组合。
根据本发明的实施例,所述核酸载体具有SEQ ID NO:6~SEQ ID NO:20所示的核苷酸序列。
需要说明的是,以天然鱼腥藻I类内含子Ana PIE载体为基础,本申请的申请人进行了a~z的至少一种处理,a~z处理(在本申请中也称为突变类型)的含义如下所述:
a.在第1位核苷酸前插入体外转录模板序列(SEQ ID NO:2)且在在第312位核苷酸后插入体外转录模板序列(SEQ ID NO:3);
b.在第182位核苷酸后插入克隆载体序列(SEQ ID NO:4);
c.缺失第160~182位核苷酸;
d.缺失第156~189位核苷酸;
e.缺失第141~189位核苷酸;
f.缺失第136~194位核苷酸;
g.缺失第132~197位核苷酸;
h.缺失第135~194位核苷酸;
i.缺失第134~194位核苷酸;
j.缺失第133~194位核苷酸;
k.缺失第132~194位核苷酸;
l.第135位核苷酸突变为任意与原来不同的氨基酸,如突变为G;
m.第134位核苷酸突变为任意与原来不同的氨基酸,如突变为G;
n.第133位核苷酸突变为任意与原来不同的氨基酸,如突变为G;
o.第132位核苷酸突变为任意与原来不同的氨基酸,如突变为G;
p.第195位核苷酸突变为不与第208位核苷酸配对的核苷酸,如突变为G;
q.第196位核苷酸突变为不与第207位核苷酸配对的核苷酸,如突变为G;
r.第197位核苷酸突变为不与第206位核苷酸配对的核苷酸,如突变为G;
s.在第182位后与第183位前插入匹配长度不小于19bp第一配对茎结构,错配率不超过10%;优选的,在第182位核苷酸后插入CGCCGGAAACGCAAUAGCCG,同时在第183位核苷酸前插入CGGCUAUUAUGCGUUACCGGCG,即第一配对茎结构具有CGCCGGAAACGCAAUAGCCGCGGCUAUUAUGCGUUACCGGCG所示的核苷酸序列;
t.在第182位后与第183位前分别插入匹配长度不小于11bp第二配对茎结构,错配率不超过10%;优选的,在第182位核苷酸后插入CGCCGGAAACGC,同时在第183位核苷酸前插入GCGUUACCGGCG,即第二配对茎结构具有CGCCGGAAACGCGCGUUACCGGCG所示的核苷酸序列;
u.在第182位后与第183位前插入匹配长度不小于6bp第三配对茎结构,错配率不超过10%;优选的,在第182位核苷酸后插入CGCCGG,同时在第183位核苷酸前插入CCGGCG,即所述第三配对茎结构具有CGCCGGCCGGCG所示的核苷酸序列;
v.在第182位后与第183位前插入匹配长度不小于5bp第四配对茎结构,错配率不超过10%;优选的,在第182位核苷酸后插入CGCCG,同时在第183位核苷酸前插入CGGCG,即第四配对茎结构具有CGCCGCGGCG所示的核苷酸序列;
w.在第182位后与第183位前插入匹配长度不小于4bp第五配对茎结构;优选的,在第182位核苷酸后插入CGCC,同时在第183位核苷酸前插入GGCG,即第五配对茎结构具有CGCCGGCG所示的核苷酸序列;
x.在第182位后与第183位前插入匹配长度不小于3bp第六配对茎结构;优选的,在第182位核苷酸后插入CGC,同时在第183位核苷酸前插入GCG,即第六配对茎结构具有CGCGCG所示的核苷酸序列;
y.在第182位后与第183位前插入匹配长度不小于2bp第七配对茎结构;优选的,在第182位核苷酸后插入CG,同时在第183位核苷酸前插入CG,即所述第七配对茎结构具有CGCG所示的核苷酸序列;
z.在第182位后与第183位前插入匹配长度不小于1bp第八配对茎结构;优选的,在第182位核苷酸后插入C,同时在第183位核苷酸前插入G,即所述第八配对茎结构具有CG所示的核苷酸序列。
需要说明的是,本申请所示的核苷酸序列,如无特别说明,均是5'端到3'方向展示的。
需要说明的是,本申请所示的配对茎结构中的匹配长度是指配对的核苷酸对的数量,如匹配长度不小于1bp第八配对茎结构,是指该配对茎结构中配对的核苷酸对的数量不小于1对,例如CG所示的第八配对茎结构,匹配长度为1bp,配对的核苷酸对的数量对的数量为1。
需要说明的是,本申请所述的“在核苷酸之前”是指在核苷酸序列的5'端方向,“在核苷酸之后”是在核苷酸序列的3'端方向。如在第182位核苷酸后插入CGCCGGAAACGCAAUAGCCG,同时在第183位核苷酸前插入CGGCUAUUAUGCGUUACCGGCG,是指CGCCGGAAACGCAAUAGCCG在核酸序列的3'端方向,其5'端与第182位核苷酸相连,CGGCUAUUAUGCGUUACCGGCG在核酸序列的5'端方向,其3'端与第183位核苷酸相连。
根据本发明的实施例,所述核酸载体可以在维持PIE系统较高外显子成环效率的同时,有效缩短外显子序列长度,增加用目的基因片段替代外显子片段的可能性,降低目的基因序列设计与成环位点选择的盲目性与试错成本,实现不引入额外片段的Ana PIE成环系统。
根据本发明的实施例,所述核酸载体还具有以下附加技术特征中的至少之一:
根据本发明的实施例,所述核酸载体相对于SEQ ID NO:1所示的核苷酸序列,具有表1中成环能力为“+”的突变类型组合;
表1:相对于SEQ ID NO:1核苷酸序列突变后成环能力表
备注,表1中的“成环能力”栏中的“+”表示具有成环能力,“突变类型”栏中的“+”表示相应突变类型组中选择的突变类型。
根据本发明的实施例,所述核酸载体具有SEQ ID NO:6~SEQ ID NO:20所示的核苷酸序列。
根据本发明的实施例,所述载体为Ana PIE载体。Ana PIE载体作为两种相对成熟的PIE工程载体之一,较另一种PIE工程载体T4 td PIE载体具有更高的外显子成环效率与更低的不完全反应率。
在本发明的第二方面,本发明提出了一种Ana PIE载体。根据本发明的实施例,所述载体的上游外显子的长度为2nt,下游外显子长度为3nt,外显子成环率为超过25%。
根据本发明的实施例,所述Ana PIE载体可以在维持较高外显子成环效率的同时,有效缩短外显子序列长度,增加用目的基因片段替代外显子片段的可能性,降低目的基因序列设计与成环位点选择的盲目性与试错成本,实现不引入额外片段的Ana PIE成环系统。。此外,Ana PIE载体可以提升其在体内的稳定性,可作为工程载体稳定存在于机体中并与目的蛋白的长效表达。
根据本发明的实施例,所述载体的外显子成环率为39%。
在本发明的第三方面,本发明提出了一种构建核酸载体的方法。根据本发明的实施例,所述方法包括:将Ana PIE外显子进行突变处理,以便获得所述核酸载体。
根据本发明的实施例,采用所述方法构建的核酸载体具有外显子长度短而外显子成环效率较高的优势。
根据本发明的实施例,所述方法还具有以下附加技术特征中的至少之一:
根据本发明的实施例,所述Ana来源于鱼腥藻中I型内含子。
根据本发明的实施例,所述Ana具有如SEQ ID NO:1所示的序列。
根据本发明的实施例,所述突变处理包括截短处理或单个核苷酸突变处理。
根据本发明的实施例,所述Ana外显子包括上游外显子和下游外显子,相对于具有如SEQ ID NO:1所示的序列的Ana PIE,所述上游外显子包括第132~182位核苷酸,所述下游外显子包括第183~197位核苷酸,所述截短处理后,所述上游外显子和下游外显子具有碱基。
根据本发明的实施例,所述截短处理后所述上游外显子保留碱基位于内含子与茎环内侧配对的茎结构之间。
根据本发明的实施例,所述下游外显子保留与内含子进行配对的碱基。
根据本发明的实施例,所述截短处理后,所述下游外显子保留3位碱基。
根据本发明的实施例,所述截短处理后,所述上游外显子保留至少2位碱基。
根据本发明的实施例,所述突变处理包括c、d、e、f、h、i、o处理的至少之一。
根据本发明的实施例,第132位核苷酸突变为G。
根据本发明的实施例,进一步包括s、t、u、v、w、x、y、z处理的至少之一;
根据本发明的实施例,进一步包括a或b处理的至少之一。
根据本发明的实施例,包括对Ana PIE外显子进行表1中成环能力为“+”的任一的处理组合,即对Ana PIE外显子进行表1中成环能力为“+”的任一的突变类型组处理。
在本发明的第四方面,本发明提出了一种核酸载体。根据本发明的实施例,所述核酸载体是通过本发明第三方面所述的方法获得的。
本发明提供的核酸载体在人群中免疫原性较低,能够避免被预存中和抗体识别,具有较高的转导效率,且增强了目的基因的表达。
在本发明的第五方面,本发明提出了一种将目的基因插入核酸载体的方法。根据本发明的实施例,所述方法包括将所述目的基因从预定位点进行断开处理;断开后的目的基因包括5'端序列和3'端序列,将所述3'端序列的3'端与所述5'端序列的5'端相连,获得预插入目的基因;将所述预插入目的基因插入本发明第一方面所述的核酸载体或本发明第四方面所述的核酸载体中的外显子。
根据本发明的实施例,所述预定位点位于核苷酸CUU与AA之间。
根据本发明的实施例,将所述预插入目的基因插入前面所述的核酸载体或前面所述的Ana PIE载体中是通过如下方式进行的:
利用所述预插入目的基因替换所述核酸载体或所述Ana PIE载体中的克隆载体序列。
发明人发现,采用本方法进行目的基因成环,不要求两侧外显子序列均与内含子存在配对区域,可以与对应内含子片段组合构建PIE系统实现高效体外环化,并且可以使用目的基因片段替代实现不引入额外片段的PIE成环系统。
在本发明的第六方面,本发明提出了一种核酸药物或疫苗。根据本发明的实施例,所述核酸药物或疫苗包括本发明第一方面所述的核酸载体或本发明第四方面所述的核酸载体以及待携带核酸。
根据本发明的实施例,所述核酸药物或疫苗在人群中免疫原性较低,且不引入额外的外显子序列。因此,作为核酸药物或疫苗副作用小,在疾病的治疗中更高效、安全。
根据本发明的实施例,所述待携带核酸为mRNA。
根据本发明的实施例,所述核酸药物或疫苗进一步包括:药学上可接受的载体或辅料。
在本发明的第七方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括本发明第一方面所述的核酸载体或本发明第四方面所述的核酸载体或本发明第六方面所述的核酸药物或疫苗。
发明人发现,用作疫苗的核酸载体的优点包括易于操纵、良好的大规模可制造性以及基于多年的研究、开发、制造和临床试验经验的优异安全记录,这些临床试验使用了许多已报道的核酸载体。被用作疫苗的本发明的核酸载体通常提供细胞免疫应答。
在本发明的第八方面,本发明提出了一种前述核酸载体、核酸药物或疫苗在制备药物中的用途。根据本发明的实施例,所述药物用于治疗或预防疾病。
根据本发明的实施例,所述药物在治疗传染病、恶性肿瘤等场景具有疗效好、安全性高、成本低、周期短等方面的优势,能够为更多患者带来希望。
根据本发明的实施例,所述疾病包括肿瘤、传染病、代谢疾病、心血管疾病。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了本发明实施例1的成环位点附近预测二级结构图,A~F分别为序列SEQID NO:5~9、21;
图2显示了本发明实施例1的成环反应后毛细管电泳结果图,A~F分别为序列SEQID NO:5~9、21;
图3显示了本发明实施例2的成环位点附近预测二级结构图,A~D分别为序列SEQID NO:10、11、22、23;
图4显示了本发明实施例2的成环反应后毛细管电泳结果图,A~D分别为序列SEQID NO:10、11、22、23;
图5显示了本发明实施例3的成环位点附近预测二级结构图,A~J分别为序列SEQID NO:24~26、12、27~32;
图6显示了本发明实施例3的成环反应后毛细管电泳结果图,A~J分别为序列SEQID NO:24~26、12、27~32;
图7显示了本发明实施例4的成环位点附近预测二级结构图,A~H分别为序列SEQID NO:13~20;
图8显示了本发明实施例4的成环反应后毛细管电泳结果图,A~H分别为序列SEQID NO:13~20;
图9显示了本发明实施例5的成环位点附近预测二级结构图,A~C分别为序列SEQID NO:33~35;
图10显示了本发明实施例5的成环反应后毛细管电泳结果。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
在对本发明描述的过程中,对于本文中有关的术语进行了解释和说明,这些解释和说明仅仅是为了方便对于方案的理解,并不能看做是对本发明保护方案的限制。
在本文中,术语“包含”或“包括”为开放式表达,即包括本发明所指明的内容,但并不排除其他方面的内容。
在本文中,术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生,并且该描述包括其中发生该事件或状况的情况,以及其中未发生该事件或状况的情况。
在本发明的一方面,本发明提供了一种核酸载体。根据本发明的实施例,所述载体相对于SEQ ID NO:1所示的核苷酸序列,具有下列突变的至少之一:突变类型a~f、h、i、o、s-z。
在本文中,所述核酸载体可以指克隆载体,也可以指重组载体,可以通过将所述核酸与商购的载体(如质粒或病毒载体)可操作地连接而获得。
在本发明的一方面,本发明提出了一种Ana PIE载体。根据本发明的实施例,所述载体的上游外显子的长度为2nt,下游外显子的长度为3nt,成环率为超过25%。
根据本发明的实施例,所述载体的外显子成环率为39%。
在本文中,所述置换内含子外显子(PIE,permuted intron exon)系统是指一种环状排列的I型内含子前体RNA。由端对端融合的外显子组成,这些外显子中断半个内含子序列,自我剪接以产生环状RNA。
在本文中,发明人发现,PIE系统中的外显子片段会保留在所形成的环上,故保留的外显子片段越短,成环后引入的额外片段就越短,而使用目的基因片段替代的可能性就越高,但保留的外显子片段过短也可能影响PIE系统的成环能力。
基于此,发明人通过对Ana PIE系统两侧外显子与内含子间形成的茎环结构进行预测,并结合大量试验,来确定需要保留的外显子序列长度。发明人发现,在保留Ana PIE系统较高的成环能力情况下,下游外显子需要保留与内含子配对的3nt,而上游需要在内含子与环内侧配对茎结构之间保留至少2nt,且环内部其它序列不会与内含子序列形成其它茎环结构而干扰内含子特定结构的形成。基于上述方法指导Ana PIE系统外显子序列截短改造,可以在维持PIE系统较高外显子成环效率的同时,有效缩短外显子序列长度,增加用目的基因片段替代外显子片段的可能性,降低目的基因序列设计与成环位点选择的盲目性与试错成本,实现不引入额外片段的Ana PIE成环系统。
在本发明的又一方面,本发明提出了一种构建核酸载体的方法。根据本发明的实施例,所述方法包括:将Ana外显子进行突变处理,以便获得所述核酸载体。
根据本发明的实施例,所述Ana来源于鱼腥藻中I型内含子。
根据本发明的实施例,所述Ana具有如SEQ ID NO:1所示的序列。
根据本发明的实施例,所述Ana外显子包括上游外显子和下游外显子,所述截短处理后,所述上游外显子和下游外显子具有碱基。
根据本发明的实施例,所述截短处理后所述上游外显子保留碱基位于内含子与茎环内侧配对的茎结构之间。
根据本发明的实施例,所述下游外显子保留与内含子进行配对的碱基。
根据本发明的实施例,所述截短处理后,所述下游外显子保留3位碱基。
根据本发明的实施例,所述截短处理后,所述上游外显子保留至少2位碱基。
根据本发明的实施例,所述突变处理包括选自下列的至少之一:突变类型c~f、h、i、o。
根据本发明的实施例,所述方法进一步包括s、t、u、v、w、x、y、z处理的至少之一。
根据本发明的实施例,所述方法进一步包括a或b处理的至少之一。
在本文中,所述I型内含子是一类具有酶催化功能的内含子,转录成RNA后,可以进行自我剪接。此类内含子转录后可以形成9个由碱基配对形成的特定二级结构,分别命名为P1至P9。其中,P1和P7是保守的。I型内含子具有自我剪接的功能,在剪接反应中,有一种鸟嘌呤核苷(含有游离的3'-OH)G-OH参与反应。G首先结合到内含子的5'端,当线性的内含子成为环状时,会将原来的5'端和15个碱基(或以上)的节段(包括G)切除出去。这种自我剪接,是由RNA的特定序列的核酸内切酶的活性所催化。
根据本发明的实施例,所述I型内含子的天然成环机制依赖于与体系中相关盐离子相耦合的剪接位点附近序列所形成的特定RNA二/三级结构。除外显子与内含子配对区域外,两侧外显子间形成的茎环结构也可能具有两方面的作用。一方面,两侧外显子间形成稳定性高的茎环结构有利于两侧剪接位点靠近,从而促进外显子-内含子特定结构形成与稳定;另一方面,两侧外显子间所形成的部分茎环结构可能也参与了外显子-内含子所形成的特定结构。
在本发明的又一方面,本发明提出了一种核酸药物或疫苗。根据本发明的实施例,所述核酸药物或疫苗包括本发明第一方面所述的核酸载体或本发明第四方面所述的核酸载体以及待携带核酸。
根据本发明的实施例,所述待携带核酸为mRNA。
根据本发明的实施例,所述核酸药物或疫苗进一步包括:药学上可接受的载体或辅料。
在本文中,术语“药学上可接受的”表明药物组合物能够给药至受试者而不产生妨碍药物组合物给药的不良生理反应。例如,“药学上可接受的辅料”是指在制备一般安全、无毒、可取的药物组合物中有用的辅料,优选地,这些辅料或稀释剂的实例包括但不限于:水、盐水、林格氏溶液、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3%甘油、透明质酸、乙醇、聚亚烷基二醇如聚丙二醇、甘油三酯、5%人血清白蛋白,也可以使用脂质体和非水媒介物,例如不挥发油。
在本文中,术语“药学上可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的一种或多种。有许多情况下,药物组合物中包括等渗剂,例如糖类、多元醇(如甘露醇、山梨醇)或氯化钠等。当然药学上可接受的载体还可包括微量的辅助物质,例如润湿剂或乳化剂、防腐剂或缓冲剂,用来延长抗体的保存限期或效力。
在本发明的再一方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括本发明第一方面所述的核酸载体或本发明第四方面所述的核酸载体或本发明第六方面所述的核酸药物或疫苗。
本发明所述的核酸药物或疫苗的给药剂量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的药物有效量可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于:所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等;患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如,由治疗状况的迫切要求,可每天给予若干次分开的剂量,或将剂量按比例地减少。
下面将结合实施例对本公开的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本公开,而不应视为限定本公开的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:Ana PIE系统两侧外显子片段二级结构预测分析与外显子截短成环测试
本发明实施例基于RNA二级结构预测软件预测的Ana PIE系统两侧外显子所形成的二级结构,对两侧外显子进行截短以逐步截去两侧外显子间形成的茎环结构,分别构建质粒体外转录(SEQ ID NO:5~9、21)进行PIE成环测试,以测试两侧外显子所形成茎环结构对Ana PIE系统外显子成环效率的影响。
具体步骤如下:
1、取20ug质粒37℃过夜酶切线性化,获得反应产物;
2、向反应产物中加入10% SDS至终浓度为0.5%,加入1/10体积的蛋白酶K(1ug/ul),混匀后,50℃30min;65℃10min热失活;每管加入1/10体积3M乙酸钠,等体积酚氯仿,震荡混匀后,静置3min;4℃,12000rpm离心15min,小心转移上清至新管中;重复一次酚氯仿提取;加入等体积异丙醇,混匀后置于-20℃沉淀30min,4℃,12000rpm离心20min;弃上清,加入1ml 70%乙醇,清洗沉淀,4℃,12000rpm离心10min;重复乙醇清洗3次;弃上清,晾干沉淀;每管加入10ul nuclease-free water溶解沉淀,作为IVT(全自动体外转录)模板;
3、使用IVT试剂盒进行IVT反应,反应后加入2ul DNase I(1U/ul),37℃孵育30min;
4、加入5ul的10mM GTP,55℃孵育15min进行PIE成环反应;
5、加入30μl的7.5M LiCl沉淀溶液,30μl Nuclease-free water,轻弹混匀,-20℃静置30min;12000rpm,4℃,离心20min;弃上清,加入1ml预冷的70%乙醇漂洗沉淀,颠倒混匀。12000rpm,4℃,离心10min,重复3次;弃上清,12000rpm,4℃简短离心2min,用枪头吸尽残余液体,在冰上晾干沉淀;加入150μl Nuclease-free water轻弹溶解;
6、按要求配制Buffer S、P、C、D,用1x Dilution buffer稀释样品浓度为20ng/ul(共20ul),混匀,80℃,2min,冰浴3-5min。进行毛细管电泳检测;
成环位点附近预测二级结构如图1所示,高亮(黑色字体)区域为外显子区域,成环反应后毛细管电泳结果如图2所示,外显子成环效率如表2所示,其中,SEQ ID NO:5~9所对应的突变类型参照表1。
表2:外显子成环效率统计结果
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCACGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:5)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCUUAAACGGUCGUGUCGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQID NO:6)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCUUAAACGGUCCGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:7)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUCGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:8)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:9)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGCGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:21)。
结果表明,外显子截短会使Ana PIE系统外显子成环效率稍微降低,但截短至第一段互补配对区域前的部分时,即上游外显子截短至4nt(AAAA)、下游外显子截短至3nt(CUU),Ana PIE系统仍具有较高的外显子成环效率,而外显子完全去除后Ana PIE系统失去成环能力。
实施例2:Ana PIE系统外显子片段进一步截短成环测试
在Ana PIE系统两侧外显子仅保留至第一段互补配对区域前的部分的基础上,分别对上游外显子进行进一步截短,构建质粒体外转录(SEQ ID NO:10、11、22、23)进行PIE成环测试,以测试未形成配对结构的上游外显子截短序列长度对Ana PIE系统外显子成环效率的影响。实验步骤同实施例1。
成环位点附近预测二级结构如图3所示,高亮(黑色字体)区域为外显子区域,成环反应后毛细管电泳结果如图4所示,外显子成环效率如表3所示,其中,SEQ ID NO:10、11、所对应的突变类型参照表1。
表3:外显子成环效率统计结果
样品 | 描述 | 环状(%) | 缺口(%) | 前体(%) |
SEQ ID NO:10 | 上游外显子截短至3nt | 39 | 5 | 26 |
SEQ ID NO:11 | 上游外显子截短至2nt | 39 | 6 | 27 |
SEQ ID NO:22 | 上游外显子截短至1nt | 14 | 28 | 42 |
SEQ ID NO:23 | 无上游外显子 | 5 | 27 | 54 |
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:10)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:11)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:22)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGCGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:23)。
结果表明,上游外显子继续截短至1nt时才导致Ana PIE系统外显子成环效率明显下降。
实施例3:Ana PIE系统外显子片段进一步突变成环测试
在Ana PIE系统上游外显子截短至4nt(AAAA)、下游外显子截短至3nt(CUU)的基础上,对外显子关键位点进行不同突变,分别构建质粒体外转录(SEQ ID NO:12、24~32)进行PIE成环测试,以测试成环位点局部二级结构变化对Ana PIE系统外显子成环效率的影响。具体实验步骤参见实施例1。
成环位点附近预测二级结构如图5所示,高亮(黑色字体)区域为外显子区域,成环反应后毛细管电泳结果如图6所示,外显子成环效率如表4所示,其中,SEQ ID NO:12所对应的突变类型参照表1。
表4:外显子成环效率统计结果
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAGCGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:24)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAGCGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:25)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAGCGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:26)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGGACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:12)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGGUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:27)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGGGUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:28)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGGGGAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:29)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCGUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:30)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUGAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:31)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGUCGACGGCAGACUGGCUGUGUAUAAGGGAGCCUGACAUUUAUAUUCCCCAGAACAUCAGGUUAAUGGCGUUUUUGAUGUCAUUUUCGCGGUGGCUGAGAUCAGCCACUUCUUCCCCGAUAACGGACACCGGCACACUGGCCAUAUCGGUGGUCAUCAUGCGCCAGCUUUCAUCCCCGAUAUGCACCACCGGGUAAAGUUCACGGGAGACUUUAUCUGACAGCAGACGUGCACUGGCCAGGGGGAUCACCAUCCGUCGCCCGGGCGUGUCAAUAAUAUCACUCUGUACAUCCACAAACAGACGAUAACGGCUCUCUCUUUUAUAGGUGUAAACCUUAAACUGCAUUUCACCAGCCCCUGUUCUCGUCAGCAAAAGAGCCGUUCAUUUCAAUAAACCGGGCGACCUCAGCCAUCCCUUCCUGAUUUUCCGCUUUCCAGCGUUCGGCACGCAGACGACGGGCUUCAUUCUGCAUGGUUGUGCUUACCAGACCGGAGAUAUUGACAUCAUAUAUGCCUUGAGCAACUGAUAGCUGUCGCUGUCAACUGUCACUGUAAUACGCUGCUUCAUAGCAUACCUCUUUUUGACAUACUUCGGGUAUACAUAUCAGUAUAUAUUCUUAUACCGCAAAAAUCAGCGCGCAAAUACGCAUACUGUUAUCUGGCUUUUAGUAAGCCGGAUCCGGUCUCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCGGAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:32)。
结果表明,Ana PIE系统下游外显子与内含子配对的3nt中任1nt突变均会使外显子成环效率明显下降,而上游外显子序列突变后与内含子形成其它茎环结构而干扰内含子特定结构的形成进一步会使外显子成环效率明显下降。
实施例4:Ana PIE系统环内侧配对茎截短测试
在Ana PIE系统上游外显子截短至2nt(AA)、下游外显子截短至3nt(CUU)的基础上,对具有辅助成环作用的环内侧配对茎结构进行不同程度的截短,分别构建质粒体外转录(SEQ ID NO:13~20)进行PIE成环测试,以测试是否截短的外显子所保留的外显子成环效率依赖于环内侧配对茎结构的辅助。实验步骤同实施例1。
成环位点附近预测二级结构如图7所示,高亮(黑色字体)区域为外显子区域,成环反应后毛细管电泳结果如图8所示,外显子成环效率如表5所示,其中,SEQ ID NO:13~20所对应的突变类型参照表1。
表5:外显子成环效率统计结果
样品 | 描述 | 环状(%) | 缺口(%) | 前体(%) |
SEQ ID NO:13 | 环内侧配对茎结构截短至12bp | 34 | 7 | 23 |
SEQ ID NO:14 | 环内侧配对茎结构截短至6bp | 38 | 7 | 21 |
SEQ ID NO:15 | 环内侧配对茎结构截短至5bp | 36 | 7 | 20 |
SEQ ID NO:16 | 环内侧配对茎结构截短至4bp | 32 | 8 | 21 |
SEQ ID NO:17 | 环内侧配对茎结构截短至3bp | 29 | 10 | 33 |
SEQ ID NO:18 | 环内侧配对茎结构截短至2bp | 32 | 13 | 23 |
SEQ ID NO:19 | 环内侧配对茎结构截短至1bp | 34 | 11 | 23 |
SEQ ID NO:20 | 环内侧配对茎结构去除 | 28 | 12 | 32 |
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCCGGAAACGCAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAAGCGUUACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:13)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCCGGAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAACCGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:14)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCCGAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAACGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:15)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCCAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAAGGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:16)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGCAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAAGCGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:17)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACGAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAACGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQID NO:18)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAAGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQID NO:19)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAAAACAAAAAACAAAAAAACGGAGACCGTCGACGGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGACACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCGGTCTCGAAAAAACAAAAAACAAAACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ IDNO:20)。
结果表明,辅助成环的环内侧配对茎结构的截短会使Ana PIE系统的外显子成环效率稍微降低,但即使环内侧配对茎结构完全去除,外显子截短的Ana PIE系统仍有较高外显子成环效率。
实施例5:Ana PIE系统不引入额外片段成环测试
在目的基因片段IRES-EGFP上可以找到3个CUU-AA位点,分别作为成环位点,将位点3'端序列置于5'端而将位点5'端序列置于3'端连接,再在上下游添加体外转录模板序列与Ana PIE系统内含子序列构建质粒体外转录(SEQ ID NO:33~35)进行PIE成环测试。具体实验步骤参考实施例1。
成环位点附近预测二级结构如图9所示,高亮(黑色字体)区域为CUU-AA位点,成环反应后毛细管电泳结果如图10所示,外显子成环效率如表6所示。SEQ ID NO:33外显子成环效率较低而SEQ ID NO:34~35外显子成环效率较高。
表6:外显子成环效率统计结果
样品 | 描述 | 环状(%) | 前体(%) |
SEQ ID NO:33 | Ana PIE内含子特定结构被干扰 | 22.9 | 63.3 |
SEQ ID NO:34 | Ana PIE内含子特定结构未被干扰 | 30.7 | 54.2 |
SEQ ID NO:35 | Ana PIE内含子特定结构未被干扰 | 35 | 48.5 |
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACUUUAGUCACGACCUUAACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUUAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAAAUUCUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:33)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUUAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAAAUUCUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCUUAAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACUUUAGUCACGACCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:34)。
GGGACACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAAAUUCUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCUUAAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACUUUAGUCACGACCUUAACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAU(SEQ ID NO:35)。
结果表明,下游外显子保留与内含子配对的3nt,而上游内含子与环内侧配对茎结构之间保留至少2nt,且环内部其它序列未与内含子序列形成其它茎环结构干扰内含子特定结构的形成,可以实现不引入额外片段的Ana PIE成环系统。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
Claims (24)
1.一种核酸载体,其特征在于,相对于SEQ ID NO:1所示的核苷酸序列,具有下列突变的至少之一:
缺失第134~194位核苷酸之间的任一或多个核苷酸;或
第132位核苷酸发生突变。
2.根据权利要求1所述的核酸载体,其特征在于,相对于SEQ ID NO:1所示的核苷酸序列,具有下列突变的至少之一:
c.缺失第160~182位核苷酸;
d.缺失第156~189位核苷酸;
e.缺失第141~189位核苷酸;
f.缺失第136~194位核苷酸;
h.缺失第135~194位核苷酸;
i.缺失第134~194位核苷酸;
o.第132位核苷酸发生突变;
任选地,第132位核苷酸突变为G。
3.根据权利要求1所述的核酸载体,其特征在于,进一步包括:
s.在第182位后与第183位前插入第一配对茎结构,所述第一配对茎结构匹配长度不小于19bp,错配率不超过10%;优选的,所述第一配对茎结构具有CGCCGGAAACGCAAUAGCCGCGGCUAUUAUGCGUUACCGGCG所示的核苷酸序列;或
t.在第182位后与第183位前插入第二配对茎结构,所述第二配对茎结构匹配长度不小于11bp,错配率不超过10%;优选的,所述第二配对茎结构具有CGCCGGAAACGCGCGUUACCGGCG所示的核苷酸序列;或
u.在第182位后与第183位前插入第三配对茎结构,所述第三配对茎结构匹配长度不小于6bp,错配率不超过10%;优选的,所述第三配对茎结构具有CGCCGGCCGGCG所示的核苷酸序列;或
v.在第182位后与第183位前插入第四配对茎结构,所述第四配对茎结构的匹配长度不小于5bp,错配率不超过10%;优选的,第四配对茎结构具有CGCCGCGGCG所示的核苷酸序列;或
w.在第182位后与第183位前插入第五配对茎结构,所述第五配对茎结构的匹配长度不小于4bp;优选的,所述第五配对茎结构具有CGCCGGCG所示的核苷酸序列;或
x.在第182位后与第183位前插入第六配对茎结构,所述第六配对茎结构的匹配长度不小于3bp;优选的,所述第六配对茎结构具有CGCGCG所示的核苷酸序列;或
y.在第182位后与第183位前插入第七配对茎结构,所述第七配对茎结构的匹配长度不小于2bp;优选的,所述第七配对茎结构具有CGCG所示的核苷酸序列;或
z.在第182位后与第183位前插入第八配对茎结构,所述第八配对茎结构的匹配长度不小于1bp;优选的,所述第八配对茎结构具有CG所示的核苷酸序列。
4.根据权利要求1~3任一项所述的核酸载体,其特征在于,进一步包括:
a.在第1位核苷酸前插入体外转录模板序列,且在第312位核苷酸后插入体外转录模板序列;
任选地,所述体外转录模板序列具有SEQ ID NO:2所示的核苷酸序列;
任选地,所述体外转录模板序列具有SEQ ID NO:3所示的核苷酸序列。
5.根据权利要求1或2所述的核酸载体,其特征在于,进一步包括:
b.在第182位后与第183位前插入克隆载体序列。
6.根据权利要求3或5所述的核酸载体,其特征在于,进一步包括在配对茎结构的中间位置插入克隆载体序列,所述中间位置两边的配对茎结构的核苷酸长度差异不超过2nt;
任选地,进一步包括在第一配对茎结构的CGCCGGAAACGCAAUAGCCG核苷酸序列后,CGGCUAUUAUGCGUUACCGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第二配对茎结构的CGCCGGAAACGC核苷酸序列后,GCGUUACCGGCG前插入克隆载体序列;或
进一步包括在所述第三配对茎结构的CGCCGG核苷酸序列后,CCGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第四配对茎结构的CGCCG核苷酸序列后,CGGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第五配对茎结构的CGCC核苷酸序列后,GGCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第六配对茎结构的CGC核苷酸序列后,GCG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第七配对茎结构的CG核苷酸序列后,CG核苷酸序列前插入克隆载体序列;或
进一步包括在所述第八配对茎结构的C核苷酸序列后,G核苷酸序列前插入克隆载体序列。
7.根据权利要求5或6所述的核酸载体,其特征在于,所述克隆载体序列具有SEQ IDNO:4所示的核苷酸序列。
8.根据权利要求1~3任一项所述的核酸载体,其特征在于,相对于SEQ ID NO:1所示的核苷酸序列,具有表1中成环能力为“+”的任一的突变类型组。
9.根据权利要求1所述的核酸载体,其特征在于,所述核酸载体具有SEQ ID NO:6~SEQID NO:20所示的核苷酸序列。
10.根据权利要求1所述的核酸载体,其特征在于,所述载体为Ana PIE载体。
11.一种Ana PIE载体,其特征在于,所述载体的上游外显子的长度为2nt,下游外显子长度为3nt,外显子成环率超过25%。
12.一种构建核酸载体的方法,其特征在于,包括:
将Ana PIE外显子进行突变处理,以便获得所述核酸载体。
13.根据权利要求12所述的方法,其特征在于,所述Ana PIE来源于鱼腥藻中I型内含子;
任选地,所述Ana PIE具有如SEQ ID NO:1所示的序列;
任选地,所述突变处理包括截短处理或单个核苷酸突变处理。
14.根据权利要求13所述的方法,其特征在于,所述Ana PIE外显子包括上游外显子和下游外显子,相对于具有如SEQ ID NO:1所示的序列的Ana PIE,所述上游外显子包括第132~182位核苷酸,所述下游外显子包括第183~197位核苷酸,所述截短处理后,所述上游外显子和下游外显子具有碱基;
任选地,所述截短处理后所述上游外显子保留碱基位于内含子与茎环内侧配对的茎结构之间;
任选地,所述截短处理后所述下游外显子保留与内含子进行配对的碱基。
15.根据权利要求13或14所述的方法,其特征在于,所述截短处理后,所述下游外显子保留3位碱基;
任选地,所述截短处理后,所述上游外显子保留至少2位碱基。
16.根据权利要求13所述的方法,其特征在于,所述突变处理包括c、d、e、f、h、i、o的至少之一。
17.根据权利要求13所述的方法,其特征在于,第132位核苷酸突变为G;
任选地,进一步包括s、t、u、v、w、x、y、z处理的至少之一;
任选地,进一步包括a或b处理的至少之一。
18.根据权利要求14所述的方法,其特征在于,包括对Ana PIE外显子进行表1中成环能力为“+”的任一的处理组合。
19.一种核酸载体,其特征在于,是根据权利要求12~18任一项所述的方法构建获得的。
20.一种将目的基因插入核酸载体的方法,其特征在于,包括:
将所述目的基因从预定位点进行断开处理;
断开后的目的基因包括5'端序列和3'端序列,将所述3'端序列的3'端与所述5'端序列的5'端相连,获得预插入目的基因;
将所述预插入目的基因插入权利要求1~10或19所述的核酸载体或权利要求11所述的Ana PIE载体中;
任选地,所述预定位点位于核苷酸CUU与AA之间;
任选地,将所述预插入目的基因插入权利要求1~10或19所述的核酸载体或权利要求11所述的Ana PIE载体中是通过如下方式进行的:
利用所述预插入目的基因替换所述核酸载体或所述Ana PIE载体中的克隆载体序列。
21.一种核酸药物或疫苗,其特征在于,包括权利要求1~10或19所述的核酸载体或权利要求11所述的Ana PIE载体以及待携带核酸;
任选地,所述待携带核酸为mRNA。
22.一种药物组合物,其特征在于,包括权利要求1~10或19所述的核酸载体或权利要求11所述的Ana PIE载体或权利要求21所述的核酸药物或疫苗;
任选地,进一步包括:药学上可接受的载体或辅料。
23.权利要求1~10或19所述的核酸载体或权利要求11所述的Ana PIE载体或权利要求21所述的核酸药物或疫苗在制备药物中用途,所述药物用于治疗或预防疾病。
24.根据权利要求23所述的用途,其特征在于,所述疾病包括肿瘤、传染病、代谢疾病或心血管疾病。
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