毛细管柱上反应研究氨基酸修饰肽核酸与蛋白质的相互作用

Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

肽核酸(PNA)是一种由碱基和电中性的肽骨架组成的核酸类似物。相比核酸,PNA在体内具有更高的生物稳定性,具有成为蛋白质探针的潜能。在PNA骨架上引入氨基酸,可以提高其水溶性和细胞通透性,但也可能影响其与蛋白质的相互作用。本研究以含有凝血酶15-mer适配体相同碱基序列的15-mer PNA为模型,分别在其骨架N-末端修饰两个赖氨酸和谷氨酸形成(Lys)_2-PNA和(Glu)_2-PNA、建立了毛细管柱上反应方法,用于快速比较(Lys)_2-PNA和(Glu)_2-PNA与3种蛋白质人凝血酶(THB)、单链DNA结合蛋白(SSB)和人血清白蛋白(HSA)的相互作用,评估修饰的PNA与蛋白结合的亲和力和特异性。并比较了(Lys)_2-PNA和(Glu)_2-PNA与含有互补碱基序列的(Lys)_2-c PNA和(Glu)_2-c PNA与THB的相互作用差异。毛细管柱上反应结果表明,(Lys)_2-PNA和(Glu)_2-PNA与3种蛋白的相互作用强弱均为THB>SSB>HSA,但(Lys)_2-PNA和(Lys)_2-c PNA与THB的结合能力相近,(Glu)_2-PNA与THB的结合较(Glu)_2-c PNA更强。利用亲和毛细管电泳法测定了(Glu)_2-PNA与3种蛋白的亲和常数Kb。毛细管柱上反应无需样品孵育,分析快速简便,成本低,可用于蛋白质的PNA探针相互作用分析,以及辅助PNA探针的设计表征。

Peptide nucleic acid(PNA)is a kind of nucleic acid analog which consists of purines,pyrimidines bases,and a neutrally charged peptide backbone.PNA has the potential to be a very useful biological probe for proteins analysis since it has more in vivo biological stability as compared to DNA-or RNA-based aptamers.Usually,the addition of amino acids or peptide to PNA backbone is used to improve its water-solubility and cell-permeability,but these modifications may affect the interaction between PNA and proteins.In this research,we designed two types of amino acid modified PNAs:(Lys)2-PNA and(Glu)2-PNA which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid units on N-terminal of the peptide backbone,respectively.To rapidly assess the binding affinity and specificity of modified PNA and proteins,the online CE reaction method was developed to analyze interactions of(Lys)2-PNA/(Glu)2-PNA and three proteins including thrombin(THB),single-stranded DNA-binding protein(SSB)and human serum albumin(HSA).Meanwhile,the interactions of(Lys)2-PNA/(Glu)2-PNA and thrombin were compared with that of the corresponding complementary base sequence(Lys)2-cPNA/(Glu)2-cPNA and thrombin.The online CE reaction results showed that the interaction of(Lys)2-PNA and(Glu)2-PNA with three proteins was in the order of THB>SSB>HSA.However,(Lys)2-PNA and(Lys)2-cPNA showed similar binding affinity with thrombin;while the binding affinity of(Glu)2-PNA with thrombin was stronger than that of(Glu)2-cPNA with thrombin.Moreover,the binding constant K b of(Glu)2-PNA and three proteins was determined by affinity capillary electrophoresis(ACE).The K b was calculated as 8.39×10 6 L/mol for thrombin and 5.91×10 5 L/mol for SSB,while the binding affinity of(Glu)2-PNA with HSA was too weak to be calculated.The quantitative results of ACE showed agreement with the online CE reaction results,verifying the reliability of online CE reaction method.Besides,the online CE reaction eliminated the requirement of incubation and thus it was faster in detection,simpler in operation with low testing cost.The presented method was particularly suitable for the interaction studies of expensive modified PNAs and proteins,and would assist the design of PNA probe that binds to proteins.