蛋白质/核酸相互作用研究方法进展

蛋白质和核酸是构成生命体最为重要的两类生物大分子,蛋白质与核酸的相互作用是分子生物学研究的中心问题之一,它是许多生命活动的重要组成部分。研究蛋白质/核酸相互作用近期采用的新技术有:核酸适体技术、生物信息学方法、蛋白质芯片技术以及纳米技术等。本文就这些新的研究方法进行综述。     

JACS：核酸与蛋白质相互作用机理研究获进展

近期来自吉林大学的研究人员分别与清华大学和中科院等处的研究人员在高分子结晶态分子间相互作用和RNA与蛋白质间相互作用研究方面取得重要进展。所获得的跨学科研究新成果公布在著名的国际期刊《美国化学会志》（J．Am．Chem．Soc．）上。获得了Nature Materials等多个杂志的推荐。     

JACS：核酸与蛋白质相互作用机理研究获进展

近期来自吉林大学的研究人员分别与清华大学和中科院等处的研究人员在高分子结晶态分子间相互作用和RNA与蛋白质问相互作用研究方面取得重要进展。所获得的跨学科研究新成果公布在著名的国际期刊《美国化学会志》（J．Am．Chem．Soc．）上,获得了Nature Materials等多个杂志的推荐。     

核酸条形码技术：扩展蛋白质-蛋白质相互作用检测通量的新方法

蛋白质-蛋白质相互作用(protein-protein interaction, PPI)几乎参与了机体内所有重要的生物学过程,在细胞的基本生命过程中扮演了至关重要的角色,开发高通量的PPI检测新方法具有重要的生物学意义。目前,下一代测序技术(next-generation sequencing, NGS)发展快速,能在几天内测定超过10亿个模板的DNA序列。由于并行DNA测序技术所特有的敏感性、特异性、高通量和多路复用优势,其已被用作广谱分子计数器,应用于基因组测序和转录物组测序等领域。核酸条形码技术通过将寡核苷酸标签与目标蛋白质连接起来,从而标记编码蛋白质。之后,利用高通量的测序方法检测相互作用的蛋白质,实现了PPI的高通量检测。这一技术推动了PPI检测方法的飞速发展,提升了单次实验检测的通量,为构建PPI网络提供了强有力的技术支持。本文详细阐述了核酸条形码在PPI检测方法中的设计、生成和读取;通过分析核酸条形码技术在PPI研究中的应用范例,探讨了各自的优势和不足,并评估了数据的可靠性,讨论了基于核酸条形码技术的PPI检测方法未来的发展趋势。     

蛋白质-蛋白质、蛋白质-核酸相互作用研究方法及在人肠道病毒A71型研究中的应用

研究蛋白质-蛋白质相互作用的方法主要有酵母双杂交、噬菌体展示、免疫共沉淀、谷光苷肽巯基转移酶沉淀、细胞内共定位、亲和印迹、病毒铺覆蛋白结合技术、表面等离子共振、荧光共振能量转移等技术。检测蛋白质-核酸相互作用的方法主要包括酵母单杂交、染色质免疫沉淀、电泳迁移率实验、DNA-蛋白质印迹、报告基因、免疫共沉淀、谷光苷肽巯基转移酶沉淀、噬菌体展示等技术。在我们实验室对人肠道病毒A71型(EV-A71)的研究中经常会用到这些方法,但在该研究领域尚未有中文综述发表,因此本文对EV-A71研究中这些方法的应用进行了综述,该综述对蛋白质-蛋白质、蛋白质-核酸在其他病毒研究中的应用同样具有重要启示。     

蛋白质-蛋白质、蛋白质-核酸相互作用研究方法及在人肠道病毒A71型研究中的应用北大核心CSCD

研究蛋白质-蛋白质相互作用的方法主要有酵母双杂交、噬菌体展示、免疫共沉淀、谷光苷肽巯基转移酶沉淀、细胞内共定位、亲和印迹、病毒铺覆蛋白结合技术、表面等离子共振、荧光共振能量转移等技术。检测蛋白质-核酸相互作用的方法主要包括酵母单杂交、染色质免疫沉淀、电泳迁移率实验、DNA-蛋白质印迹、报告基因、免疫共沉淀、谷光苷肽巯基转移酶沉淀、噬菌体展示等技术。在我们实验室对人肠道病毒A71型(EV-A71)的研究中经常会用到这些方法,但在该研究领域尚未有中文综述发表,因此本文对EV-A71研究中这些方法的应用进行了综述,该综述对蛋白质-蛋白质、蛋白质-核酸在其他病毒研究中的应用同样具有重要启示。     

DNA甲基化作用对HL-60细胞中蛋白质-核酸相互作用的影响

以S-腺苷酰-L-甲硫氨酸(SAM)为诱导物,在10μmol/L的最佳浓度下,可诱导16%的HL-60细胞分化.HPLC法检测碱基含量,发现在细胞分化过程中伴有基因组DNA甲基化水平升高.选择对5-甲基胞嘧啶敏感的限制性核酸内切酶切割DNA,证实基因组DNA对HaeⅢ,SmaⅠ,SalⅠ,XhoⅠ和HindⅢ的切割产生阻抗作用.以凝胶滞留法检测DNA与核蛋白的结合状况,表明DNA与胞内DNA结合蛋白的结合能力发生改变.     

顽拗性黄皮胚脱水过程中核酸、蛋白质代谢的变化

黄皮种子发育晚期,胚内核酸、蛋白质合成能力增强,而花生胚的核酸、蛋白质合成能力在发育晚期则呈下降趋势。黄皮胚的发育在达到生理成熟后维持着活跃的生理代谢并转入萌发状态;而花生胚的代谢活性逐步降低并转入生理静止状态。脱水处理引起生理成熟期黄皮胚核酸、蛋白质合成能力急剧下降,核酸水解酶活性增强。不同程度脱水的黄皮胚吸胀２４ｈ,核酸、蛋白质的合成能力随脱水程度的加深而降低;生物大分子代谢能力的变化是顽拗性     

Biological Microchips with Hydrogel-Immobilized Nucleic Acids,Proteins, and Other Compounds: Properties and Applications in Genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single-nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, protein microchips have been created, containing immobilized antibodies, antigens, enzymes, and many other substances, as well as microchips with gel-immobilized live cells.     

Interaction of Keratin K1 with Nucleic Acids on the Cell Surface

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.     

Simple and Rapid Procedure Suitable for Quantitative Isolation of Low and High Molecular Weight Extracellular Nucleic Acids

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100–10000 b.p. DNA fragments and 50–10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost‐effective.     

核酸探针中放射性同位素的快速检测

利用尼龙膜作为介质,以0．4mol／L的NaOH为层析液,进行膜上层析,分离经放射性同位素标记的核酸探针和未掺入的放射性游离单核苷酸,再经放射性强度测定即可计算出标记后的核酸探针的放射性比活。方法简单快捷,产生的放射性废物少,完全可以替代经典的三氯乙酸沉淀法及滤膜吸附法。     

Crystallographic Studies of Chemically Modified Nucleic Acids: A Backward Glance

Chemically modified nucleic acids (CNAs) are widely explored as antisense oligonucleotide or small interfering RNA (siRNA) candidates for therapeutic applications. CNAs are also of interest in diagnostics, high‐throughput genomics and target validation, nanotechnology and as model systems in investigations directed at a better understanding of the etiology of nucleic acid structure, as well as the physicochemical and pairing properties of DNA and RNA, and for probing protein–nucleic acid interactions. In this article, we review research conducted in our laboratory over the past two decades with a focus on crystal‐structure analyses of CNAs and artificial pairing systems. We highlight key insights into issues ranging from conformational distortions as a consequence of modification to the modulation of pairing strength, and RNA affinity by stereoelectronic effects and hydration. Although crystal structures have only been determined for a subset of the large number of modifications that were synthesized and analyzed in the oligonucleotide context to date, they have yielded guiding principles for the design of new analogs with tailor‐made properties, including pairing specificity, nuclease resistance, and cellular uptake. And, perhaps less obviously, crystallographic studies of CNAs and synthetic pairing systems have shed light on fundamental aspects of DNA and RNA structure and function that would not have been disclosed by investigations solely focused on the natural nucleic acids.     

一种快速有效提取植物和真菌DNA和RNA的简易方法

本文利用一种真菌核酸的快速提取方法提取了3种真菌的DNA和RNA,并将该法略作改进用于烟草DNA和RNA的提取.同时,通过低温保藏试验评价核酸提取液和提取产物的稳定性和有效性;利用凝胶电泳、PCR和RT-PCR等方法比较分析核酸质量;并将提取效果与传统方法或试剂盒的提取效果进行比较,以分析其有效性.结果表明,改进后的提取方法是一种简单、方便和有效的实验方法,不仅可用于真菌也可用于植物DNA和RNA提取,用这种方法提取得到的DNA和RNA在低温保存条件下比较稳定,能较长时间保持其原有活性.     

A m6A Sensing Method by Its Impact on the Stability of RNA Double Helix

N⁶‐Methyladenosine (m⁶A) is one of the most important RNA modifications in epigenetics. The development of detection method for m⁶A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m⁶A modification and target RNAs without m⁶A modification. We verified the fact that the presence of m⁶A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m⁶A detection methods in the future.     

Conformational Studies of Nucleic Acids I. A Rapid and Direct Method for Generating Furanose Coordinates from the Pseudorotation Angle

Abstract

The greatest difficulty in modeling a nucleic acid is generating the coordinates of its furanoses. This difficulty arises from constraints imposed by the closed ring geometries of these sugars. We have developed a new method for modeling these furanose rings. Using this method, the coordinates of a sugar can be obtained quickly and unambiguously for any point on the pseudorotational pathway from one parameter: the phase angle of pseudorotation P. The significant difference between this and previous sugar modeling schemes is that here the endocyclic bond lengths of the five-membered sugar ring are allowed to vary a small amount according to simple, explicit, and experimentally reasonable analytic functions of P. The coefficients of these functions follow from the empirical behavior of the endocyclic bond angles and from geometrical constraints due to ring closure. The ability to model the sugars directly from one parameter greatly facilitates carrying out the global conformational studies on nucleic acid constituents which will be attempted in subsequent papers of this series.     

New Pyrosequencing Method to Analyze the Function of the Klenow Fragment (EXO−) for Unnatural Nucleic Acids: Pyrophosphorolysis and Incorporation Efficiency

The incorporation of deoxynucleoside triphosphates (dNTPs) catalyzed by polymerases is conventionally examined using gel electrophoresis autoradiography. Here, we studied an alternative method, pyrosequencing, to verify the incorporation of dNTPs containing unnatural nucleotides by polymerases. We found that the pyrosequencing method more rapidly and easily confirmed the incorporation of dNTPs than the conventional method, especially in the presence of low-efficiency dNTP polymerases. Furthermore, the method can detect the pyrophosphorolysis reaction just before the position of the unnatural nucleic acid, and the efficiency of incorporation just after it.