【作者】 周亮；

【导师】 朱伟；

【作者基本信息】 华南理工大学 ， 生物学， 2023， 博士

【摘要】 基因—脱氧核糖核酸(DNA)分子上具有遗传效应的特定核苷酸序列的总称,是核酸中储存信息的遗传单位,控制着生物体内各种蛋白质的合成及生物体的运作,是遗传和物质延续的基础。1985年,美国科学家率先提出“人类基因组计划”,其本意在于破译人类全部遗传信息,使人类在分子水平上全面地认识自我。随着人类基因组序列图绘制完成,基因在疾病治疗、新药研发、遗传病分析、个体识别和刑事侦查等领域的作用越发显著。然而,基因作为生物信息的物质载体,其本身具有脆弱性和可降解性,开发新技术实现其稳定、长效和低成本保存,进而拓展现有基因信息的保存模式和基因库的构建方式具有重要意义或国家层面的战略意义。生物硅化,也即二氧化硅组成的生物结构的形成过程,该过程普遍存在于自然界(如硅藻等),也被广泛用于构建有机-无机复合材料。通过对硅氧烷前驱体的选择和水解-缩聚条件的精确控制,该技术可有效对自然界中化石的形成过程进行模仿。人体由40-60万亿个细胞组成,绝大多数含有细胞核,里面富含30亿个DNA碱基对,是理想的基因存储载体。通过模拟自然界化石形成的过程,对细胞进行处理,加速“细胞化石”的形成,从而有可能实现对细胞中基因组的原位封装和长效储存。本文以细胞中基因组的长效存储为目标,通过调控硅氧烷前驱体的入胞机制,深入研究无机材料与细胞及内部基因组的作用模式,建立“原位封装,按需提取”的细胞内基因组存储理念,以期发展出生物信息(基因组、转录组和蛋白组)存储的新范式。具体研究内容包括:1.开发出“冷冻硅化”技术,使二氧化硅前驱体在休眠状态和冰晶辅助下扩散到细胞和细胞核中,随后在细胞蛋白的催化下水解缩合,以无定形二氧化硅形式沉积至整个细胞中,实现了对细胞内DNA的完整封装。区别于传统甲醛保存模式,冷冻硅化技术不会对DNA造成损伤和片段化,保证了细胞中基因组的完整性。基因组在细胞内的完好封装不仅可极大提升细胞基因组对极端条件(如紫外、高温和氧自由基等环境)的抵抗能力,同时可极大延长全血基因组的存储年限。研究显示在20 ~oC时处理后的基因组存储半衰期约为1208年,与未保护血样相比,稳定性增加了167倍。该技术成本低,可拓展性强,易于与不同媒介结合,如纸基载体或3D打印技术,从而极大扩展技术的应用场景。2.开发出“深度硅化”技术,使二氧化硅前驱体在少量渗透性溶剂二甲基亚砜(DMSO)的辅助下,在室温条件下提升无机硅元对细胞的渗透和细胞内沉积,从而提升无机材料对细胞的封装效果。研究显示,在渗透性小分子DMSO的辅助下,硅氧烷前驱体在细胞内的沉积量是原有“冷冻硅化”技术水平下的30倍,达到27.2 pg/细胞;经加速老化1天后,凝胶电泳显示深度硅化处理的样品与新鲜细胞样品具有相同的明亮且清晰的DNA条带,而甲醛固定样品DNA条带几乎不可见;即使老化21天后,深度硅化仍可以完整扩增所有的目标基因组片段;“深度硅化”技术在室温下的开展,避免了对低温的需求,使技术在应用端更为便捷。3.开发出“靶向硅化”技术,通过分子结构的理性设计,赋予硅氧烷前驱体靶向线粒体行为,结合“冷冻硅化”技术,实现在空间上对细胞内线粒体DNA(mt DNA)的精准封装。研究显示,“靶向硅化”技术切实可行,可以在短时间内实现对mt DNA的快速封装;同样,经过加速老化后,未保护样品在7天内都失去了它们的特征性DNA条带,但靶向硅化的样品仍然保持稳定可见的DNA条带;线粒体在细胞内完好封装极大提升mt DNA对极端条件(如紫外、高温和氧自由基等环境)的抵抗能力。细胞内DNA的时空精准封装和长效存储提供了与时空相关的多维层面的信息,对认识与基因相关的疾病的发生和发展有积极的推动作用。本论文旨在发展“原位封装,按需提取”的细胞基因组存储理念,通过在分子水平深入理解无机硅氧烷前驱体的入胞机制,在时空不同维度下精准调控无机材料与细胞及细胞内DNA的作用模式,建立加速老化模型,揭示加速老化或极端条件下DNA结构的变化规律,从而形成较为完整的基于细胞内基因组存储的“分子理性设计-入胞机制研究-细胞与材料复合方式调控-DNA结构变化规律解析”科学关键体系,进而推动新技术在基因库领域的实际应用。更多还原

【Abstract】 Gene,a general term for specific nucleotide sequences with genetic effects on deoxyribonucleic acid(DNA)molecules,is the genetic unit that stores information in nucleic acid,controls the synthesis of various proteins and the operation in organisms,which is the basis of heredity and material continuation.In 1985,"Human Genome Project" was first proposed by American scientists,aiming at deciphering all human genetic information and enabling human beings to fully understand themselves at the molecular level.With the completion of the human genome project,the role of genes in the fields of disease treatment,new drug development,genetic disease diagnosis,individual identification and criminal investigation has become more and more significant.However,gene is fragile and degradable as the material carrier of biological information.It is of great significance at the national level to develop new technologies to achieve its stable,long-term and low-cost preservation,and to expand the existing mode of preservation of genetic information and the construction of gene banks.Biosilicification,namely the formation of biological structures composed of silica,is ubiquitous in nature(such as diatoms,etc.),and is also widely used to construct organicinorganic composite materials.Through the selection of siloxane precursors and the precise control of hydrolysis-condensation conditions,this technology can effectively imitate the formation of fossils in nature.The human body is composed of 40-60 trillion cells,most of which have nuclei,containing 3 billion DNA base pairs,which are ideal gene storage carriers.By simulating fossil formation in nature,the cells are processed to accelerate the formation of "cell fossils",making it possible to achieve in situ encapsulation and long-term storage of genomes in cells.In this paper,aiming at the long-term storage of genome in cells,by regulating the entry mechanism of siloxane precursors,in-depth investigating on the interaction mode of inorganic materials with cells and internal genomes,and establishing an "in situ encapsulation,on-demand extraction" intracellular,the concept of genome storage is expected to develop a new paradigm for the storage of biological information(genome,transcriptome and proteome).The specific research contents include:1.We developed the " cryosilicification " technology,with the assistance of ice crystals,which allows the silica precursor to diffuse into cells and nuclei in a dormant state.The precursor is then hydrolyzed and condensed under the catalysis of cell proteins,and deposited into the whole cell in the form of amorphous silica,realizing the complete encapsulation of intracellular DNA.Different from the traditional formaldehyde preservation mode,the cryosilicification technology will not cause damage and fragmentation to DNA,ensuring the integrity of the genome in cells.The complete packaging of the genome in the cell can not only greatly improve the resistance of the cell genome to extreme conditions(such as ultraviolet light,high temperature and oxygen free radicals,etc.),but also greatly extend the storage life of the whole blood genome.Studies have shown that the half-life of genome storage after treatment at 20 °C is about 1208 years,and the stability is increased by 167 times compared with unprotected blood samples.This technology is low-cost,highly scalable,and easy to combine with different media,such as paper-based carriers or 3D printing technology,thereby greatly expanding the application scenarios of the technology.2.We developed the "deep-silicification" technology,which enables the silica precursor to improve the penetration and intracellular deposition of inorganic silicon elements at room temperature with the assistance of a small amount of permeable solvent dimethyl sulfoxide(DMSO),thereby improving the encapsulation effect of inorganic materials on cells.Studies have shown that with the assistance of the permeable small molecule DMSO,the deposition of siloxane precursors in cells is 30 times that of the original " cryosilicification " technology,namely 27.2 pg/cell.After accelerated aging for 1 day,gel electrophoresis showed that deepsilicification samples had the same bright and clear DNA bands as fresh cell samples,while DNA bands in formaldehyde-fixed samples were almost invisible.deep-silicified samples could still fully amplify all target genome fragments even after accelerated aging for 21 days.The development of " deep-silicification " technology at room temperature avoids the need for low temperature and makes it more convenient in application.3.We developed the "targeted-silicification" technology,through the rational design of the molecular structure,endowing the siloxane precursor with the behavior of targeting the mitochondria,combining with the " cryosilicification " technology,realizing the spatial precision of the mitochondrial DNA(mt DNA)in the cell encapsulation.Studies have shown that the " targeted-silicification " technology is feasible and enables rapid encapsulation of mt DNA in a short period of time.Similarly,after accelerated aging,unprotected samples lost their characteristic DNA bands within 7 days,while the targeted-silicified sample still maintained stable and visible DNA bands.The intact packaging of mitochondria in the cell greatly improves the resistance of mt DNA to extreme conditions(such as ultraviolet light,high temperature and oxygen free radicals,etc.).The spatiotemporal precise encapsulation and long-term storage of intracellular DNA provides multi-dimensional information related to time and space,and plays a positive role in promoting the understanding of the occurrence and development of gene-related diseases.This research developed the cell genome storage concept of "in situ encapsulation,ondemand extraction",through the in-depth understanding of the cell entry mechanism of inorganic siloxane precursors at the molecular level,and the precise regulation of inorganic materials and action of cells and intracellular DNA in different dimensions of time and space.We established an accelerated aging model,based on intracellular genome storage,revealing the changing rules of DNA structure under accelerated aging or extreme conditions,thus forming a relatively complete "molecular rational design-cell entry mechanism research-cell and material composite regulation-DNA structure changing rules analysis" system,which promoted the practical application of new technologies in the field of gene banks.更多还原