Table of Content

10 March 2023, Volume 44 Issue 3

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Preface

核酸修饰与编辑的化学生物学前沿专辑

周　翔　伊成器　季泉江

2023, 44(3):  1-2. 

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Content

Cover and Content of Chemical Journal of Chinese Universities Vol.44 No.3(2023)

2023, 44(3):  1-4. 

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Review

Recent Advances in Chemical Control of CRISPR/Cas9 Genome Editing Technology

XIAO Heng, LI Yongkui, XING Xiwen

2023, 44(3):  20220410.  doi:10.7503/cjcu20220410

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As a revolutionary technology in the field of genetic engineering， clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（CRISPR/Cas9） genome editing contribute significantly to various important diseases， such as cancer， genetic diseases and infectious diseases. However， precisely spatiotemporal control of genome editing in specific cells and tissues to avoid off-target effects is still one of the major challenges for clinical translation of this technology. Recently， using chemical molecules and reactions to control the activity of CRISPR/Cas9 is becoming one of the important means to increase the gene editing efficiency. This review summarized the recent advances about chemical control of CRISPR/Cas9 gene editing and the future applications of these technologies in clinical medicine was proposed.



Study of Epigenetic Modifications of Nucleic Acids Based on Supramolecular Chemistry

ZHANG Kaisong, WANG Shaoru, ZHANG Yutong, TIAN Tian

2023, 44(3):  20220335.  doi:10.7503/cjcu20220335

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Nucleic acids are the genetic material of living organisms and play important roles in the living system. In addition to the classical bases， there are also naturally modified bases in nucleic acids， which are called epigenetic modifications of nucleic acids. Epigenetic modifications of nucleic acids play an important role in the regulation of gene expression and have a great impact on the genetic and growth processes of organisms， and the nucleic acid epigenetics are closely associated to diseases as well. Supramolecular chemistry study the intermolecular bonds， and many biomolecules perform their biological functions via supramolecular processes， and it can be said that a large number of supramolecular chemical processes naturally exist in living organisms. This paper highlighted some representative work on epigenetic modification of nucleic acids based on supramolecular chemistry.



Research Progress on Chemical Intervention of N⁶-Methyladenosine Modification

HUAN Xinyu, LAI Ganqiang, HUANG Yue, YANG Caiguang

2023, 44(3):  20220340.  doi:10.7503/cjcu20220340

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There are multiple modifications on mRNA， which includes N⁶-methyladenosine（m⁶A）， N¹-methyladenosine（m¹A）， 5-methylcytosine（m⁵C）， etc. As the most common modification on mRNA， m⁶A has an impact on the processing of the 5′ ends and 3′ ends of mRNA， as well as the procedure of localization， degradation， and translation. It also regulates the expression of genes at the post-transcriptional level and participates in manifold physiological activities. This paper made a review of the mechanisms on the molecular level of the m⁶A modification and the relationship between the modification and various diseases. Then it outlined the development course of the m⁶A identification technology， emphasized on the latest research progress on m⁶A chemical intervention in order to enable the readers to form a comprehensive understanding of m⁶A modification and provide references for the development of small-molecular drugs targeting on m⁶A modification.



Sequencing Methods for Detection of Nucleic Acid Epigenetic Modifications

FANG Xin, ZHAO Ruiqi, MO Jing, WANG Yafen, WENG Xiaocheng

2023, 44(3):  20220342.  doi:10.7503/cjcu20220342

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All the somatic cells in an organism share the same genetic information， but have different RNA expression subsets. Only part of genes are expressed and perform their functions at a specific time. In recent years， breakthroughs in epigenetic research have helped people understand the regulation of gene expression to a certain extent. Three types of biological macromolecules： DNA， RNA， and protein， are chemically modified after synthesis. These modifications involve the regulation of almost all biological processes. So far， researchers have identified more than 17 and 160 chemical modifications in DNA and RNA， respectively. The interest in various biological functions of DNA and RNA modifications has promoted the development of the frontier fields of epigenomics and epigenomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome is an issue of great concern to us， and it is also a key to the research of epigenomics and epitranscriptomics. Herein， we summarize the detection methods of nucleic acid modifications， and put forward some bottlenecks in the existing technologies and possible innovative methods.



Applications of CRISPR-Cas Technologies in Microbiome Engineering

HU Yucan, CAO Zhaohui, ZHENG Linggang, SHEN Juntao, ZHAO Wei, DAI Lei

2023, 44(3):  20220362.  doi:10.7503/cjcu20220362

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Microbiome engineering aims to achieve targeted and precise manipulation of complex microbial communities. Clustered regularly interspaced short palindromic repeats（CRISPR）-CRISPR-associated protein（CRISPR-Cas） system is an emerging powerful tool for gene editing， which provides a probability to engineering microbiome from gene to ecosystem level. In this review， we summarize recent developments in the emerging area of microbiome engineering， focusing on the applications of CRISPR-Cas technologies in genetic manipulation of microbiome. To begin with， we introduce the concept of microbiome engineering. We propose to classify microbiome engineering by： （1） manipulation at the ecological level， i. e.， alterations in community composition； （2） manipulation at the genetic level， i. e.， editing of metagenomes. Furthermore， manipulation of mcirobiomes at the genetic level can be achieved via different approaches： （1） genome engineering of cultured microbial isolates； （2） metgenome engineering in situ. CRISPR-Cas tools have important applications in microbiome engineering， including strain-specific deletion， transcriptome regulation， site-specific insertion in complex communities， and dissecting the contribution of a single gene in microbiome-host interaction. The complexity of microbiomes requires the development of assistive technologies， such as highly efficient delivery methods， controllable genetic regulatory elements， etc. Finally， we discuss the challenges and opportunities in microbiome engineering， an emerging research area at the intersection of synthetic biology， chemical biology and microbiome. Breakthroughs in microbiome engineering will greatly advance our understanding of microbiome and transform many fields such as human health， agriculture， and environmental protection.



Applications of Gene Editing in Mitochondrial Diseases

CHANG Liying, LING Xinyu, CHEN Heqi, WANG Xue, LIU Tao

2023, 44(3):  20220363.  doi:10.7503/cjcu20220363

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Mitochondria， as energy factories of cells， play an important role in maintaining cell energy metabolism and human life activities. The mutations of mitochondrial genome have led to a series of mitochondrial diseases， which seriously threaten human life and health. The development of gene editing methods targeting mitochondria is of great significance for the treatment of mitochondrial diseases. In recent years， a series of gene editing methods inclu-ding restriction nuclease， zinc finger nuclease， transcriptional activator like effector nuclease， clustered regularly interspaced short palindromic repeats（CRISPR） and mitochondrial base editor have been developed. This article reviewed the recent progresses， deficiencies and prospects of the applications of gene editing tools for mitochondria gene editing. We hope it could provide reference for the development of mitochondrial disease treatment.



Progress on the Recognition， Complex Structure and Intracellular Detection of Nucleic Acid G-quadruplex

LIU Wenting, LIU Liuyi, ZHU Bochen, MAO Zongwan

2023, 44(3):  20220419.  doi:10.7503/cjcu20220419

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DNA or RNA guanine-rich sequences can form non-canonical secondary structures known as G-quadruplexes. The structures of G-quadruplex are rich and diverse， and they exist dynamically in organisms. G-quadruplex is involved in key genomic functions such as transcription， replication， genomic stability and epigenetic regulation and is closely related to cancer biology. The studies on the structural and functional mechanisms of G-quadruplex have promoted G-quadruplex-targeted tumor intervention and treatment. This paper reviews the specific recognition， cell detection and biological functional regulation of G-quadruplex， summarizes the research progress in designing small molecules recognizing and targeting of G-quadruplex and their complex structures. The possibility of G-quadruplex-targeted intervention and disease treatment is discussed. Finally， the challenges and opportunities in the future research of G-quadruplex field are prospected.



Progress on the Stereocontrolled Synthesis of Phosphorothioate Oligonucleotides

CAO Shujie, LI Hongjun, GUAN Wenli, REN Mengtian, ZHOU Chuanzheng

2023, 44(3):  20220304.  doi:10.7503/cjcu20220304

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Phosphorothioate（PS） oligonucleotides have found a wide range of applications in biochemical mechanism studies， biomedicine， materials and related fields. The stereo configuration of PS has a remarkable effect on the biochemical properties of PS nucleic acids， which has aroused wide interest in developing methods for efficient and stereoselective synthesis of PS oligonucleotides in the past 30 years. This review summarized the methods for stereocontrolled synthesis of PS oligonucleotides， with a focus on the research progress in the past decade. The advantages and disadvantages of different methods were comparatively analyzed. Finally， the prospect of stereocontrolled synthesis of PS oligonucleotides was briefly discussed.



Research Progress of CRISPR-Cas9 Functional Regulation System Based on Small Molecule Reaction Tools

KONG Hao, XU Feiyang, WANG Yixiang, ZHANG Yan

2023, 44(3):  20220346.  doi:10.7503/cjcu20220346

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CRISPR is one of the major research focus of gene editing at present， and has been widely used in research areas like disease treatment and crop improvement. Among these CRIPSR systems， CRISPR-Cas9 system is the most deeply studied one. How to reduce the off-target effect in mammalian cells is one of the major challenges of CRISPR-Cas9. The functional regulation system of CRISPR-Cas9 can be constructed by using light or active molecule induced small molecule reaction tools. By controlling the function of sgRNA， Cas9 or Cas9/sgRNA complex， spatial-temporal regulation of the gene editing function of CRISPR-Cas9 can be achieved， and the probability of non-specific action can be greatly reduced. At the same time， small molecules have little interference with the original system， thus small molecule reactions have becoming more and more important tools for the CRISPR-Cas9 regulation systems. In this review， we summarize the main research progress of CRISPR-Cas9 functional regulation system based on small molecule reaction tools， and prospected the future development of this field.



Applications of Nanopore Sequencing Technology in the Detection of Nucleic Acid Modifications

CHEN Jialu, HUANG Shuo

2023, 44(3):  20220333.  doi:10.7503/cjcu20220333

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A wide variety of chemical modifications have been found in DNA and RNA. These nucleic acid modifications play critical roles in the regulation of gene expression， affect biological processes such as growth and development， and cause diseases including cancer. Accurate identification and localization of nucleic acid modifications can help to understand their functional mechanism， and contributes to diagnosis and treatment of relevant diseases. Nanopore sequencing is an emerging single-molecule sequencing technology and can directly detect nucleic acid modifications. It can simultaneously detect multiple modifications based on the difference in pore blockage signals between modified and canonical bases. This review briefly summarizes the principle and recent development of nanopore sequencing， computer algorithms for nucleic acid modification identification and applications of nanopore sequencing. By the end of this review， prospects of future development of nanopore sequencing is also proposed.



In-solution Selection Methods of DNA-encoded Library

WANG Yingying, LI Xiaomin, CAI Yahui, LI Xiaoyu, SHI Bingbing

2023, 44(3):  20220438.  doi:10.7503/cjcu20220438

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DNA-encoded library（DEL） has become a nearly indispensable technology platform for the development of new drugs in the pharmaceutical industry. The DEL technology has become matured over the past three decades and the rapid development of DEL-compatible chemistries has tremendously improved the chemical diversity of DELs and promoted its application in drug discovery. DEL technology has been widely adopted by pharmaceutical companies， and numerous clinical candidates have been identified from DEL selection. Usually， the selection of DEL is mainly affinity-based selection on solid-phase. The purified target protein is immobilized on the matrix， and then the binders are separated from non-binders by physical washes. In recent years， a series of in-solution selection methods for DEL have been developed， which has further expanded the target scope suitable for DEL and revealing the potential of DEL as an effective tool to explore fundamental biology. This review mainly introduces the selection methods of DEL in-solution phase and their applications， and finally gives a brief outlook on the application of DEL technology to complex biological targets and functional selection.



Functions of Plant RNA Modifications and Their Analytical Methods

TANG Xiaomeng, YUAN Bifeng, FENG Yuqi

2023, 44(3):  20220265.  doi:10.7503/cjcu20220265

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Apart from the canonical nucleobases， RNA molecules contain a variety of chemical modifications. So far， over 150 different types of RNA modifications have been identified in the three-domain system of life. These chemical modifications do not alter the sequence of RNA， but they can change the structures and biochemical properties of RNA， which thereby can control and regulate the spatiotemporal expression of genes. As an important epigenetic regulation layer， RNA modifications also play crucial roles in regulating plant growth and development and respond to stresses. In recent years， with the rapid advancement of analytical methods， especially the technologies for RNA modification sequencing， the functions and mechanisms of plant RNA modifications have gained in-depth understanding. In the current review， we introduce the functions of plant RNA modifications and summarize the analytical methods for plant RNA modifications. We believe that this review will provide useful information for the systematic study of plant RNA modifications in the future.



Non-viral Delivery of CRISPR/Cas9 Genome Editing

SHENG Jinhan, ZHENG Qizhen, WANG Ming

2023, 44(3):  20220344.  doi:10.7503/cjcu20220344

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Genome editing based on the clustered regularly interspaced short palindromic repeats（CRISPR）/ CRISPR-associated protein 9（CRISPR/Cas9） system provides an efficient and rapid tool for precise control and regulation of mammalian cell genome. Its chemical biology and biomedical application is however challenged by the delivery of the CRISPR/Cas9 gene editing tool that composed of Cas9 protein and gRNA into mammalian cells. In recent years， a large variety of non-viral delivery vectors have been designed for CRISPR/Cas9 gene editing delivery in the form of DNA， messenger RNA（mRNA） and ribonucleoprotein complex（RNP）. In this article， we summarize the most recent progress of non-viral CRISPR/Cas9 genome editing tool， and the application prospect of CRISPR/Cas9 genome editing technology in chemical biology.



Research Advances of Detection Approaches Towards N⁴-Acetylcytidine（ac⁴C） RNA

HE Yinming, KONG Sudong, LIN Jianguo, XIE Minhao, CHENG Liang

2023, 44(3):  20220319.  doi:10.7503/cjcu20220319

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A large number of epigenetic chemical modifications have been detected in cellular mRNA and non-coding RNAs. Among these modifications， N⁴-acetylcytidine（ac⁴C） is a relatively unique one， which has widely been discovered in tRNA， rRNA and tRNAs in eukaryotes and prokaryotes. Current research have shown that ac⁴C RNA may possess a variety of biological functions， including regulating the translation process of proteins， affecting the stability of RNA， and modulating RNA-protein interactions. However， only one ac⁴C acetyltransferase， NAT10， has been identified so far. Besides， current technical methods for the detection and sequencing of ac⁴C RNA are quite limited. This review comprehensively summarized current advances in the exploration of distribution of ac⁴C RNA， its roles in gene expression regulation and its detection methods. Finally the opportunities and shortcomings of the related research on ac⁴C were summarized and prospected.



Article

Effects of Exogenous N⁶-methyladenosine Incorporation on the Expression of Cellular mRNA Transcripts

ZHANG Qingyi, CAO Jie, SHU Xiao, LIU Jianzhao

2023, 44(3):  20220173.  doi:10.7503/cjcu20220173

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N⁶-methyladenosine（m⁶A） is the most prevalent and reversible internal mRNA modification in eukaryotic cells， and plays an essential role in RNA fate determination. In this work， we studied the exogenous incorporation of m⁶A into cellular mRNA through salvage pathway and evaluated its resultant biological effects. First， we found that feeding of HeLa cells with m⁶A nucleoside significantly altered cell morphology and viability. We then synthesized isotope-labelled d₃-m⁶A（N⁶-methyl-d₃-adenosine） and examined the d₃-m⁶A incorporation rate in cellular mRNA along with incubation time using mass spectrometry. Next， RNA sequencing（RNA-seq） was used to study the biological effects of exogenous m⁶A incorporation. Finally， thousands of genes were found to be differentially expressed， and were dramatically enriched in pathways such as ribosome biogenesis， mRNA metabolic process and cell morphogenesis differentiation. All these results suggested that exogenous m⁶A could be incorporated into mRNAs through a metabolic pathway to influence cellular gene expression.



Characterization of an RNA-Cleaving TNA Enzyme and Application in MicroRNA Cleavage

SUN Xin, GAO Mingmei, ZHANG Ze, WANG Haiyan, YU Hanyang

2023, 44(3):  20220246.  doi:10.7503/cjcu20220246

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In this work， we systematically characterized a previously identified RNA-cleaving threose nucleic acid（TNA） enzyme 6-29 including the substrate sequence requirement and reaction rate constant， and further demonstrated its application in microRNA cleavage. It was revealed that 6-29 possessed a relatively broad substrate sequence scope and was able to cleave RNAs with distinct sequences and cleavage sites. Moreover， TNA enzyme 6-29 remained active in the presence of a variety of metal ions such as Mg²⁺ and Mn²⁺， and functioned under a multi-turnover condition. Lastly， it was demonstrated that 6-29 catalysed site-specific cleavage reactions on severaldifferent microRNA sequences. This work highlights catalytic TNA as a promising molecular tool， which could be developed to act as a key functional element in disease theranostics.