Intracellular selection of trans-cleaving hammerhead ribozymes

被引：22

作者：

Huang, Xin ^{[1
]}

Zhao, Yongyun ^{[2
]}

Pu, Qinlin ^{[1
]}

Liu, Getong ^{[1
]}

Peng, Yan ^{[1
]}

Wang, Fei ^{[1
]}

Chen, Gangyi ^{[1
]}

Sun, Meiling ^{[1
]}

Du, Feng ^{[1
]}

Dong, Juan ^{[1
]}

Cui, Xin ^{[1
]}

Tang, Zhuo ^{[1
]}

Mo, Xianming ^{[2
]}

机构：

[1] Chinese Acad Sci, Nat Prod Res Ctr, Chengdu Inst Biol, Chengdu 610041, Sichuan, Peoples R China

[2] Sichuan Univ, West China Hosp, Lab Stem Cell Biol, State Key Lab Biotherapy, Chengdu 610041, Sichuan, Peoples R China

来源：

NUCLEIC ACIDS RESEARCH | 2019年 / 47卷 / 05期

基金：

中国科学院西部之光基金;

关键词：

CLEAVAGE; DISCOVERY; HIV; RNA; VARIANTS; CELLS; TRIAL;

D O I：

10.1093/nar/gkz018

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.

引用

页码：2514 / 2522

页数：9

共 38 条

[11] Repression of small toxic protein synthesis by the Sib and OhsC small RNAs [J].

Fozo, Elizabeth M. ;

Kawano, Mitsuoki ;

Fontaine, Fanette ;

Kaya, Yusuf ;

Mendieta, Kathy S. ;

Jones, Kristi L. ;

Ocampo, Alejandro ;

Rudd, Kenneth E. ;

Storz, Gisela .

MOLECULAR MICROBIOLOGY, 2008, 70 (05) :1076-1093

[12] Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing [J].

Fukuda, Masatora ;

Kurihara, Kei ;

Yamaguchi, Shota ;

Oyama, Yui ;

Deshimaru, Masanobu .

RNA, 2014, 20 (03) :392-405

[13] Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA [J].

Hendrix, C ;

Anne, J ;

Joris, B ;

VanAerschot, A ;

Herdewijn, P .

BIOCHEMICAL JOURNAL, 1996, 314 :655-661

[14] Structure-based Search Reveals Hammerhead Ribozymes in the Human Microbiome [J].

Jimenez, Randi M. ;

Delwart, Eric ;

Luptak, Andrej .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2011, 286 (10) :7737-7743

[15] Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity [J].

Khvorova, A ;

Lescoute, A ;

Westhof, E ;

Jayasena, SD .

NATURE STRUCTURAL BIOLOGY, 2003, 10 (09) :708-712

[16] KINETIC CHARACTERIZATION OF INTRAMOLECULAR AND INTERMOLECULAR HAMMERHEAD RNAS WITH STEM-II DELETIONS [J].

LONG, DM ;

UHLENBECK, OC .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (15) :6977-6981

[17] Tertiary contacts distant from the active site prime a ribozyme for catalysis [J].

Martick, Monika ;

Scott, William G. .

CELL, 2006, 126 (02) :309-320

[18] A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene [J].

Mok, Wendy W. K. ;

Li, Yingfu .

CHEMBIOCHEM, 2013, 14 (06) :733-738

[19] Effective targeted gene 'knockdown' in zebrafish [J].

Nasevicius, A ;

Ekker, SC .

NATURE GENETICS, 2000, 26 (02) :216-220

[20] An in vivo selection method to optimize trans-splicing ribozymes [J].

Olson, Karen E. ;

Mueller, Ulrich F. .

RNA, 2012, 18 (03) :581-589

← 1 2 3 4 →