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

[21] RNA Mimics of Green Fluorescent Protein [J].

Paige, Jeremy S. ;

Wu, Karen Y. ;

Jaffrey, Samie R. .

SCIENCE, 2011, 333 (6042) :642-646

[22] Identification of Hammerhead Ribozymes in All Domains of Life Reveals Novel Structural Variations [J].

Perreault, Jonathan ;

Weinberg, Zasha ;

Roth, Adam ;

Popescu, Olivia ;

Chartrand, Pascal ;

Ferbeyre, Gerardo ;

Breaker, Ronald R. .

PLOS COMPUTATIONAL BIOLOGY, 2011, 7 (05)

[23]

Persson T, 2002, CHEMBIOCHEM, V3, P1066, DOI 10.1002/1439-7633(20021104)3:11<1066::AID-CBIC1066>3.0.CO

[24]

2-G

[25] AUTOLYTIC PROCESSING OF DIMERIC PLANT-VIRUS SATELLITE RNA [J].

PRODY, GA ;

BAKOS, JT ;

BUZAYAN, JM ;

SCHNEIDER, IR ;

BRUENING, G .

SCIENCE, 1986, 231 (4745) :1577-1580

[26] SEQUENCE REQUIREMENTS OF THE HAMMERHEAD RNA SELF-CLEAVAGE REACTION [J].

RUFFNER, DE ;

STORMO, GD ;

UHLENBECK, OC .

BIOCHEMISTRY, 1990, 29 (47) :10695-10702

[27] Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures [J].

Saksmerprome, V ;

Roychowdhury-Saha, M ;

Jayasena, S ;

Khvorova, A ;

Burke, DH .

RNA, 2004, 10 (12) :1916-1924

[28] RIBOZYMES AS POTENTIAL ANTI-HIV-1 THERAPEUTIC AGENTS [J].

SARVER, N ;

CANTIN, EM ;

CHANG, PS ;

ZAIA, JA ;

LADNE, PA ;

STEPHENS, DA ;

ROSSI, JJ .

SCIENCE, 1990, 247 (4947) :1222-1225

[29] HIV and Ribozymes [J].

Scarborough, Robert J. ;

Gatignol, Anne .

GENE THERAPY FOR HIV AND CHRONIC INFECTIONS, 2015, 848 :97-116

[30] Ribozyme- and deoxyribozyme-strategies for medical applications [J].

Schubert, S ;

Kurreck, J .

CURRENT DRUG TARGETS, 2004, 5 (08) :667-681

← 1 2 3 4 →