Tài liệu Báo cáo khoa học: An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities

An allosteric DNAzyme with dual RNA-cleaving and

DNA-cleaving activities

Dazhi Jiang*, Jiacui Xu*, Yongjie Sheng, Yanhong Sun and Jin Zhang

Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, China

Introduction

DNAzymes are efficient biological catalysts that

strengthen the catalytic power of nucleic acids [1,2]. To

date, a series of DNAzymes with RNA-cleaving (or

DNA-cleaving) activity have been obtained by in vitro

selection. Some investigations have focused on the

improvement of specific characteristics and functions

of these DNAzymes through rational design, including

the following: using oligo-DNAs [3,4] or different

wavelengths of light [5–8] as effectors to control the

catalytic activity of the DNAymes; engineering DNA￾zyme-based sensors for Mg2+ [9,10], Cu2+ [11], Hg2+

[12,13], Pb2+ [14], and UO2

2+ [15]; and constructing

molecular logic gates and nanomotors [16–20]. How￾ever, engineering an allosteric DNAzyme with dual

RNA-cleaving and DNA-cleaving activities is very

challenging. To our knowledge, such a DNAzyme has

not been reported.

In this article, we report on a new catalytic activity

in a DNAzyme scaffold generated by rational recon￾struction, and the regulation of catalytic activity by a

conformational transition. We prepared a DNA-cleav￾ing DNAzyme, using a deoxyribonucleotide residue

grafting strategy, as a model system for designing a

bifunctional DNAzyme that undergoes the self-cleav￾age reaction, but also possesses the ability to catalyze

the cleavage of an RNA substrate (RS). An oligo￾RNA molecule played a double role as both the

substrate for the RNA-cleaving activity of the recon￾structed DNAzyme and as a ‘negative’ effector for

controlling the self-cleavage activity of the DNAzyme.

Keywords

activity; allosteric; DNAzyme; grafting;

regulation

Correspondence

J. Zhang, Key Laboratory for Molecular

Enzymology and Engineering of Ministry of

Education, Jilin University, Changchun,

130021 China

Fax: +86 431 88980440

Tel: +86 431 88980440

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 4 November 2009, revised 21

March 2010, accepted 1 April 2010)

doi:10.1111/j.1742-4658.2010.07669.x

A series of RNA-cleaving or DNA-cleaving DNAzymes have been

obtained by in vitro selection. However, engineering an allosteric

DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very

challenging. We used an in vitro-selected pistol-like (PL) DNAzyme as a

DNA scaffold for designing a DNAzyme with dual catalytic activities. We

prepared the 46-nucleotide DNAzyme with DNA-cleaving activity

(PL DNAzyme), and then grafted the deoxyribonucleotide residues from

an 8–17 variant DNAzyme into the region of stem–loop I and the catalytic

core of the PL DNAzyme scaffold. This deoxyribonucleotide residue graft￾ing resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving

activities (DRc DNAzyme). Drc DNAzyme has properties different from

those of the original PL DNAzyme, including DNA cleavage sites and the

required metal ion concentration. Interestingly, the RNA substrate and

RNase A can act as effectors to mediate the DNA cleavage. Our results

show that RNA-cleaving and DNA-cleaving activities simultaneously coex￾ist in DRc DNAzyme, and the DNA cleavage activity can be reversibly

regulated by a conformational transition.

Abbreviations

DRc DNAzyme, DNA-cleaving and RNA-cleaving DNAzyme; PL, pistol-like; RS, RNA substrate.

FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2543