Tài liệu Báo cáo khoa học: Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering

Efficient RNA ligation by reverse-joined hairpin ribozymes

and engineering of twin ribozymes consisting of

conventional and reverse-joined hairpin ribozyme units

Sergei A. Ivanov, Ste´phanie Vaule´on and Sabine Mu¨ ller

Ruhr-Universita¨t Bochum, Bochum, Germany

In recent years RNA has become the focus of develop￾ment into new diagnostic and therapeutic schemes.

Antisense-RNA, ribozyme, aptamer and siRNA tech￾nologies have been developed and have found applica￾tion in molecular medicine [1–7]. Signalling aptamers

and aptazymes have been constructed that can sense a

number of molecules in real time and thus are valuable

diagnostic tools [8–10]. Furthermore, recently discov￾ered riboswitches that regulate gene expression in vivo

in response to specific metabolites [11–13] or tempera￾ture [14] may lead to new RNA-based therapeutic

strategies.

Elucidation of the molecular principles of RNA

functioning in a specific context has led to the engi￾neering of RNA molecules with new functions. Two

complementary strategies can be used in RNA engi￾neering: rational design and directed evolution.

Whereas directed molecular evolution relies on the cre￾ation of a repertoire of modified RNAs from which

beneficial variants are filtered, in a rational design

experiment, defined changes in the nucleotide sequence

and ⁄ or secondary structure of a specific RNA are

planned on the basis of a preconceived idea. This

requires detailed structural and mechanistic informa￾tion on the parent RNA. In cases where this informa￾tion is available, rational design has contributed to the

development of new functional RNA, for example, sig￾nalling aptamers and aptazymes [8–10].

Work in our laboratory has focused on the rational

design of functional RNA, in particular on the

development of hairpin-derived twin ribozymes for

site-specific alteration of RNA sequences, and fluores￾cent and affinity labelling of large RNA molecules

[15–18]. The hairpin ribozyme catalyses the reversible

site-specific cleavage of suitable RNA substrates, gen￾erating fragments with a 2¢,3¢-cyclic phosphate and,

respectively, a free 5¢-OH terminus [19,20]. In the

reverse reaction, the oxygen atom of the free 5¢-OH

group of one RNA fragment attacks the phosphorous

of the cyclic 2¢,3¢-phosphate group of another, result￾ing in ligation of the two fragments. In contrast to the

hammerhead ribozyme, the conformation of the hair￾pin ribozyme–substrate complex does not change signi￾ficantly upon cleavage: the two cleavage fragments

Keywords

rational design; RNA catalysis; RNA ligation;

sequence alteration; twin ribozyme

Correspondence

S. Mu¨ller, Ruhr-Universita¨t Bochum,

Fakulta¨t Chemie, Universita¨tsstrasse 150,

D-44780 Bochum, Germany

Fax: +49 234 321 4783

Tel: +49 234 322 7034

E-mail: [email protected]

(Received 13 June 2005, accepted 15 July

2005)

doi:10.1111/j.1742-4658.2005.04865.x

In recent years major progress has been made in elucidating the mechanism

and structure of catalytic RNA molecules, and we are now beginning to

understand ribozymes well enough to turn them into useful tools. Work in

our laboratory has focused on the development of twin ribozymes for site￾specific RNA sequence alteration. To this end, we followed a strategy that

relies on the combination of two ribozyme units into one molecule (hence

dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes

that are structurally optimized and which, in addition to cleavage, catalyse

efficient RNA ligation. The most efficient variant ligated its appropriate

RNA substrate with a single turnover rate constant of 1.1 min)1 and a

final yield of 70%. We combined a reverse-joined hairpin ribozyme with a

conventional hairpin ribozyme to create a twin ribozyme that mediates the

insertion of four additional nucleotides into a predetermined position of a

substrate RNA, and thus mimics, at the RNA level, the repair of a short

deletion mutation; 17% of the initial substrate was converted to the inser￾tion product.

4464 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS