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

How-ever, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging.. An oligo-RNA molecule played a double role as both the substrate for

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 UO22+ [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, usDNA-cleav-ing 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: zhangjin@jlu.edu.cn

*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-cleavgraft-ing and DNA-cleavgraft-ing 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.

(Fig 1A) can efficiently catalyze Cu -dependent

self-cleavage, and is composed of a catalytic core spanning

nucleotides 27–46 and two base-paired structural

ele-ments (stems I and II) flanked by regions of ssDNA

[21] The 5¢-arm of the enzyme binds the cleavable

sequence via Watson–Crick base pairs and the 3¢-arm

through formation of a DNA triplex

The catalytic residues were derived from the 8–17

variant DNAzyme (Fig 1B) The original 8–17

DNA-zyme was isolated by in vitro evolution; this enDNA-zyme

can efficiently cleave RNA to provide 2¢,3¢-cyclic

phos-phate and the 5¢-hydroxyl termini of RNA fragments

[22] In its catalytic core, the dinucleotides A6G7 of a

terminal AGC loop and C13G14 of a bulge loop are

essential, and serve as the key deoxyribonucleotide

residues involved in the cleavage of the RNA

phospho-diester bond [23–25] Deoxyribonucleotides A12 and

A15 of a bulge loop are not conserved A12 can be

changed to T12, and A15 can be changed to G15

When 8–17 DNAzyme binds its substrate, a gÆT

wobble pair can be formed, and is considered to be

significant and crucial for the catalytic activity

Like the parent PL DNAzyme, DRc DNAzyme was shown to catalyze self-cleavage in the presence of

a Cu2+ (Fig 2A) Other metal ions, including

Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cd2+, Zn2+, and

Ba2+, failed to facilitate the cleavage activity The reconstructed DNAzyme was shown to use divalent copper ions with high specificity, despite replacement

of the right domain of the DNAzyme scaffold Incuba-tion of DRc DNAzyme yielded two distinct DNA cleavage products (Pa and Pb) To map the cleavage site of the self-cleaving DNAzyme, we used denaturing PAGE and ran the gel until the reaction products were clearly separated (Fig 2B) The Pa and Pb cleavage fragments were produced upon DRc DNAzyme scission at C14 and C24, respectively The control,

PL DNAzyme, displayed four cleavage fragments (Pa, Pb1, Pb2, and Pb3) which were cleaved at A14, T28, G29, and G30, respectively

To study the rate of DRc cleavage directly, the DNAzyme was incubated at pH 7.0 and 23C The DRc DNAzyme exhibited a narrow functional range for the concentration of Cu2+, with optimum activity

A

C

B

5′

5′

Fig 1 Sequence and predicted secondary structures of original DNAzymes and the reconstructed DNAzyme (A) Sequence and secondary structure of a 46-nucleotide self-cleaving PL DNAzyme A triple helix interaction (dots) occurs between the four base pairs of stem II and four consecutive pyrimidine residues near the 5¢-DNA The major site of DNA cleavage is indicated by the black arrowhead (B) Sequence and secondary structure of 8–17 variant DNAzyme The capital letters represent deoxyribonucleotides, and the small letters represent ribonucleotides (C) Sequence and secondary structure of the reconstructed DRc DNAzyme The DRc DNAzyme can form the DNA or RNA cleavage folded motifs under different reaction conditions.

being reached at 100 lm The rate of DNA cleavage

was highly dependent on the concentration of Cu2+

used in the reaction mixture When the concentration

of Cu2+ was higher or lower than 100 lm, the

cleav-age activity rapidly decreased (Fig 3A) The original

PL DNAzyme showed a bell-shaped dependence on

Cu2+ concentration from 10 mm to 1 nm, with

cleav-age essentially going to completion at 10 lm DRc

DNAzyme and PL DNAzyme were incubated at 23C

in the presence of 100 lm Cu2+and buffers of varying

pH (5.0–8.5) The DRc DNAzyme cleavage product

increased to a maximum yield of 47% at pH 7.5

(Fig 3B) A similar pattern was observed for PL

DNAzyme To evaluate the effect of temperature on

DNA cleavage, we incubated DRc DNAzyme under

standard cleavage conditions while varying the

temper-ature As compared with PL DNAzyme, DRc

DNA-zyme function seemed to have decreased sensitivity to

the reaction temperature DRc DNAzyme exhibited a

broad functional range for temperature, with optimum

activity being reached at 23C (Fig 3C)

After characterizing the DNA-cleaving activity of

DRc DNAzyme, we continued to study its

RNA-cleaving activity (Fig 3D–F) Improved cleavage activity has been observed upon replacement of Mg2+

with Mn2+ To obtain RNA-cleaving rates over a broad range of metal concentrations, cleavage reac-tions in the presence of Mn2+ (100–200 mm) were performed at pH 7.5 The cleavage activity exhibited a sharp metal concentration dependence, with maximal activity at 1 mm (Fig 3D) DRc DNAzyme was mod-erately perturbed in its RNA-cleaving activity relative

to the 8–17 variant Although the Cu2+and ascorbate were important for the DNA-cleaving activity of DRc DNAzyme, they did not support the RNA-cleaving activity of DRc DNAzyme under our reaction conditions (100 lm Cu2+, 10 lm ascorbate, 10 mm

Mn2+, and 50 mm Tris⁄ HCl, pH 7.5)

To investigate the effect of pH on RNA cleavage, the pH dependence of DRc DNAzyme was analyzed between pH 4.92 and pH 9.18 in the presence of 1 mm

Mn2+ (Fig 3E), and was very similar to that of the 8–17 variant DNAzyme It was not feasible to obtain

a quantitatively meaningful rate versus pH, because,

at high pH, Mn2+ precipitation occurred The RNA-cleaving activity of DRc DNAzyme was assayed at

C

D

Fig 2 The DNA-cleaving activity and cleavage sites of DRc DNAzyme (A) 5¢- 32 P-labeled DRc DNAzyme was incubated in buffer A at 23 C with 10 l M various divalent metal ions, which generated two labeled products (P a and P b ) in the presence of Cu2+ Control reactions were incubated in the absence of metal ions Reaction products were separated by 20% denaturing PAGE and imaged by autoradiography (B) Trace amounts of 5¢- 32 P-labeled PL DNAzyme or DRc DNAzyme were incubated in buffer A, containing 10 l M CuCl2, 0.3 M NaCl, 10 l M

L -ascorbate (except for PL–), and 30 m M Hepes at 23 C PL+ and PL ) represent the presence and absence, respectively, of L -ascorbate in the reaction Lanes M I and M II were loaded with 5¢- 32 P-labeled synthetic DNAs of different lengths as indicated, each with a sequence that corresponded to the respective 5¢-terminus of the substrate DNA The letters indicate the 3¢-termini of these radiolabeled marker DNAs (C, D) Schemes for the substrate cleavage sites of PL DNAzyme and DRc DNAzyme, respectively The arrowheads and asterisks denote the positions of cleavage sites.

different temperatures The DNAzyme (Fig 3F)

showed a linear temperature dependence between 25.8

and 53.7C

When evaluating the optimal design for a DNAzyme

with respect to DNA-cleaving and RNA-cleaving

activities, we found that a number of nucleotides within

the catalytic core and substrate-binding arm of PL

DNAzymes were not highly conserved and could be

substituted by the structural domain derived from the

8–17 variant DNAzyme We wanted to combine our

optimization of the arm design and modification of the

catalytic domain to yield an enhanced DNAzyme

Unfortunately, other designed DNAzymes (DA1–DA3)

suggested that DNA-cleaving and RNA-cleaving

activi-ties could not coexist within a DNA motif, and the

double activities competed with each other Because

DRc DNAzyme has comparatively high activities, we

focused substantial effort on its characterization The

DRc DNAzyme with new catalytic activity can arise

from an existing DNAzyme scaffold, indicating that a

single DNA sequence can catalyze the two respective

reactions and assume either of two DNAzyme folds RNAzymes previously investigated have shown similar properties [26,27] The characterization data collected (Fig 3) provide a number of indications and constraints for future modeling studies on the active structure and evolution of the DNAzymes

The regulating effects of RS and RNase on the DNA cleavage of DRc DNAzyme

After characterizing the DNA-cleaving and RNA-cleaving activities of DRc DNAzyme, we found that

an RS could act as an effector to control the DNA cleavage of DRc DNAzyme (Fig 4A) Regulation proceeds via an effector-generated rearrangement of the active site, where the DNA-cleaving active site

of DRc DNAzyme is hindered through the binding of

RS The self-cleaving DRc DNAzyme was incubated

in reaction buffer B, containing 100 lm CuCl2, 0.3 m NaCl, 10 lm l-ascorbate, and 30 mm Hepes (pH 7.0)

at 23C The self-cleavage of DRc DNAzyme was

Fig 3 Characterization of the DNA-cleaving and RNA-cleaving reactions catalyzed by DRc DNAzyme (A–C) Analyses of DNA cleavage at dif-ferent Cu 2+ concentrations, pH values, and temperatures, respectively All reactions were conducted using trace amounts of 5¢- 32 P-labeled DRc DNAzyme In (A), the CuCl2concentration was varied from 1 to 10 m M The reactions were conducted at pH 7.0 (30 m M Hepes) and

23 C with 0.3 M NaCl and 10 l M L -ascorbate In (B), the reactions were conducted under different pH conditions with 0.3 M NaCl, 10 l M

L -ascorbate, and 10 l M CuCl 2 , and were incubated at 23 C In (C), the effect of reaction temperature on DRc DNAzyme function was assessed with cleavage assays conducted as described in (A), except that 10 l M CuCl2was present and the temperature was varied from

12 to 40.7 C (D–F) Analyses of RNA cleavage at with different Mn 2+ concentration, reaction pH values, and temperatures, respectively All reactions were conducted using 20 n M DNAzyme and 2 n M 5¢- 32

P-labeled RS In (D), the MnCl 2 concentration was varied from 0.1 to

200 m M The reactions were conducted at 37 C and pH 7.5 (50 m M Tris ⁄ HCl) In (E), reactions were conducted under different pH condi-tions with 10 m M Mn 2+ , and were incubated at 37 C In (F), the reaction temperature was varied from 25.8 to 53.7 C The reactions were conducted at pH 7.5 (50 m M Tris ⁄ HCl) with 10 m M Mn2+.

measured in the presence of various concentration of

RS regulator, and the percentage cleavage versus RS

concentration yielded a sigmoidal curve (Fig 4B) The

presence of a 12-nucleotide RNA regulator that is

complementary to the RS recognition domain

dramati-cally decreased the rate of DNA self-cleavage We

systematically varied the length of the RS to determine

the effects on regulation of the DNA self-cleaving

activity Enlarging the RS to 18 bp essentially

abol-ished the catalytic activity, as expected This might be

attributed to steric interference in the DNA-cleaving

catalytic domain of DRc DNAzyme Reducing the RS

to 6 bp increased the rate of DNA cleavage, which

approached the self-cleaving rate of DRc DNAzyme in

the absence of RS RSs with greater lengths were more

efficient at decreasing the DNA self-cleavage of DRc

DNAzyme

Here, the RS actually acted as a ‘negative’ effector

to regulate the cleaving activity of DRc DNA-zyme We were interested in constructing a reversible control for catalytic activity RNase is a type of nucle-ase that catalyzes the degradation of RNA into smaller components Bishop and Klavins reported that RNase H had been used to reverse binding in a deoxy-ribozyme nanomotor [20] In our study, RNase A and RNase H were selected as ‘positive’ effectors to elimi-nate the RS regulation As shown in Fig 4C, RNase A was more efficient than RNase H in triggering the DNA-cleaving activity of DRc DNAzyme Under the conditions of the DNA-cleaving regulated system, most RSs exist in a single-stranded state, and a few RSs can form a DNAÆRNA duplex with the RNA substrate recognition domain of DRc DNAzyme RNase A cleaved ssRNA and RNase H specifically

A

C B

Fig 4 The regulating effects of RS and RNase on DNA cleavage (A) Scheme for reversible modulation of DNA self-cleavage (B) The RS acted as a ‘negative’ effector to decrease the DNA cleavage The data fit the Boltzmann equation and the curve is sigmoidal (C) RNase A or RNase H acted as ‘positive’ effectors to renew the DNA cleavage.

property for DNAzymes that have various applications.

In conclusion, we have described a key residue

graft-ing strategy for generatgraft-ing RNA-cleavgraft-ing activity in a

self-cleaving DNAzyme The DNAzyme with

DNA-cleaving and RNA-DNA-cleaving activities was constructed

by incorporating the catalytic domain of 8–17 variant

DNAzyme into the right domain of the secondary

structure of PL DNAzyme, demonstrating that dual

activities can coexist in a small DNA scaffold Using

the RNA substrate and RNase A, we constructed a

simple conformational switch to control the

DNA-cleaving activity of DRc DNAzyme The generation of

a new active site within a DNAzyme scaffold and

reg-ulation of the catalytic activity provide further insights

into the engineering of DNAzymes

Experimental procedures

Materials

The DNA sequences (PL DNAzyme, 5¢-GAATTCTAATAC

GACTCAGAATGAGTCTGGGCCTCTTTTTAAGAAC-3¢;

8–17 variant DNAzyme, 5¢-AATACTCCGAGCCGGTCG

GGCCTC-3¢; DRc DNAzyme, 5¢-GAATTCTAATACTCC

GAGCCGGTCGGGCCTCTTTTTAAGAAC-3¢) were

pre-pared by automated synthesis, and purified by 16%

denaturing PAGE (Sangon, Shanghai, China) The RS

(5¢-gaggcagguauu-3¢) was also prepared by automated

syn-thesis and purified by HPLC (TaKaRa, Dalian, China)

kinase was purchased from TaKaRa RNase A and RNase

H were purchased from MBI All chemical reagents were

purchased from BBI

Activity assays for the DNAzyme

To assess the cleavage activity of the DNAzyme,

radiola-beled RNA or DNA were first generated by enzymatically

tagging the 5¢-termini of synthetic RSs or self-cleaving

5 mm dithiothreitol, 2 lm RS or self-cleaving DNAzyme,

was terminated after a designated period of time by the addition of stop solution containing 60 mm EDTA, 8 m urea,

blue solution Cleavage products were separated by 20% denaturing PAGE and visualized by autoradiography

Regulation of the DNA cleavage of the DNAzyme

For ‘negative’ regulation assays, reactions were initiated by

and 10 ng of RNase A (or 5 U of RNase H) Cleavage products were separated by denaturing PAGE and imaged

by autoradiography

Acknowledgement

This work was supported by grants from the National Natural Science Foundation of China (General Pro-gram No 30770479)

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