Site-selective DNA hydrolysis induced by a metal-free peptide nucleic acid-cyclen conjugate

Article abstract of DOI:10.1039/c1cc14185f

A metal-free artificial restriction DNA cutter which is composed of cyclen and classical peptide nucleic acid (PNA) was synthesized. Analysis of DNA cleavage products indicates the site-selective hydrolysis.

Full text of DOI:10.1039/c1cc14185f

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Site-selective DNA hydrolysis induced by a metal-free peptide
nucleic acid–cyclen conjugatew
Ming-Qi Wang,a Jun-Liang Liu,a Jing-Yi Wang,a Da-Wei Zhang,b Ji Zhang,a
Frank Streckenbach,c Zhuo Tang,*c Hong-Hui Lin,*b Yan Liu,d Yu-Fen Zhaod and Xiao-Qi Yu*a
Received 12th July 2011, Accepted 11th August 2011
DOI: 10.1039/c1cc14185f
A metal-free artificial restriction DNA cutter which is composed
of cyclen and classical peptide nucleic acid (PNA) was synthesized.
Analysis of DNA cleavage products indicates the site-selective
hydrolysis.
DNA-cutting agent.4g Recently, peptide nucleic acid (PNA)
based artificial nucleases have achieved remarkable success.4–7
Kramer and co-workers described a family of PNA–metal
¨
chelating conjugates whose Zr(IV) complexes could site-
selectively hydrolyze single-stranded DNA.6 Very recently,
double-stranded DNA cleaving has been achieved by artificial
restriction DNA cutters (ARCUTs), which combine Ce(IV)/
EDTA with two pseudo-complementary peptide nucleic acid
(pcPNA) additives, in Komiyama’s laboratory.4 However, in
the previous work, DNA cleavage was typically observed
under non-physiological conditions (e.g., 22 1C or 50 1C).
And meanwhile, most efforts were focused on the metal
complexes, which may cause metal dissociation and uncontrolled
redox chemistry.8 In the absence of metal ions, cleavage is
considered to be much safer for applications. Herein, we
present a study on the water soluble 1,4,7,10-tetraazacyclodo-
decane (cyclen) tethered PNA tetramer conjugate possessing
features that are required for metal-free site-selective hydrolysis
of DNA under physiological conditions (Fig. 1).
DNA is one of the most important biomacromolecules in all
living organisms, which contains the genetic information. Human
diseases such as cystic fibrosis, Alzheimer’s, sickle cell anemia,
and certain cancers are associated with mutations in the
sequence of particular genes.1 However, the half-life of DNA
by spontaneous hydrolysis under physiological conditions is
estimated as thousands to billions of years which is primarily
due to the repulsion between the negatively charged backbone
and potential nucleophiles.2 Therefore, studies of DNA cleaving
agents are of great interest and importance for their potential
biotechnology applications. In this field, site-selectivity is an
especially challenging goal even though it is common in
nature. To date, it has been achieved mainly by three systems:
(1) restriction enzymes; (2) DNAzymes; (3) chemistry-based
artificial nucleases. Although restriction enzymes can recognize
DNA sequences and are commercially available, they have
significant drawbacks such as short recognition sequences
which cannot ensure high specificity of cleaving large DNA
molecules. DNAzymes refer to single-stranded DNA with DNA/
RNA cleaving ability which is obtained through in vitro selection
from random DNA libraries.3 All the DNA-cleaving DNAzymes
obtained up to now need specific metal ions as cofactors for
sequence-specific cleavage.
The target conjugate of the PNA tetramer and cyclen was
prepared by a standard liquid-phase peptide synthesis protocol as
previously described9 and has been characterized by MALDI-
TOF mass spectrometry and HPLC. It was found to be water soluble
and quite stable. A detailed synthesis route is given in the ESIw (S-1).
Chemistry-based artificial nucleases are basically composed
of two key components, a sequence-recognizing moiety and a
a Key Laboratory of Green Chemistry and Technology (Ministry of
Education), College of Chemistry, Sichuan University, Chengdu,
610064, P. R. China. E-mail: xqyu@scu.edu.cn;
Fax: +86 28 85415886
b Key Laboratory of Bio-Resources and Eco-Environment (Ministry of
Education), College of Life Sciences, Sichuan University, Chengdu,
Sichuan, 610064, P. R. China
c Natural Products Research Center, Chengdu Institute of Biology,
Chinese Academy of Sciences, 610041, P. R. China
d College of Chemistry and Chemical Engineering, Xiamen University,
Xiamen, 361005, P. R. China
w Electronic supplementary information (ESI) available: Experimental
procedures for the preparation of the compound, the DNA binding
and cleavage studies. See DOI: 10.1039/c1cc14185f
Fig. 1 Structure and schematic view of the PNA–cyclen conjugate
used for site-selective cleavage of the DNA.
c
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DNA binding ability is a critical step for subsequent cleavage,
and DNA melting experiment is a straightforward way to
investigate the relative binding ability of PNA–cyclen that inter-
acts with calf thymus DNA (CT-DNA).10 As shown in Fig. S1
(ESIw), the duplex CT-DNA alone melted at 76.4 1C, whereas the
duplex gains a stabilization of 9 1C in the presence of the
PNA–cyclen conjugate. As cyclen itself did not stabilize CT-DNA
apparently,10 the absorbance changes observed at 260 nm
clearly revealed the increased thermal stability of PNA–DNA
duplexes relative to the corresponding DNA–DNA duplexes. On
the other hand, the binding constant was further determined
by evaluating the fluorescence emission intensity of the ethidium
bromide (EB)–DNA system (Fig. S2w). Addition of the conjugate
to the DNA-bound EB solution caused an appreciable reduction
in the emission intensity. The quenching constant Ksv given by
the slope of the plot was estimated as 0.39 ꢀ 103 Mꢁ1. Upon
comparison, cyclen itself showed almost no change in the
fluorescence intensity (Fig. S2bw). Moreover, the apparent
binding constant Kapp was also calculated as 4.8 ꢀ 103 Mꢁ1
for the PNA–cyclen conjugate. This binding constant implies
the stability of PNA–DNA duplex.
migration (data not shown). The lack of electrostatic repulsion
between DNA and neutral PNA leaves the binding affinity
independent of salt concentration.11 Therefore, increasing the
ionic strength of reaction buffer would not influence the cleavage
activity (Fig. S6w). The PNA–cyclen conjugate catalyzed cleavage
in pH range 6.0–9.0 was also studied (Fig. S7w), and the results
showed that the amount of cleaved DNA increased with the pH
of the solution, which could be the result of more active nucleophiles
formation by the deprotonation of water.12,13 The process of
DNA cleavage perhaps was related to H2O molecules activated
by the cyclen motif.14 Furthermore, time-course study of
DNA cleavage was carried out. Data shown in Fig. S8 (ESIw)
were fit to the integrated equation of pseudo first order reaction
to obtain the rate of DNA cleavage, kobs = 0.185 ꢂ 0.014 hꢁ1
.
It is about 107 times higher than that of DNA natural degradation.2
To verify the mechanism of DNA cleavage promoted by the
PNA–cyclen conjugate, typical reactive oxygen species (ROS)
scavengers13 including hydroxyl radical scavengers (DMSO
and t-BuOH), singlet oxygen scavenger (NaN3) and super-
oxide scavenger (KI) were added to the system. As shown in
Fig. 3 and Fig. S9 (ESIw), no evident inhibition effect on the
DNA cleavage was observed in the presence of any scavenger.
The results ruled out the involvement of cleavage by ROS
species, indicating that the DNA cleavage may occur via a
hydrolytic pathway. In addition, the ligation experiment of
cleaved pUC 19 DNA further provided the evidence for a
hydrolytic mechanism. After the cleaved DNA fractions were
recycled by cutting off the gel and then incubated with T4
ligase for 12 h at 25 1C, most of the DNA fractions were
re-ligated successfully (Fig. 3b, lane 4). The results clearly
suggested that the PNA–cyclen conjugate did hydrolyze DNA.
The denaturing polyacrylamide gel electrophoresis (PAGE)
experiment was performed with the aim of investigating the
sequence selectivity. A 24-nt hairpin oligonucleotide incorpor-
ating only one potential binding and cleavage site chosen as
The DNA cleaving ability was assayed by using supercoiled
pUC19 plasmid DNA as a substrate in Tris-HCl (50 mM, pH 7.4)
in the absence of any external agents or metal ions and deter-
mined by agarose gel electrophoresis. Under these conditions,
after 24 h of incubation at 37 1C, a 0.57 mM PNA–cyclen
conjugate completely cleaved supercoiled DNA to open
circular form (Fig. 2, Lane 6). In order to avoid any effect
due to residual metal contamination in the sample, a potent
metal chelating agent (6 mM EDTA) was introduced into the
system. No evidence indicated that inhibition of DNA cleavage
occurred (Lane 5). However, a similar assay using cyclen alone did
not show any apparent cleavage (Lane 4). For further demon-
strating that the DNA cleavage was because of the metal-free
cyclen motif domain, supplementary experiments were carried
out by using a 3Boc-protected cyclen tethered PNA tetramer
as cleaving agent (Fig. S4, ESIw). Almost no cleavage activity
was observed. Results demonstrate that the DNA cleavage is
initiated by the cyclen derivative when PNA was introduced.
Further investigations of the cleaving ability have been carried
out through a series of optimization experiments including
concentration, ionic strength, pH value and reaction time. The
percentages of supercoiled DNA decreased with the increasing
compound concentration (Fig. S5, ESIw). Higher concentration
would condense DNA due to its strong binding affinity and
resulted in partial block of the plasmid in the electrophoresis
Fig. 3 (a) Agarose gel electrophoresis assays for the effect of ROS
scavengers on the cleavage reaction. Lane 1, DNA control; Lane 2,
0.57 mM PNA–cyclen conjugate control; Lanes 3–6, 0.57 mM PNA–
cyclen conjugate in the presence of DMSO (1 M), NaN3 (1 M), t-BuOH
(1 M), KI (1 M), respectively. (b) Agarose gel for ligation of pUC 19
DNA linearized by the PNA–cyclen conjugate. Lane 1, DNA markers;
Lane 2, pUC 19 linearized by PNA–cyclen without T4 DNA ligase;
Lane 3, DNA control; Lane 4, pUC 19 linearized by PNA–cyclen with
T4 DNA ligase; Line 5, pUC 19 cleaved by PNA–cyclen conjugate.
Fig. 2 Agarose gel of pUC 19 (7 mg mlꢁ1) incubated in Tris-HCl buffer
(50 mM, pH 7.4) at 37 1C in the dark for 24 h. Lane 1, DNA marker;
Lane 2, DNA control; Lane 3, 6 mM of EDTA; Lane 4, 0.57 mM of
cyclen; Lane 5, 0.57 mM of PNA–cyclen conjugate + 6 mM EDTA;
Lane 6, 0.57 mM of PNA–cyclen conjugate.
c
11060 Chem. Commun., 2011, 47, 11059–11061
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reasonably site-selective hydrolysis of the DNA targets under
physiological conditions. Owing to the significant sequence
selectivity of PNAs, cleavage agents conjugated to PNAs have
been an ongoing research area in our group, and they could
regulate efficiently DNA cleavage with well-defined sequence
selectivity. So far the system has displayed modest rate of cleavage,
and in most cases, the cleaver has been used in excess amounts.
In future experiments we will aim at finding reasonably high rate
class of PNA-based artificial nucleases which could display multiple
turnover of site-selective hydrolysis of the DNA targets.
This work was financially supported by the National Science
Foundation of China (Nos. 20725206, 20732004 and 20972104),
Program for Changjiang Scholars and Innovative Research
Team in University, the Key Project of Ministry of Education
in China and Scientific Fund of Sichuan Province for Outstanding
Young Scientist. We gratefully acknowledge the award of a
fellowship to F.S. by the DAAD (program Modern Applications
of Biotechnology in China). We also thank Sichuan University
Analytical & Testing Center for NMR spectra analysis.
Fig. 4 (a) Schematic view of sequence-selective cleavage of 24-nt hairpin
DNA by PNA–cyclen conjugate. (b) Phosphoimager picture of a 15%
polyacrylamide/7 M urea gel showing the cleavage products of the
32
oligonucleotide P-labeled at the 50-end. Line 1, 16–21 nt markers;
Line 2, DNA alone, Line 3, as in Line 2 with the addition of PNA–cyclen
conjugate (0.57 mM), Lines 4–8, 21–17 nt markers respectively.
Notes and references
a substrate was accordingly phosphorylated at its 50-end with
[g-32P] ATP in the presence of T4 polynucleotide kinase and
further purified by PAGE. The cleavage reaction of the oligo-
nucleotide was carried out in Tris-HCl (50 mM, pH 7.4) at 37 1C
for 48 h by adding 0.57 mM PNA–cyclen conjugate. As shown in
Fig. 4, two new faster moving bands were observed (Lane 3) and
the new bands were identified to be 19-mer (Lane 6) and 17-mer
(Lane 8), respectively, according to the weight markers. All
cleavages were located inside the loop region which was in close
proximity to the terminal PNA modification. While in the
absence of the PNA–cyclen conjugate, no cleavage products
were observed in the labeled DNA strand (Lane 2) under the
same conditions. The result demonstrated the cleaving sites were
in close proximity to the binding site on the DNA strand of the
PNA-cyclen conjugate. However, when alterations of nucleotides
in the stem (e.g. from AQT to TQA or GRC to CRG) of the
oligonucleotide were made, the resulting PNA–cyclen conjugate
exhibited no cleavage activity (Fig. S10, ESIw). These observa-
tions indicated that the cleavage of PNA–cyclen relies selectively
on the binding sequence for its activity.
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We have also examined DNA site-selective cleavage by using the
MALDI-TOF analysis. A similar 12-nt hairpin oligonucleotide
substrate (50-OH-CCCTATATAGGG-30-OH) was used and the
obtained molecular masses of the cleaved fragments are shown in
Fig. S11 (ESIw). Cleavage fragments at m/z =1546, 1746, 2139 and
3092 (detailed analyses are shown in Table S1w) addressed to the
products 50-OH-ATAGGG-30-OH binding with the PNA–cyclen
conjugate ([M + H]+, calc. 3092; [M + 2H]2+, calc. 1546), 50-OH-
CCCTAT-30-OH ([M + H3O]+, calc. 1745), and 50-OH-
CCCTATA-30-phosphate ([M + H3O]+, calc. 2138). The results
were consistent with the PAGE experiments. In addition, it was
further proved that phosphodiester bonds of DNA were cleaved
via the hydrolysis pathway. According to the results and some
reports from the literature,8b,14,15 a plausible mechanism for
DNA cleavage is schematically depicted in Scheme S1.w
In summary, a water soluble PNA-based compound was
designed and characterized to serve as a nuclease mimic. This
new class of metal independent artificial nucleases gives
15 M. Kodama and E. Kimura, J. Chem. Soc., Dalton Trans., 1980,
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Chem. Commun., 2011, 47, 11059–11061 11061