Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA

Article abstract of DOI:10.1080/07391102.2018.1441071

The facile construction of metal–DNA complexes using ‘Click’ reactions is reported here. A series of 2′-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through ‘Click’ reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal–DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal–DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.

Full text of DOI:10.1080/07391102.2018.1441071

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Construction and structure studies of DNA-
bipyridine complexes as versatile scaffolds for site-
specific incorporation of metal ions into DNA
Rui Wang, Srivathsan V. Ranganathan, Phensinee Haruehanroengra, Song
Mao, Matteo Scalabrin, Daniele Fabris, Alan Chen, Hehua Liu, Abdalla E.A.
Hassan, Jianhua Gan & Jia Sheng
To cite this article: Rui Wang, Srivathsan V. Ranganathan, Phensinee Haruehanroengra, Song
Mao, Matteo Scalabrin, Daniele Fabris, Alan Chen, Hehua Liu, Abdalla E.A. Hassan, Jianhua Gan
&
Jia Sheng (2018): Construction and structure studies of DNA-bipyridine complexes as versatile
scaffolds for site-specific incorporation of metal ions into DNA, Journal of Biomolecular Structure
and Dynamics, DOI: 10.1080/07391102.2018.1441071
To link to this article: https://doi.org/10.1080/07391102.2018.1441071
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Journal of Biomolecular Structure and Dynamics, 2018
https://doi.org/10.1080/07391102.2018.1441071
Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for
site-specific incorporation of metal ions into DNA
a,b
b
a,b
a,b
a,b
Rui Wang , Srivathsan V. Ranganathan , Phensinee Haruehanroengra , Song Mao , Matteo Scalabrin ,
a,b
a,b
c
d
c
a,b
Daniele Fabris , Alan Chen , Hehua Liu , Abdalla E.A. Hassan , Jianhua Gan * and Jia Sheng *
a
Department of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA;
b
c
The RNA Institute, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA; State Key
Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and
d
Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China; Applied Nucleic Acids Research Center, Faculty of
Science, Zagazig University, Zagazig, Egypt
Communicated by Ramaswamy H. Sarma
(
Received 6 November 2017; accepted 12 February 2018)
The facile construction of metal–DNA complexes using ‘Click’ reactions is reported here. A series of 2′-propargyl-modi-
fied DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through ‘Click’ reac-
tion to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the
DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchange-
able using a simple EDTA washing-and-filtration step. The constructed metal–DNA complexes were found to be ther-
mally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and
installing the bipyridine ligands by molecular modeling and simulation. These metal–DNA complexes could have wide
applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.
Keywords: metallic DNA; click reaction; propargyl-DNA; DNA material; DNA scaffold
1
.
Introduction
as an excellent biofunctional framework for developing
cell imaging probes, biocatalysts, drug deliver systems,
and therapeutics (Chen, Groves, Muscat, & Seelig, 2015;
Garg, Rath, & Goyal, 2015; Garg, Singh, Arora, &
Murthy, 2012; Gupta, Tiwari, & Vyas, 2012; Wang,
Huang, Yang, He, & Liu, 2013; Xu et al., 2014). These
functional DNA molecules can be further used to con-
struct small molecule–DNA hybrids that will largely
increase the scope and versatility of their functional
applications (Hong et al., 2015; McLaughlin, Hamblin,
DNA, the fundamental biomolecule of life, has increas-
ingly emerged over the past few decades as a key build-
ing block and structural framework creating a myriad of
functional materials (Cutler, Auyeung, & Mirkin, 2012;
Macfarlane, O’Brien, Petrosko, & Mirkin, 2013; Rothe-
mund, 2006; Seeman, 1982, 2010). These DNA-based
materials are very attractive in both fundamental research
and practical applications. For instance, owing to the
unique base pairing specificity, predictability, and versa-
tile programmability, DNA self-assembly has become an
important strategy for the construction of nano-materials
such as carbon nanotubes, nanobricks, metal nanoparti-
cles, and quantum dots, etc. through a bottom-up method
with precise design and control (Deng, Samanta,
Nangreave, Yan, & Liu, 2012; Douglas, Dietz, et al.,
&
Sleiman, 2011; Thaner, Eryazici, Farha, Mirkin, &
Nguyen, 2014). It is well known that single-stranded
DNA/RNA molecules, so-called aptamers, can be
selected through SELEX technology (Systematic Evolu-
tion of Ligands by EXponential enrichment) (Ellington
&
Szostak, 1990; Robertson & Joyce, 1990; Tuerk &
Gold, 1990) to bind almost every bimolecular target
including proteins, carbohydrates, lipids, nucleic acids,
and whole cells with extraordinarily high binding speci-
ficity and affinity. These DNA aptamers can serve as
good structure scaffolds for the site-specific incorporation
of diverse functionalities into any desired positions of
the binding targets. In this sense, the hybrid complex of
DNA-small molecules represents a general strategy to
2
2
009; Douglas, Marblestone, et al., 2009; Kuzyk et al.,
012; Maune et al., 2010). In addition, the electrostatic
properties and electrical conductivity of DNA as a highly
charged polymer makes it attractive nanowire to fabricate
nanoscale electro devices (Fink & Schönenberger, 1999;
Klotsa, Römer, & Turner, 2005; Liu, Diao, & Nishi,
2
008; Roche, 2003). More importantly, DNA is
biodegradable and biocompatible, and therefore can work
*
Corresponding authors. Email: ganjhh@fudan.edu.cn (Jianhua Gan); jsheng@albany.edu (Jia Sheng)
©
2018 Informa UK Limited, trading as Taylor & Francis Group

2
R. Wang et al.
diversify the DNA building blocks and holds great
promising to construct novel DNA based functional
materials. However, although this DNA hybrid strategy
has gained increasing popularity in nanostructure con-
structions, its applications in other areas, such as bio-
catalysis and organic synthesis, are yet to be explored
probably due to the lack of feasible synthetic methods
and the detailed structure information about small mole-
cule–DNA interactions.
An interesting application that we are currently explor-
ing involves constructing metallic DNA molecules as
novel artificial ribonucleases to cleave target pathogenic
RNA sequences, and as chiral catalysts to facilitate stere-
oselective organic transformations (Gjonaj & Roelfes,
building blocks, which are well compatible with solid
phase synthesis and post-synthetic treatments. The
minor-groove located propargyl groups have been
demonstrated to produce modest perturbation of DNA
biophysical properties such as duplex stability; and have
been used to generate several fluorescent DNA probes
(Berndl et al., 2009; Holzhauser, Rubner, & Wagen-
knecht, 2013; Yamada et al., 2011). For proof of princi-
ple, we synthesized a few single- and double-stranded
DNA oligos containing each of the four 2′-propargyl
(2′-prop)-A, C, G, and U in different sequence contexts.
The modified products were purified by reverse phase
HPLC and characterized by electrospray ionization mass
spectrometry (ESI-MS, Table 1).
2
013; Oltra & Roelfes, 2008; Park et al., 2014; Sakamoto
The selection of the metal binding ligands fell on a
typical bipyridine structure, which has been previously
applied to construct DNA-based asymmetric catalysts
and functional nano-materials (Heck, Dumarcay, & Mar-
sura, 2002; Ipe, Yoosaf, & Thomas, 2006), as the start-
ing material to install the ‘Click’ active azido tags. As
shown in Scheme 2, the treatment of 5,5′-dimethyl-bipyr-
idine with N-bromosuccinimide (NBS) and azobisisobu-
tyronitrile (AIBN) in a heated CCl4 solution for 17 h
produced a clean mixture of mono- and di-brominated
bipyridine compounds with ~1:1 ratio, which can be sep-
arated by column chromatography. The mono-brominated
product 6 was subsequently transferred to the azido-
bipyridine 2 through the substitution of bromine by azide
in a heated DMF solution with quantitative yield.
et al., 2003). These two functions can be achieved by the
site-specific incorporation of metal ions into specific posi-
tions of DNA scaffolds that serve as either RNA targeting
reagents (Hall, Husken, & Haner, 1996; Sakamoto et al.,
2
&
eral methods have been developed for the site-specific
introduction of metal ions into nucleic acids mainly
through nucleobase–metal chelating or ‘click’ chemistry, a
widely used bioorthogonal strategy to label macro-
molecules (Dey & Jäschke, 2015; Duprey, Takezawa, &
Shionoya, 2013; Gierlich, Burley, Gramlich, Hammond,
003; Williams et al., 2015) or chiral environments (Bos
Roelfes, 2014; Rioz-Martínez & Roelfes, 2015). Sev-
&
2
Carell, 2006; Richters, Krug, Kösters, Hepp, & Müller,
014; Scharf & Müller, 2013; Stubinitzky et al., 2014;
Takezawa & Shionoya, 2012). However, the construction
of a general scaffold or template that can bind a variety of
metal ions for different functional applications has not
been fully explored. In this paper, we demonstrated that
the ‘click’ conjugation could be used to build this type of
general platform for metal binding in a site-specific way.
We first synthesized a series of DNA strands with previ-
ously reported 2′-propargyl modified nucleotide building
blocks at different positions (1, Scheme 1), which can be
subsequently linked to a metal chelating bipyridine ligand
with a preinstalled azido group (2, Scheme 1) to make
DNA–ligand–metal complexes (3, Scheme 1). These site-
specifically located bipyridines can work as versatile plat-
forms to bind different metal ions that are easily
exchangeable using an EDTA-washing-and-filtration step
With these building blocks in hand, we carried out
the ‘Click’ reaction using the standard CuBr catalyst in
the presence of acetonitrile as Cu chelating reagent.
+
Although only moderate synthesis yields could be
achieved for this Click conjugation, the products were
easily separated from the substrates by our reverse phase
HPLC conditions (see Section 4 for conditions). Exem-
plified in Figure 1, the incorporation of bipyridine ligand
into DNA oligonucleotides decreased their retention time
by about 2 min in our chromatogram, which suggested
that these bipyridine–DNA complexes possessed a
Table 1. 2′-Propargyl modified DNA oligonucleotides.
Entry
1
DNA sequence
CCAA(2′-prop-A)
MS (Calcd.)
(
4, Scheme 1). In addition, the 3D structure information of
this type of DNA–metal complex was also elucidated by
X-ray crystallography and molecular simulation studies.
5610.64 (5610.72)
GGTTACAGGTAAG
2
CCAAAGGTTACA(2′-prop-G)
GTAAG
5610.64 (5610.72)
3
4
5
6
7
8
9
TCGGGT(2′-prop-A)CCCGA
TCGGGTAC(2′-prop-C)CGA
TCG(2′-prop-G)GTACCCGA
TCGGG(2′-prop-U)ACCCGA
CGCGAAT(2′-prop-U)CGCG
G(2′-OMe-U)GTA(2′-prop-C)AC
G(2′-OMe-U)GT(2′-prop-A)CAC
3700.67 (3700.45)
3700.67 (3700.45)
3700.67 (3700.45)
3686.38 (3686.45)
3686.39 (3686.45)
2479.47 (2479.64)
2479.47 (2479.64)
2
.
Results and discussion
2
.1. Synthesis of metallic DNA through ‘Click’
reaction
Based on the synthetic feasibility of installing a reactive
group into flexible locations of DNA, we chose the
2
′-propargyl modified ribonucleotides as ideal DNA

Construction and structure studies of metal-DNA
3
Interestingly, the ESI-MS data clearly showed the
presence of copper ion in the complex (Table 2, entry 1)
after both reaction and purification, which can be
explained by the strong binding of copper ion to the
bipyridine ligand. The copper ion was subsequently
washed out using 10 equivalents of EDTA through an
ultrafiltration device with a 3 K-cut-off membrane. The
concentrated solution was subsequently washed with
water to obtain a metal-free species that could serve as a
general template for the binding of other metal ions
2
+
2+
2+
2+
including Cu , Ni , Zn , and Cd . The corresponding
metal–DNA complexes were shown to be very stable
under the conditions employed for ESI-MS analysis
(Table 2 and Figures S1–7). In order to further test their
chemical stability, the metal–DNA oligonucleotide com-
plexes were heated in 10 mM phosphate buffer (pH 7)
under 60°C for 24 h and monitored by HPLC for possi-
ble strand cleavage. As exemplified in Figure S8 for
2
+
DNA6-Cu , the metallic DNA oligonucleotides were
thermally stable. These data demonstrated that bipyri-
dine-directed chelation represents a general and versatile
strategy to achieve site-specific incorporation of metal
ions into different sites of DNA structure.
2.2. Thermal denaturation studies of metallic DNA
duplexes
It has been shown that the propargyl modifications at the
2
′-positions of DNA strand could increase the duplex
stability when the DNA strand is hybridized with com-
plementary RNA, whereas it can slightly destabilize the
duplex when hybridized with DNA (Grøtli, Douglas,
Eritja, & Sproat, 1998; Pujari, Leonard, & Seela, 2014).
Consistent with these observations, as exemplified using
DNA 2 in Table 1, the 2′-propargyl modified DNA
duplex provided nearly the same Tm as the unmodified
native species (black and blue curves in Figure 2),
whereas the overall thermal stability decreased by 6.6°C
for the duplex containing the additional bipyridine ligand
(red curve in Figure 2), indicating certain extent of struc-
tural perturbation.
Table 2. Molecular Masses of DNA Oligonucleotide [5′-
TCGGG(2′-prop-U)ACCCGA-3′] after ‘Click’ reaction and
EDTA-aided ions exchange. The analysis was performed by
ESI-MS as described in the experimental section.
Figure 1. Analytical HPLC chromatograms obtained from 2′-
propargyl-DNA 1 [5′-CCAA(2′-prop-A)GGTTACAGGTAAG]
before (A) and after (B) ‘Click’ reaction with the bipyridine
ligand. Panel (C) is the chromatogram obtained from the co-in-
jected samples.
Entry
Metal Ions
Calcd MS
Found MS
1
2
3
4
5
6
After Click
After Wash
3986.47
3925.54
3986.47
4036.14
3987.48
3981.48
3986.67
3925.75
3986.66
4036.64
3987.67
3981.67
2
+
+
+
Cu
Cd
2
slightly higher overall polarity than the initial propargyl
modified species. The separated substrate could be
reused for the next cycle of ‘Click’ reaction.
2
Zn
2
+
Ni

4
R. Wang et al.
rotated for ~100 degree as observed in the base pairing
pattern (Figure 3(C)). The 2′-propargyl groups were
located in the minor groove of the duplex and turned to
the 3′-direction of each strand. This arrangement leaves
plenty of space for further modifications of the propargyl
group within the minor groove.
2.4. Molecular simulation studies of metallic DNA
duplex
The next step aimed at the characterization of the com-
plete bipyridine conjugates to elucidate how the metal
chelating ligands aligned in the DNA duplex. Unfortu-
nately, all crystals obtained under many different condi-
tions showed very weak and disordered diffraction
patterns. This outcome suggested that, although the
ligand might not affect the overall duplex structure, it
was likely to adversely affect the molecular packing. As
a possible alternative, we employed computational mod-
eling to generate structures, in which two bipyridine
ligands containing two copper ions were attached to the
propargyl groups in the initial crystal structure. The final
model of the whole complex was energy minimized and
then subjected to molecular dynamic simulations. The
comparison between the duplexes with and without the
ligands revealed that the ligands did not significantly dis-
truct the helical structure of the duplex during the course
of the simulation (see Supplementary Figure S9). How-
ever, it was interesting to note that the ligands adopted
two distinct types of orientations as shown in Figure 4.
The dominant configuration (~40%) adopted by the mod-
ified duplex involves one of the ligands reaching across
the minor groove to form a salt bridge between the che-
lated copper ion and the phosphate group of two nucleo-
tides to the 3′ side of the complementary strand
(Figure 4(A) and (C)). This across-the-groove binding by
one of the ligands restricts the ability of the other ligand
to simultaneously form another salt bridge, and hence is
accomadated in the minor groove with the copper ion
facing outward and solvated. The second configuration
(~30%) involves both ligands simultaneously bound to
phosphate groups, which are two nucleotides to the 3′
side of their respective strands (Figure 4(B) and (D)).
While this configuration allows for simulataneous forma-
tion of two salt bridges, we speculate that the entropic
penalty associated with this state makes it less favorable
than the single salt bridge state of the duplex, which also
possesses a twofold symmetry due to the symmetrical
nature of the duplex. Even though we observed both the
symmetrical states of the dominant configuration, we did
not observe a transition between the two due to the large
energy barrier between the states. We surmise that the
multiple orientations adopted by the ligands might have
resulted in the poor resolution of their positions in the
crystal structure.
Figure 2. UV-melting temperature studies of native, 2′-propar-
gyl modified, and dipyridine ‘Clicked’ DNA duplexes.
Sequences: DNA-2 in Table 1, 5′-CCAAAGGTTACAXG-
TAAG-3′, where X represents native dG (black), 2′-prop-G
(
blue) and 2′-dipyridine-G (red), respectively, pairs with their
native complementary strand 5′-CTTACCTGTAACCTTTGG-
′. Their T values are 55.4, 55.1, and 48.8°C, respectively.
3
m
2
.3. Crystal structure studies of 2′-propargyl modified
DNA duplex
In order to explore the possible effects of these propargyl
and bipyridine modifications on the DNA duplex struc-
ture, we successfully crystallized and solved a high-reso-
lution structure of the self-complementary octamer
duplex containing two 2′-propargyl-A residues (DNA-9
in Table 1) [5′-G(2′-OMe-dU)GT(2′-prop-A)CAC-3′] . In
2
this construction, the 2′-OMe-dU residue was used to
facilitate crystallization and drive the DNA sequence to
fold into an A-form duplex without major structural per-
turbation, in analogy with the 2′-SeMe-dU functionality
(
Jiang, Sheng, Carrasco, & Huang, 2007; Sheng, Jiang,
Salon, & Huang, 2007). This modified DNA oligonu-
cleotide could crystallize under several different condi-
tions of Nucleic Acid Mini-Screening and Natrix buffer
kit within a few days. The crystals with best diffraction
were grown under buffer conditions containing 10% 2-
methyl-2,4-pentanediol (MPD), 40 mM sodium cacody-
late (pH 7.0), 12 mM spermine tetra-HCl, 40 mM LiCl,
8
0 mM strontium chloride, and 20 mM magnesium chlo-
ride. The best crystal diffracted to 1.6 Å and the data
collection/structure refinement statistics are summarized
in Table 3.
The overall duplex structure and local 2′-prop-A:U
pairing pattern are shown in Figure 3. Overall, there is
no major structural perturbation caused by these two
propargyl groups by comparing the modified DNA
duplex with its native counterpart (Figure 3(A) and (B)),
although the backbone of 2′-propargyl-A residue is

Construction and structure studies of metal-DNA
5
Table 3. X-ray data collection and structural refinement statistics of 2′-propargyl modified DNA 8mer duplex [5′-G(2′-OMe-dU)GT
2′-prop-A)CAC-3′]
(
2
.
DNA-9 (PDB ID: 5E36)
Data collection
Space group
C2221
Unit cell (Å, °)
32.0, 55.5, 75.7, 90, 90, 90
Resolution range/last shell (Å)
Unique reflections
Completeness (%)
30–1.6 (1.66–1.60)
9231
98.9
7.2
R
merge (%)
<
I/σ(I)>
30.9
5.7
Redundancy
Refinement
Molecules per asymmetric unit
Resolution range (Å)
Number of reflections
Completeness (%)
3 single strands
27.76–1.60
8752
98.61
13.7
R
work (%)
Rfree (%)
16.1
Bond length r.m.s. (Å)
Bond angle r.m.s.
Overall B-factor (Å )
0.048
1.398
10.87
2
Note: Rmerge ¼ RjI ꢀ \I [ j=RI.
Figure 3. Structural features of octamer DNA duplex [5′-G(2′-OMe-dU)GT(2′-prop-A)CAC-3′] . (A) and (B) 180° rotation views of
2
duplex superimposed comparison between propargyl modified 8mer (red) and native 8mer (cyan) (PDB ID: 1DNS), with the two 2′-
propargyl groups stick into minor grooves. The observed r. m. s. d is 1.38 Å. (C) Base pair comparison of propargyl-A:T (red) and
native A:T (cyan). The oxygen and carbon atoms are showed as red and blue spheres, respectively.

6
R. Wang et al.
2
Figure 4. Computational models of bipyridine modified octamer DNA duplex [5′-G(2′-OMe-U)GT(2′-prop-A)CAC-3′] after ‘click’
reaction. (A,B) Surface view of the complex with the bipyridine rings extending cross and within the minor groove. (C,D) Stick view
of the overall duplex structures with the two configurations of ligands and copper ions.
5'-DNA
(A,C,G,T)
O
O
N
N
N
N
O
O
Metal ions
'Click'
N
N
N
M
N
3
'-DNA
1
N
N
Cu2+, Ni2+, Fe2+, Zn2+, Cd2+, Pb2+ etc
+ N
N
3
4
N
3
2
Scheme 1. Construction of metal–DNA complex by the site-specific incorporation of metal ions into DNA through a ‘click’ reaction
between the propargyl modified DNA1 and an azido modified bipyridine 2 as the metal chelating ligand.
Me
Me
Me
N
NBS, AIBN, CCl4
NaN
, DMF
3
N
N
N
N
80 °C, 12h
N
77 °C, 17h
Me
Br
N3
5
6
2
Scheme 2. Synthesis of azido-bipyridine ligand 2 starting from 5,5′-dimethyl-bipyridine compound 5.

Construction and structure studies of metal-DNA
7
It is worthy to note that we performed the molecular
dynamics simulations in 1 M NaCl solution. At such
concentrations of salt, the copper ion is not expected to
strongly coordinate with chloride ions (Jauniaux,
Mawissa, Peellaerts, & Rodesch, 1992), which is what
we observed in our simulations. Instead, we found that
phosphate-bound copper ion coordinates with three water
molecules, maintaining a six-membered coordination
sphere (see Supplemental Figure S10).
ambient temperature was added catalytic AIBN (0.092 g,
0.56 mmol). The mixture was heated to reflux at 77°C
for 17 h until the starting material was completely con-
sumed. The mixture was filtered through silica gel when
hot. The solution was cooled and filtered again to
remove most of the di-bromo product. The filtrate was
concentrated and purified by silica gel chromatography
with 5% MeOH in CH Cl (contain 0.5% NEt ) to pro-
2
2
3
vide the product 0.59 g in 40% isolate yield. Rf 0.8, 5%
1
MeOH in CH Cl (contain 0.5% NEt3). H NMR
2
2
(
400 MHz, CDCl ): δ 8.68 (s, 1H), 8.62 (s, 1H), 8.37 (d,
3
3
.
Conclusion
1H, J = 8.2 Hz), 8.30 (d, J = 8.2 Hz, 1H), 7.85 (d,
J = 8.2 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 4.55 (s, 2H),
In summary, we have described an efficient and facile
method for constructing metallic DNA oligonucleotides
through a ‘Click’ conjugation between 2′-propargyl-mod-
ified DNAs and a bipyridine metal chelating ligand. The
bipyridine-modified DNA can serve as ideal structural
scaffolds to bind different metal ions, which can be
easily exchangeable in our DNA contexts. These com-
plexes are thermally stable and do not cause major struc-
tural perturbations to the DNA duplex. In order to obtain
structural insights, we solved the crystal structure of a
propargyl modified DNA duplex and modeled two bipyr-
idine ligands with two copper ions into the propargyl
groups by molecular simulation. We observed potential
interactions between coordinated copper and oxygen
atoms in the DNA backbone. Such DNA-based metal
chelating systems will be expected to have wide applica-
tions in the construction of artificial ribonucleases and
chiral catalysis, as well as the delivery of metal ions.
1
3
2
.41 (s, 3H). C NMR (CDCl , 100 MHz) δ 156.27,
3
153.07, 149.7, 137.44, 133.68, 120.7, 29.65, 25.14 ppm.
4.3. Synthesis of the azido-bipyridine compound 2
To a stirred solution of 5-(bromomethyl)-5′-methyl-2,2′-
bipyridine, 6 (1.31 g, 5.0 mmol) in MeOH (25 mL) was
added sodium azide (1.01 g, 15.39 mmol) and the mix-
ture was heated to 70°C for overnight. When the starting
material was completely consumed, the solvent was con-
centrated, diluted with CH Cl (20 mL) and the mixture
2
2
was washed with brine, dried over sodium sulfate. The
solvent was concentration and purified by silica gel chro-
matography using eluent 5% MeOH in CH Cl (with 1%
2
2
NEt ) to give the product 1.06 g in 94% yield. Rf 0.4,
3
1
5
% MeOH in CH Cl (with 1% NEt3). H NMR
2 2
(
400 MHz, CDCl ) δ 8.63 (s, 1H), 8.53 (s, 1H), 8.43 (d,
3
J = 8.2 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 7.80 (d,
J = 8.2 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 4.45 (s, 2H),
+
2
[
.42 (s, 3H). ESI-MS [M + H] 226.111 (Calculated
4
.
Experimental
+
M + H] : 226.101) (Heck et al., 2002).
4
.1. Materials and instruments
The organic reagents and solvents were purchased from
Sigma-Aldrich and used directly without further purifica-
tion. All solid reagents were dried under a high vacuum
line prior to use. Air sensitive reactions were carried out
under nitrogen. Analytical TLC plates pre-coated with
silica gel F254 (Dynamic Adsorbents) were used for mon-
itoring reactions and visualized by UV light. Flash col-
umn chromatography was performed using silica gel
4.4. DNA oligonucleotide synthesis
The 2′-propargyl modified phosphoramidites were pur-
chased from ChemGenes Corporation. All the DNA
oligonucleotides were synthesized at 1.0-μmol scales by
solid phase synthesis using a MerMade MM8 synthe-
sizer. All the phosphoramidites were dissolved in ace-
tonitrile to a concentration of 0.07 M. 0.02 M I in THF/
2
1
13
31
(
32–63 μm). All H, C, and P NMR spectra were
Py/H O solution was used as oxidizing reagent. All the
2
recorded on a Brucker 400 spectrometer. Chemical shift
other reagents are standard solutions obtained from
ChemGenes or Glen Research. Synthesis was performed
on the appropriate nucleoside immobilized via a succi-
nate linker to control pore glass (CPG-500). All oligonu-
cleotides were prepared in DMTr-on form. After
synthesis, the oligos were cleaved from the solid support
and fully deprotected with AMA (ammonium hydroxide:
methylamine = 1:1) at 65°C for 30 min. The amines
were removed by Speed-Vac concentrator before HPLC
purification. After the DMTr-on purification, the
detritylation was carried out by the treatment of 3%
1
3
values are in ppm. C NMR signals were determined
using APT technique. High-resolution MS data were
achieved by ESI mass spectrometry at University at
Albany, SUNY.
4
.2. Synthesis of compound 6
To a stirred solution of 5,5′-dimethyl-2,2′-bipyridine, 5
1.03 g, 5.60 mmol) and N-bromosuccinimide (1.02 g,
.60 mmol) in anhydrous CCl (50 mL) under argon at
(
5
4

8
R. Wang et al.
trichloroacetice acid for 3 min, followed by the neutral-
ization with triethylamine to pH 7.0. The DMTr-off
oligonucleotides were purified again by HPLC.
4.8. UV-melting temperature Tm study
Solutions of the duplex DNAs (0.5 μM) were prepared
by dissolving the purified DNAs in sodium phosphate
(10 mM, pH 6.5) buffer containing 100 mM NaCl. The
solutions were heated to 85°C for 3 min, then cooled
down slowly to room temperature, and stored at 4°C for
4
.5. HPLC purification and analysis
The oligonucleotides were purified by reverse phase
HPLC using a Zorbax SB-C18 column at a flow rate of
6
ate, pH 7.1; buffer B contains 50% acetonitrile in 20
mMtriethylammonium acetate, pH 7.1. A linear gradient
from buffer A to 80% buffer B in 25 min was used to
elute the oligos. The analysis was carried out using the
same type of analytical column with the same eluent gra-
dient. All the DNA oligos were analyzed by ESI-MS.
2
h before Tm measurement. Thermal denaturation was
performed in a Cary 300 UV–Visible Spectrophotometer
with a temperature controller. The temperature reported
is the block temperature. Each denaturizing curves were
acquired at 260 nm by heating and cooling from 5 to
mL/min. Buffer A was 20 mMtriethylammonium acet-
80°C for four times in a rate of 0.5°C/min. All the melt-
ing curves were repeated for at least four times.
4
.9. Crystallization and diffraction data collection
DNA samples (0.5 mM duplex) were heated to 80°C for
min, cooled slowly to room temperature, and placed at
4
.6. DNA-click reaction
The propargyl modified DNA oligonucleotides
0.38 mM, 200uL in H O) and azido-bipyridine com-
3
4°C overnight before crystallization. Nucleic Acid Mini
Screen and Natrix Kits (Hampton Research) were used
to screen crystallization conditions at different tempera-
tures using the hanging-drop vapor diffusion method.
Perfluoropolyether was used as cryoprotectant for the
crystal mounting. Data were collected under a liquid
nitrogen stream at −174°C. The diffraction data were
collected at beam lines ALS 8.2.2 in Lawrence Berkeley
National Laboratory. Data were collected at a wavelength
of 1.0 Å. Crystals were exposed for 1 s per image with
a 1 degree oscillation angle. All data were processed
using HKL2000 and DENZO/ SCALEPACK (Otwi-
nowski & Minor, 1997).
(
2
pound 2 (10 mM, 114 uL, H O) were placed in a
2
1
.5 mL vial. In a separate vial, 17 uL CuBr solution
(
100 mM in DMSO/tBuOH 3:1) and 34 uL CH CN
3
solution (100 mM in DMSO/tBuOH 3:1) were mixed
and added to the DNA solution. The mixture was shaken
at room temperature for overnight before being evapo-
rated to near dryness in a Speed-Vac under 65°C.
Sodium acetate (0.3 M, 100 uL) was then added and the
suspension was stirred for 1 h before 1 mL of ethanol
was added. The vial was vortexed well and stored in a
freezer (−80°C) for 1 h before centrifugation for 15 min
at 13,000 rpm. The supernatant was carefully removed
from the DNA pellet. Seventy per cent cold ethanol
4.10. Structure determination and refinement
(
−20°C) was used to wash the pellet for three times.
Finally, the pellet was left drying on air and dissolved in
water for HPLC purification.
The 2′-propargyl modified DNA structure presented here
was solved by molecular replacement with PHASER
using PDB structure 1Z7I as the search model, followed
by the refinement using Refmac. The usual refinement
protocol includes positional refinement, restrained B-fac-
tor refinement, and bulk solvent correction. The stereo-
chemical topology and geometrical restraint parameters
of DNA/RNA were applied (Parkinson, Vojtechovsky,
Clowney, Brünger, & Berman, 1996). After several
cycles of refinement, a number of highly ordered waters
were added. Cross-validation (Brünger et al., 1998) with
a 5% test set was monitored during the refinement. The
σA-weighted maps (Read, 1986) of the (2m|Fo| − D|Fc|)
and the difference (m|Fo| − D|Fc|) density maps were
computed and used to build the 2′-propargyl group.
4
.7. Mass spectrometric nucleic acid analysis
Samples were analyzed by direct infusion ESI on a Ther-
moFisher Scientific (Waltham, MA) Velos LTQ-Orbi-
trapVelos mass spectrometer. All analyses were
performed in nanoflow mode using quartz emitters pro-
duced in house with a Sutter Instruments Co. (Novato,
CA) P2000 laser pipette puller. Up to 5 μL samples were
typically loaded onto each emitter using a gel-loader pip-
ette tip. A stainless steel wire was inserted in the back-
end of the emitter to supply an ionizing voltage that ran-
ged between 0.8 and 1.2 kV. Source temperature and
desolvation conditions were adjusted by closely monitor-
ing the incidence of ammonium adducts and water clus-
ters. The instrument was calibrated using a mixture of
4.11. Molecular simulation study
0
3
.5 mg/ml of CsI in 50% methanol, which provided 1–
ppm mass accuracy.
We performed extensive MD simulations of the DNA
duplex with and without the modifications totalling over

Construction and structure studies of metal-DNA
9
a microsecond of simulation data. The crystal structure
of DNA duplex with two 2′-propargyl groups was used
as the template to which the two bipyridine ligands with
copper ions are added. The ligands (one per strand) was
constructed in an ‘open’ type conformation, i.e. pointing
away from the duplex. We attached copper ions to the
ligands imparting a + 2 charge, followed by MD simula-
tions to predict the structural preferences of the ligand
with respect to the rest of the duplex. Molecular dynam-
ics simulations were performed using Gromacs-4.6.3
Acknowledgments
The X-ray diffraction data were collected at the Advanced
Light Source (ALS) beamlines 8.2.2.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the start-up fund from University
at Albany, SUNY. The Berkeley Center for Structural Biology
is supported in part by the National Institutes of Health,
National Institute of General Medical Sciences, and the Howard
Hughes Medical Institute. The Advanced Light Source is sup-
ported by the Director, Office of Science, Office of Basic
Energy Sciences, of the US Department of Energy [contract
number DE-AC02-05CH11231] and the authors also thank the
Key Research and Development Project of China
(
Hess, Kutzner, van der Spoel, & Lindahl, 2008). The
simulation system included the DNA duplex with/
without ligands in a solution of 1 M NaCl solution in a
3
3
D periodic box. The box size was 5 × 5 × 5 nm
+
−
containing 85 Na ions, 75 Cl ions and 3769 water
molecules. The systems were subjected to energy mini-
mization to prevent any overlap of atoms, followed by a
(2016YFA0500600) for the financial support.
1
2
0 ns equilibration run. Configurations were chosen at
ns intervals from the equilibration run, for five inde-
pendent 100 ns production runs, totalling 0.5 microsec-
ond of trajectory for analysis. The MD simulations
incorporated leapfrog algorithm with a 2 fs time step to
integrate the equations of motion. The system was main-
tained at 300 K using the velocity rescaling thermostat
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