Nucleic acid template-directed assembly of metallosalen-DNA conjugates [13]

Full text of DOI:10.1021/ja0162212

8618
J. Am. Chem. Soc. 2001, 123, 8618-8619
Scheme 1. Template-Directed Metallosalen-DNA Assemblya
Nucleic Acid Template-Directed Assembly of
Metallosalen-DNA Conjugates
Jennifer L. Czlapinski and Terry L. Sheppard*
Department of Chemistry, Northwestern UniVersity,
2145 Sheridan Road, EVanston, Illinois 60208-3113
ReceiVed May 17, 2001
ReVised Manuscript ReceiVed July 14, 2001
Watson-Crick base pairing organizes DNA duplex formation
necessary for genetic information storage in biological systems.
DNA and RNA templates also direct the specific binding of
nucleotide substrates during diverse enzyme-catalyzed reactions
in replication, transcription, and DNA repair pathways. Recently,
nucleic acid recognition properties have been extended to non-
biological systems, where DNA base pairing has been used to
drive the template-directed chemical ligation of oligonucleotides,1
and the assembly of nanostructures and novel materials.2
a 3a (DNA), SPACER ) TT or 3b (RNA), SPACER ) UU.
Scheme 2a
We have been interested in expanding the versatility of nucleic
acid base pairing for the addressable synthesis of bioconjugates
in aqueous solution. Metallosalen-DNA (4, Scheme 1) represents
an ideal system to demonstrate the concept of nucleic acid
template-directed molecular synthesis. Salens, which are con-
structed from two salicylaldehydes and a diamine, serve as ligands
for a broad range of metal ions. Many metallosalens are
compatible with aqueous conditions3 and have demonstrated utility
as DNA cleavage reagents4 and versatile catalysts for enantiose-
lective transformations.3a,5 Template-directed synthesis of metal-
losalen-DNA conjugates offers a unique approach to a new class
of metal-DNA hybrids. Metal-DNA conjugates previously have
been employed as probes of DNA structure and electron transfer,6
“chemical nucleases” for targeted nucleic acid cleavage,7 and
a Key: (a) HO(CH2)3OH, acidic alumina, PhCH3, reflux; (b) NaOH,
BzCl, THF; (c) [(i-Pr)2N]2POCH2CH2CN, (i-Pr)2NH2‚CHN4, CH2Cl2.
scaffolds for metal-mediated base pairing motifs.8 Thus, metal-
losalen-DNA may offer a new bioconjugate platform for DNA-
organized materials, nucleic acid cleavage and detection strategies,
and in vitro evolution of novel ribozymes and deoxyribozymes.9
Our approach to template-directed synthesis of metallosalen-
DNA is illustrated in Scheme 1. The DNA-metallosalen building
blocks consist of two DNA oligonucleotides modified, at either
the 3′ or 5′ end, with salicylaldehyde moieties (1 and 2). The
modified strands are aligned on a complementary nucleic acid
template (3), bringing the salicylaldehyde groups into proximity
in a duplex. The metallosalen conjugate then is assembled by
addition of an appropriate metal and diamine. Herein we report
the efficient DNA and RNA template-directed synthesis and
characterization of purified metallosalen-DNA conjugates.
A salicylaldehyde phosphoramidite (8, Scheme 2) was syn-
thesized as a precursor to salicylaldehyde-DNA conjugates 1
and 2, necessary for metallosalen-DNA assembly. The protecting
groups for 8, including a benzoate ester for the phenol and a 1,3-
dioxane for the aldehyde, were chosen for their compatibility with
DNA synthesis and postsynthetic deprotection. Starting from
known salicylaldehyde derivative 5,10 dioxane 6 was prepared
by alumina-catalyzed acetalization. Direct benzoylation of 6 with
benzoyl chloride afforded 7, which was converted to phosphor-
amidite 8 by standard methods.11
Oligonucleotide 2 was synthesized by DNA phosphoramidite
chemistry (3′-to-5′), using 8 in the final coupling step. Oligo-
nucleotide 1, bearing a 3′-terminal salicylaldehyde, was produced
by 5′-to-3′ DNA synthesis using commercial nucleoside 5′-
phosphoramidites. Standard DNA deprotection with concentrated
ammonia removed the phenolic benzoate group, and subsequent
* To whom correspondence should be addressed. E-mail:
sheppard@chem.northwestern.edu.
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10.1021/ja0162212 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/10/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 35, 2001 8619
Figure 1. Gel electrophoresis assay of metallosalen-DNA assembly.
(Lane 1) 10 bp marker. (Lanes 2-6) Mn assembly reactions at pH 8.0
for 1 h. (Lane 2) All components: Mn(OAc)2, 1, 2, and 3b, EN. (Lane
3) -EN. (Lane 4) -Mn(OAc)2. (Lane 5) -2. (Lane 6) -3b. (Lane 7)
Ni(II) assembly reaction with all components at pH 6.5 for 24 h. (Lane
8). Labeled 1. All reactions included 10 mM buffer (HEPES for Mn and
MES for Ni) and 150 mM NaCl.
Figure 2. RP-HPLC analysis of Ni-4 nucleoside/Ni-salen enzymatic
digestion products. dU, deoxyuridine (internal standard). dI, deoxyinosine.
*, peak present in the digestion blank.
template, 3b.14 Following Mn-4 assembly with 3b, selective
RNase H digestion of the RNA template strand of the DNA/RNA
hybrid facilitated the purification of Mn-4. After isolation, Mn-4
was stable in buffered aqueous solutions for several days at 25
°C. MALDI-TOF characterization of purified Mn-4 provided the
mass expected for metalated Mn-4 ([M]- calcd, 9773.35; found,
9772.83). To verify the base and salen composition, Mn-4 was
subjected to nuclease digestion and quantitative RP-HPLC
analysis.15 The component nucleosides and salen ligand were
present in the ratios expected for Mn-4.16
Metallosalen-DNA conjugates containing Ni(II) (Ni-4) were
assembled by an analogous template-directed strategy. Ni-4 was
synthesized using 2 µM each of 1, 2, and 3a (or 3b), 300 µM
Ni(OAc)2, and 150 µM EN at pH 6.5. Ni-4 assembly proceeded
in 74% yield at 37 °C in 24 h (Figure 1, lane 7).17 MALDI-TOF
MS characterization ([M]- of Ni-4 calcd, 9777.06; found,
9777.64) verified the identity of the metal-DNA conjugate. Ni-4
was further characterized by enzymatic digestion to deoxynucleo-
sides and Ni-salen, which were present in the correct ratios for
4 as shown by quantitative RP-HPLC analysis in Figure 2.
We have demonstrated the efficient nucleic acid template-
directed synthesis and characterization of a new class of metal-
DNA conjugates, metallosalen-DNA. The chemical diversity of
metallosalen-DNA assembly processes and the applications of
these metal-DNA hybrids currently are under investigation.
Nucleic acid template-directed molecular synthesis offers a
powerful approach for the addressable synthesis of new DNA
bioconjugates, which may offer significant potential for applica-
tions including targeted nucleic acid cleavage, biosensors, and
catalysis.
incubation in 15 mM NaOAc/HOAc buffer (pH 4.0) for 2 h at
37 °C cleaved the dioxane group to the aldehyde. Oligonucleotides
1 and 2 were purified by denaturing polyacrylamide gel electro-
phoresis (PAGE) or reverse-phase HPLC (RP-HPLC), and their
identities were confirmed by matrix-assisted laser desorption time-
of-flight (MALDI-TOF) mass spectrometry (for 1: [M]- calcd,
4860.17; found, 4860.86; for 2: [M]- calcd, 4835.15; found,
4835.37).
The DNA template-directed assembly of a Mn-metallosalen-
DNA conjugate (Mn-4) is demonstrated in Figure 1. Assembly
reactions were monitored by gel electrophoresis using radiolabeled
1 strand as a tracer; metallosalen-DNA formation was reflected
in a shift of the labeled strand to lower gel mobility upon
conjugation. When DNA 1 and 2 were annealed to template 3a
and incubated for 1 h at 37 °C in the presence of 100 µM
ethylenediamine (EN) and 400 µM Mn(OAc)2, a new DNA
complex, corresponding to Mn-4, was produced in ∼65% yield
(Lane 2). Removal of assembly components EN (lane 3) or strand
2 (lane 5) prevented complex formation. In the absence of
manganese (lane 4), ∼4% maximum yield of unmetalated 4 was
observed. Most importantly, DNA template 3a (lane 6) was
required for the assembly reaction. While the templated reaction
was complete in 1 h, untemplated complex formation was
detectable only after 8 h. Taken together, the results in Figure 1
demonstrate that metallosalen-DNA formation is DNA template-
and metal ion-dependent.12 Two additional contributors to Mn-4
assembly efficiency were the identity of the template spacer units
and the pH of the reaction. Two nucleotide residues (TT) provided
an optimal spacer for Mn-4 conjugate formation, consistent with
our molecular modeling of DNA-metallosalen duplexes. Optimal
yields of Mn-4 assembly were observed at pH 8 but decreased
with increasing pH as predicted by metal speciation data and the
limited solubility of manganese ions with increasing pH.13
Purified metallosalen-DNA conjugate Mn-4 was characterized
by two complementary methods: MALDI-TOF MS and nucleo-
side composition analysis. Preparative synthesis of metallosalen-
DNA (∼4 nmol) was performed using a complementary RNA
Acknowledgment. We acknowledge funding from the ACS Petroleum
Research Fund Grant 34740-G4 and MALDI-TOF facilities supported
by NIH Grant S10 RR13810. J.L.C. was supported by an Institutional
NRSA Training Grant in Molecular Biophysics (GM08382).
Supporting Information Available: Experimental procedures, spec-
troscopic and analytical data for compounds 1-8, HPLC and MALDI-
TOF data for compounds 1, 2, and 4, base composition data for 4 (PDF).
This material is available free of charge via the Internet at http://pubs.acs.org.
JA0162212
(12) The excess Mn(OAc)2 necessary for Mn-4 assembly is attributed to
the reversible binding of Mn(II) by DNA phosphate and nucleobase groups.
For a review, see: Kazakov, S. A.; Hecht, S. M. In Encyclopedia of Inorganic
Chemistry; King, R. B., Ed.; John Wiley & Sons: Chichester, 1994; Vol. 5,
pp 2697-2720.
(13) Pourbaix, M. Atlas of Electrochemical Equilibria in Aqueous Solutions;
Pergamon Press: Oxford, 1966; pp 286-413.
(14) Assembly reactions with 3a and 3b provided metallosalen-DNA in
65 and 63%, respectively.
(15) Eadie, J. S.; McBride, L. J.; Efcavitch, J. W.; Hoff, L. B.; Cathcart,
R. Anal. Biochem. 1987, 165, 442-447.
(16) By analogy to other systems, Mn-4 is believed to contain Mn(III):
Hobday, M. D.; Smith, T. D. Coord. Chem. ReV. 1973, 9, 311-317.
(17) Untemplated Ni-salen DNA assembly was undetectable until 48 h.