Protein recognition and denaturation by self-assembling fragments on a DNA quadruplex scaffold

Article abstract of DOI:10.1002/anie.200603479

Recognize this face? Quadruplex DNA has been used as a noncovalent scaffold to project binding groups for protein recognition and denaturation, with the best quadruplex decreasing the melting temperature of cytochrome c by about 45 K. (Figure Presented).

Full text of DOI:10.1002/anie.200603479

Angewandte
Chemie
DOI: 10.1002/anie.200603479
Self-Assembly
Protein Recognition and Denaturation by Self-Assembling Fragments
on a DNA Quadruplex Scaffold**
Debarati M. Tagore, K. Ingrid Sprinz, Steven Fletcher, Janarthanan Jayawickramarajah, and
Andrew D. Hamilton*
Molecular self-assembly can be defined as a process by which
small molecules associate through defined and designed
interactions to form a larger aggregate with properties not
possessed by the individual components.[1] Nature presents
innumerable examples of large multiprotein aggregates that
assemble through complementary exterior surface interac-
tions and carry out critical functions in different organisms,
from the complex photosynthetic reaction center[2] to simpler
tetrameric hemoglobin[3] and from multisubunit enzymes such
as polyketide synthases[4] to viral coat assemblies.[5] Similarly,
complex oligonucleotide assemblies exemplified by the
ribosome and lipid aggregates in bilayer membranes, repre-
sent functional assemblies formed by other biomolecules.
However, outside the field of synthetic membrane chemistry,
examples of artificial molecules that self-assemble into func-
tional aggregates, particularly under aqueous conditions, are
rare.[6]
stability of the protein is attributed to preferential binding of
the anionic porphyrins, through charge complementarity and
hydrophobic interactions, to unfolded states of the protein.
The fourfold symmetrical structure of these functional-
ized tetracarboxyphenylporphyrins suggested that an alter-
native self-assembling scaffold might be provided by one end
of an assembled guanosine tetraplex. These hydrogen-bonded
tetramers project on one end a p surface comparable in size to
tetraphenylporphyrin and which, when appropriately func-
tionalized, should present four synthetic fragments in a
similar manner (Scheme 1).
There is great potential in the use of complementary
oligonucleotide sequences to control the formation of aggre-
gates that in turn can act as scaffolds for the formation of
functional assemblies.[7] We,[8] and others,[9] have recently
shown that functionalized and complementary DNA strands
can form a duplex that presents two synthetic fragments on
one end for binding to proximal sites on a target protein.
These noncovalently assembled fragments mirror the strong
protein binding properties of covalently linked bidentate
ligands. In a further development of this strategy, we have
now constructed scaffolds that present as many as four
synthetic fragments for binding to a protein surface. We had
previously reported that copper(II) complexes of polyanionic
functionalized porphyrins are effective protein binding agents
Scheme 1. Representation of the relative sizes of a G quartet and a
tetraphenylporphyrin derivative.
Quadruplexes arise from the p stacking of cyclic arrays of
four hydrogen-bonded guanines (G quartet) in intermolecu-
lar and/or intramolecular Hoogsteen base-pair associations of
one, two, or four strands in parallel and/or antiparallel
orientations.[12] A single stretch of contiguous guanine bases
has the propensity to form a four-stranded intermolecular
parallel quadruplex in the presence of potassium ions.[13]
Herein, we describe the use of quadruplexes to generate
versatile supramolecular assemblies that orient, in a well-
defined manner, four organic fragments on one end of a
parallel quadruplex for protein recognition. An earlier,
although nonfunctional example of a functionalized tetraplex
was demonstrated by Aoyama and co-workers.[14]
The 5’-end of the quadruplex-forming oligonucleotides
was functionalized with various anionic and hydrophobic
fragments to target both the hydrophobic patch and the
invariant lysine residues near the heme edge domain of cyt c
(Figure 1). A fully covalent version of this approach has
employed synthetic porphyrins containing a variety of the
same anionic substituents that show strong binding to, and
denaturation of, cyt c.[11]
and trigger
a conformational change in cytochrome c
(cyt c).[10] The resultant cyt c adduct has markedly lowered
thermal stability characterized by a decrease in the melting
temperature of about 50 K.[11] This dramatic reduction in the
[*] D. M. Tagore, K. I. Sprinz, S. Fletcher, J. Jayawickramarajah,
Prof. A. D. Hamilton
Department of Chemistry
Yale University
P. O. Box 208017, New Haven, CT 06520-8107 (USA)
Fax: (+1)203-432-3221
E-mail: andrew.hamilton@yale.edu
Homepage: http://ursula.chem.yale.edu/~hamgrp
[**] We thank the National Science Foundation and National Institutes
of Health (GM35208) for financial support of this work. We also
thank Dr. S. Chaturvedi (PrimeSyn Inc.) and Dr. P. Gunning for their
assistance.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 223 –225
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Communications
parent unfunctionalized quadruplex 1 shows only a 9 K
decrease in the Tm (Figure 2), presumably through non-
specific oligoanionic effects of the phosphate backbone. The
results are more pronounced with the functionalized quad-
Figure 2. CD monitored thermal denaturation of cyt c at 222 nm in the
presence of functionalized quadruplexes.
Figure 1. a) Functionalized parallel quadruplex with the different
organic fragments used for the self-assembly shown as X, Y, and Z.
b) Sequences of the designed oligonucleotides wherein quadruplex 1X,
1Y, and 1Z are appended with X, Y, and Z fragments, respectively.
Oligonucleotide 1 is an unfunctionalized quadruplex, and 2Z is a
functionalized single strand that is incapable of forming higher-
ordered structures.
ruplexes 1X and 1Y in which the Tm is reduced by about 20 K
and 25 K, respectively. However, the most dramatic effect is
seen with 1Z, which in its fully self-assembled state reduces
the Tm for cyt c by about 45 K, a reduction of about 36 K and
about 45 K when compared with the unfunctionalized tetra-
plex 1 and the unassembled functionalized single strand 2Z,
respectively. The enhanced denaturation effect of quadruplex
1Z is presumably due to preferential binding to non-native
states of the protein. There is a noteworthy decrease in the
CD signal of cyt c at 958C, although the quadruplex peaks are
unaffected indicating that the quadruplex structure is main-
tained at high temperatures (see the Supporting Information).
Cyt c has a well-folded, compact structure and hence is
resistant to proteolytic digestion. However, even a partial
unfolding of the protein on denaturation would lead to an
increased susceptibility to proteolysis. Such selective degra-
dation of target proteins by destabilization of the native state
is an attractive goal. The effect of quadruplexes 1Y and 1Z in
unfolding cyt c was assessed by proteolytic digestion of the
quadruplex/cyt c complexes with trypsin at 378C. In the
presence of quadruplexes 1Yand 1Z, tryptic digestion of cyt c
is accelerated and is complete in 120 min and 30 min,
respectively (Figure 3). In contrast, there is no appreciable
digestion of cyt c after 5 h in the absence of the quadruplex.
Furthermore, the structure dependence of this effect is
evident from the significantly slower rate of proteolysis
observed in the presence of the functionalized single-stranded
oligonucleotide 2Z and the unfunctionalized quadruplex 1
(see the Supporting Information).
The organic fragments contained both an amino end for
conjugation to the oligonucleotide and a carboxylic acid end
for protein recognition. The synthesis of the carboxylic acid
precursors X and Y and their conjugation to the oligonucle-
otide is outlined in the Supporting information. The synthesis
of fragment Z has been previously reported.[11] The organic
fragments were synthesized with the carboxylic acids pro-
tected as base-labile cyanoethyl esters for a single-step
deprotection of the cyanoethyl groups along with the
protecting groups on the DNA bases, and cleavage of the
oligonucleotide from the solid support.
Functionalized parallel quadruplex formation was ach-
ieved in K+ ion containing buffer (10 mm Tris-HCl, 80 mm
KCl; pH 7.5; Tris = tris(hydroxymethyl)aminomethane) and
confirmed by using nondenaturing gel electrophoresis and
circular dichroism (CD) spectrophotometry. All the designed
functionalized quadruplexes showed a positive CD peak at
266 nm and a negative peak at 242 nm, characteristic of a
four-stranded parallel quadruplex.[15] Once formed, the quad-
ruplexes show high thermal stability as evidenced from UV
and CD denaturation studies (see the Supporting Informa-
tion). If the functionalized quadruplexes mimic the behavior
of the correspondingly functionalized porphyrins, binding to
cyt c should result in an unraveling of the protein structure
with a decrease in the a-helical content, monitored by a loss of
CD signal at 222 nm.
The denaturation and acceleration of proteolysis of cyt c
observed with oligoanionic-functionalized quadruplexes
strongly suggests a discrete interaction of the quadruplexes
based on hydrophobic and electrostatic complementarity.
This is further substantiated by the selectivity observed
against a range of other protein targets. As cyt c has a
Single-stranded functionalized oligonucleotide 2Z does
not affect the melting temperature (Tm) of cyt c, and the
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Angewandte
Chemie
component a defined stoichiometry and structure for the
aggregate. This in turn imparts the functional property of
protein binding and denaturation not seen in the individual
functionalized strands or unfunctionalized aggregates. We are
currently extending this approach to oligonucleotide aggre-
gates with different stoichiometries of strand association and
more complex symmetries of fragment positioning across the
face of the aggregate to probe the asymmetric nature of
various protein surfaces.
Figure 3. SDS-PAGE of trypsin-digested quadruplex–cyt c complexes.
Left: Cyt c treated with quadruplex 1Y causes complete proteolysis
within 120 min. Right: Proteolytic digestion is complete in 30 min for
cyt c treated with quadruplex 1Z. Cc=cyt c, M1 and M2=protein
markers, CcT=trypsin-treated cyt c for 300 min. The times for diges-
tion are shown in minutes. Proteolytic digestion was performed with
10 mm cyt c and 40 mm quadruplex.
Received: August 25, 2006
Published online: November 30, 2006
Keywords: DNA structures · enzymes · self-assembly ·
.
supramolecular chemistry · surface recognition
physiological isoelectric point (pI) of 10.5, the selectivity of
quadruplex 1Z was compared with RNAseA (pI 9.0), a-
lactalbumin (pI 5.0), and most importantly, a modified version
of cyt c (acetylated cyt c) in which certain critical lysine e-
amine residues are acetylated resulting in a different charge
distribution on the surface of the protein (Figure 4). There is
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