Nonionic side chains modulate the affinity and specificity of binding between functionalized polyamines and structured RNA

Article abstract of DOI:10.1021/ja046436m

This Communication introduces side-chain-bearing polyamines as molecules for selective recognition of folded RNA structures. The complex folded structures associated with RNA create binding pockets for proteins, and also binding sites for small molecules.

Full text of DOI:10.1021/ja046436m

Published on Web 09/18/2004
Nonionic Side Chains Modulate the Affinity and Specificity of Binding
between Functionalized Polyamines and Structured RNA
Graham R. Lawton and Daniel H. Appella*
Department of Chemistry, Northwestern UniVersity, EVanston, Illinois 60208
Received June 16, 2004; E-mail: dappella@chem.northwestern.edu
This Communication introduces a class of molecules with
characteristics for specific recognition of folded RNA structures.
The wealth of structural information available on RNA reveals the
complexity and diversity of the folded structures that RNA can
adopt to create protein-binding sites or to perform catalytic
can be readily synthesized, and that binding affinity and specificity
are sensitive to alteration of the side chains.
Polyamines were prepared using a series of reductive alkylations
between primary amines on solid support and Fmoc-protected
R-amino aldehydes derived from phenylalanine, tyrosine, serine,
or valine (Scheme 1). Sodium triacetoxyborohydride was used to
reduce the imine to the secondary amine, which was then Boc
protected. Polyamine trimers were cleaved under acidic conditions
1
functions. New molecules that recognize a three-dimensional RNA
structure and bind with high affinity and specificity could ultimately
evolve into new types of drugs that exert their biological effects
2
8
by targeting RNA.
and purified by HPLC to give the tetra-trifluoroacetate salt.
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4
5
Cationic aminoglycosides, polypeptides, and peptidomimetics
Six polyamines with different side chain sequences have been
synthesized: YYY, YSY, YVV, FFF, FSF, and FSS. Two different
RNA footprinting methods were used to view interactions at each
base, allowing the identification of binding sites and extraction of
are well-known classes of organic molecules that bind RNA. While
cationic charge is important for high-affinity binding to RNA, too
much electrostatic attraction typically decreases binding specificity
6
for one RNA over others. Developing organic molecules that can
apparent K ’s for those sites. Both assays were performed under
D
bind RNA with high affinity and specificity is a challenge that must
be overcome for RNA to be considered a viable drug target.
We are exploring whether side-chain-functionalized polyamines
can bind specifically to folded RNA structures. Polyamines with
nonionic side chains should sterically shield the cationic charges
in the backbone and reduce their capacity to engage in nonspecific
interactions. Furthermore, we are examining whether the side chains
can be optimized to direct binding specificity. To examine these
ideas, polyamine trimers (Figure 1A) have been synthesized, and
their binding to two different folded RNAs associated with HIV,
TAR, and RRE (Figure 1B,C), has been studied. TAR and RRE
represent two RNA secondary structures with similar features: both
have a stem and loop with bulges as the sites for protein binding.7
The results presented demonstrate that functionalized polyamines
high salt conditions (100 mM NaCl) to limit nonspecific electrostatic
effects and mimic physiological conditions. The first assay involved
competition with terbium cations, which are known to cleave single-
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stranded regions of RNA. Increasing concentrations of polyamine
were incubated with 32P-labeled RNA in the presence of terbium,
and the cleavage products were analyzed by denaturing PAGE.
When binding of the polyamine to the RNA occurred, such as in
the case of YYY, the fraction of RNA cleaved at the binding site
decreased with increasing polyamine concentration (Figure 2). This
decrease was fit to a simple single-site binding curve (Figure 3A,B).
For polyamines FFF, FSS, and FSF, either the curve did not fully
saturate under the concentration range tested, indicating weak
binding, or there was no effect on cleavage of the RNA.
The second RNA footprinting assay probed the effect of the
polyamines on magnesium-catalyzed hydrolytic cleavage of the
phosphodiester backbone. The rate of this cleavage is determined
by the RNA’s ability to access conformations that favor “in-line
attack” of the 2′-hydroxyl on the phosphorus.10 Typically, binding
of ligands causes a change in the conformation of the RNA, leading
to a change in the rate of cleavage at the binding sites. Most of the
polyamines had no effect on the cleavage or caused a nonspecific
decrease at millimolar concentrations. However, polyamines YYY
and YSY caused an increase in cleavage, not the typical decrease.
For YYY this increase saturated, and the data were fit to a single-
site binding curve, from which a K
D
was extracted.8
As a control, the terbium assay was performed on a YYY
tripeptide (a neutral analog) to determine if the side chains alone
were responsible for binding. Spermine, a polyamine with no side
chains, was also tested to determine whether a protonated backbone
was sufficient. Over the same concentrations as the synthesized
polyamines, no effect on cleavage was seen, suggesting that the
combination of side chains and a charged backbone is necessary
for binding.
Figure 1. (A) Polyamine scaffold and side chains. (B) Secondary structure
of TAR RNA. (C) Secondary structure of RRE IIB RNA.
Scheme 1. Solid Phase Synthesis of Polyamines
The data obtained show that binding constants for the polyamines
can range from moderate binding (4 µM) to very weak binding,
depending on the side chains present. The results indicate that the
nitrogens of the backbone do not dominate binding interactions with
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J. AM. CHEM. SOC. 2004, 126, 12762-12763
10.1021/ja046436m CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Dissociation Constants for Polyamines to the Bulge of
TAR and Internal Loop of RRE
polyaminea
RNA
Tb K
(
µM)
D
D
in-line K (µM)
YYY
YYY
YSY
YSY
YVV
YVV
TAR
RRE
TAR
RRE
TAR
RRE
4.8 ( 0.6
50 ( 7
4.1 ( 0.6
54 ( 7
>150
nm
nm
nm
>300
250 ( 50
100 ( 23
85 ( 18
Figure 2. Portions of a 20% denaturing polyacrylamide gel showing the
cleavage products for G32 (loop) and U23 (bulge) of TAR. The intensity
of each band was divided by the total activity in the lane to account for
differences in loading. The numbers below the bands are the normalized
fraction cleaved (1 ) maximum cleavage, 0 ) minimum cleavage).
a
See Figure 1A for structures of side chains. Order of letters corresponds
to order of side chains (R to R ) on polyamine backbone as illustrated in
Figure 1A. nm ) not measurable.
3
1
the polyamine displays activity that is very encouraging. The nature
of the synthesis allows a combinatorial approach to be adopted so
that optimal binding can potentially be selected from a wide variety
of polyamines.
In conclusion, a new class of functionalized polyamines has been
synthesized, and their study has revealed several promising
characteristics for development into RNA-binding molecules.
Further work will focus on optimizing the polyamines to bind tightly
and specifically to target RNAs.
Acknowledgment. We gratefully acknowledge financial support
from Northwestern University’s Weinberg College of Arts and
Sciences, Department of Chemistry, and VP of Research. We are
indebted to Dr. Olke Uhlenbeck for the use of his facilities, and to
F. V. Karginov and Dr. R. Fahlman for their help with the binding
assays.
Figure 3. Binding curves for (A) YYY vs TAR, Tb cleavage, bulge (U23-
U25) and loop (U31-G33) regions; (B) YYY vs TAR, Tb cleavage, bulge
region, and YYY vs RRE, Tb cleavage, bulge and loop regions.
the RNA. Also, binding affinity is not simply a function of the
number of aromatic groups (compare YVV to YSY in the terbium
data). Significant selectivity is demonstrated in the case of
polyamine YYY. This polyamine binds the bulge of TAR prefer-
entially over the loop of TAR (Figure 3A), and over the loop and
bulge of RRE (Figure 3B), by 1 order of magnitude. YYY is clearly
better suited to binding the bulge of TAR than any of the other
polyamines, and is not as well suited to binding the other RNA
motifs studied. The terbium assay was performed at pH 6.5, and
the “in-line” assay was performed at pH 8.0, spanning relevant
physiological pH ranges. The dissociation constants obtained from
both methods of footprinting were consistent, suggesting that
binding tolerates small changes in pH and salt concentration. Based
on binding curves that were fit to determine Hill coefficients, the
Supporting Information Available: Details for synthesis, purifica-
tion, and characterization of polyamines; conditions for footprinting
assays and binding data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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1
:1. Other polyamines, such as YVV, showed no preference for
2
TAR over RRE, suggesting that the preference of YYY for TAR
is not caused by a more accessible structure in the TAR bulge.
Generally, the binding of the polyamines was weak and
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binding affinity is still much too weak and the specificity is not
sufficient for use as an active drug. Furthermore, it is possible that
there are other weak binding sites on the RNAs that we cannot
(
(
b) Daly, T. J.; Cook, K. S.; Gray, G. S.; Maione, T. E.; Rusche, J. R.
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(
(
b) Terbium cleavage conditions: 50 mM Tris-HCl (pH 6.5), 100 µM
TbCl , 10 mM MgCl , 100 mM NaCl, [polyamine] ) 0-2 mM, 4 h, 25
3
2
°
C
(
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2
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8
[polyamine] ) 0-2 mM, 44 h, 25 °C
measure with these techniques. However, considering the simplicity
of the system and the fact that the sequence was selected randomly,
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