Screening helix-threading peptides for RNA binding using a thiazole orange displacement assay

Article abstract of DOI:10.1016/j.bmc.2008.08.066

The fluorescent intercalator displacement assay using thiazole orange has been adapted to the study of RNA-binding helix-threading peptides (HTPs). This assay is highly sensitive with HTP-binding RNAs and provides binding affinity data in good agreement w

Full text of DOI:10.1016/j.bmc.2008.08.066

Bioorganic & Medicinal Chemistry 16 (2008) 8914–8921
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Screening helix-threading peptides for RNA binding using a thiazole orange
displacement assay
Malathy Krishnamurthy a,b, Nicole T. Schirle a, Peter A. Beal a,
*
a Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
b Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The fluorescent intercalator displacement assay using thiazole orange has been adapted to the study of
RNA-binding helix-threading peptides (HTPs). This assay is highly sensitive with HTP-binding RNAs
and provides binding affinity data in good agreement with quantitative ribonuclease footprinting without
the need for radiolabeling or gel electrophoresis. The FID assay was used to define structure activity rela-
tionships for a small library of helix-threading peptides. Results of these studies indicate their RNA bind-
Received 11 June 2008
Revised 22 August 2008
Accepted 26 August 2008
Available online 29 August 2008
ing is dependent on peptide sequence,
but independent of macrocyclic ring size for the penta-, tetra- and tri-peptides analyzed.
Ó 2008 Elsevier Ltd. All rights reserved.
a-amino acid stereochemistry, and cyclization (vs linear peptides),
Keywords:
RNA recognition
Cyclic peptides
Threading intercalation
High-throughput screening
1. Introduction
it can be useful to generate libraries of compounds with structural
variations and screen for these properties.10–12 Diversity-oriented
synthesis techniques aid in rapidly generating large numbers of
molecules.13 However, rapid, high-throughput assays must be
available to evaluate specific properties of the library members.
When considering assaying the RNA-binding properties of library
components, some of the most commonly used techniques (e.g.
ribonuclease footprinting, affinity cleaving, gel-mobility shift
assays, isothermal titration calorimetry, and surface plasmon
resonance) are time consuming and, in some cases, require the
use of radioactively or fluorescently labeled RNA.2,14 For libraries
of ligands generated on solid-phase, on-bead screening techniques
have been used to screen for tight binders.15,16 Often, however,
on-bead screening techniques result in false-positive hits that
can arise as a result of non-specific interactions of the RNA probe
to the solid support or with the library members.17 To circumvent
these potential problems, we have adapted a spectroscopic, solu-
tion-phase nucleic acid binding assay to the screening of libraries
of helix-threading peptides (HTPs) prepared in our laboratory.18–21
HTPs contain a heterocyclic chromophore embedded in a peptide
backbone and can selectively bind stem-loop RNA structures that
contain a 50-PyPu-30 intercalation site flanked by at least a single
bulge on each strand, 30 to this site.18,20,22 We recently demon-
strated that a macrocyclic variant binds a naturally occurring
pre-miRNA containing such a binding site.21 In this paper, we
describe the use of the fluorescent intercalator displacement assay
to analyze the RNA-binding properties of a small collection of
HTPs.23,24 This assay is highly sensitive, simple, does not require
labeled RNA and can be carried out in homogeneous solutions in
RNA-binding small molecules have been studied in the past for
their potential as drug leads.1,2 This area of research continues to
hold promise for the development of new therapeutics, particularly
given the recent advances in defining novel regulatory roles for
RNA and advances in determining high-resolution structures of
RNA drug targets.3–5 However, small molecule/RNA interactions
are also now recognized as important for controlling gene expres-
sion through both natural and synthetic riboswitches, RNA ele-
ments within mRNA that bind small molecules. For instance,
advances from the labs of Smolke, Mulligan, Gallivan, and Yoko-
bayashi have shown how small molecule-binding RNAs can be
used as synthetic riboswitches to regulate the function of mRNAs
into which these RNA structures have been inserted.6–9 Although
small molecule control of a variety of intracellular RNAs has been
demonstrated in this way, relatively few small molecules have
been described for this purpose. To fully exploit the potential of
RNA-binding small molecules either as drug candidates or as
chemical biology tools, new classes of RNA-binding compounds
are needed. However, the design and development of new ligands
that can bind RNA inside living cells remains a challenging task.1
Several factors need to be considered including RNA target affinity
and selectivity, cell permeability, and metabolic stability. It is often
difficult to predict what structural features can result in the de-
sired properties for different classes of molecules. In such cases,
* Corresponding author. Tel.: +1 530 752 4132; fax: +1 530 752 8995.
E-mail address: beal@chem.ucdavis.edu (P.A. Beal).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.066

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
8915
multi-well plates.25 Moreover, the assay appears particularly well
suited for screening HTPs, given the large increase (up to 2300-
fold) in fluorescence signal observed when the intercalator thiazole
orange binds RNA containing a preferred HTP site. This assay was
used to evaluate structure activity relationships for HTP/RNA bind-
ing by varying HTP sequence, amino acid stereochemistry, macro-
cyclic versus linear HTPs, and macrocycle size. We found that all of
these structural parameters except ring size influence the RNA-
binding properties of HTPs to the RNA target chosen. We also re-
port an efficient synthesis of a new 2-phenylquinoline carboxylic
acid for incorporation into macrocyclic HTPs.
sequence found in human pre-miRNA 23b and also has a binding
site for HTPs.21 RNA C is a control RNA duplex that does not contain
an HTP site.
We found that when thiazole orange and RNA are present at
2.6 lM and 2 lM concentrations, respectively, the thiazole orange
fluorescence is enhanced by the presence of RNA A by 2308-fold
(Fig. 1). RNA B, which contains a lower affinity HTP site, caused a
977-fold increase in thiazole orange fluorescence at 2.6 lM con-
centration. Importantly, under these same conditions, the binding
of thiazole orange to control RNA C caused a much smaller fluores-
cence increase (96-fold). Thus, thiazole orange binding to RNAs
with intercalation ‘hot spots’ (e.g. RNAs A and B) leads to approx-
imately three orders of magnitude enhancement in fluorescence
signal, suggesting the possibility of a highly sensitive RNA-binding
assay. Thiazole orange binding to RNA A was analyzed further.
2. Results and discussion
We adapted the fluorescent intercalator displacement (FID) as-
say to the study of HTP/RNA binding. Boger and coworkers de-
scribed this assay previously for establishing the affinity and
selectivity of DNA-binding compounds.23,25–28 This assay involves
the displacement of an intercalator, such as ethidium bromide or
thiazole orange, from a nucleic acid target upon addition of a bind-
ing ligand. When a double stranded nucleic acid is treated with
certain fluorescent intercalators, there is an increase in the ob-
served fluorescence signal. Addition of a binding ligand to the com-
plex results in the displacement of the intercalator, causing a
decrease in the fluorescence. Recently, Pei and Stojanovic screened
a series of RNA and DNA aptamers in a thiazole orange displace-
ment assay and found that the assay was reliable for the study of
ligand binding to some, but not all, of the aptamers investigated.29
We tested three RNA targets in our assay, namely, RNA A, RNA B,
and RNA C (Fig. 1). RNA A is a 29-mer stem-loop RNA construct
based on an in vitro selected RNA aptamer that is predisposed to
bind HTPs.18 RNA B is a 28-mer hairpin stem structure containing
Titration of the fluorophore into a 5 lM solution of RNA A resulted
in pronounced increase in fluorescence with each addition until
one equivalent of thiazole orange had been added (Fig. 1). No addi-
tional RNA-dependent fluorescence enhancement was observed
upon further addition, suggesting the large fluorescence enhance-
ment is a result of the formation of a 1:1 complex.
The large fluorescence enhancement observed when thiazole
orange binds RNAs A and B suggested that competition experi-
ments could be carried out at low thiazole orange concentrations.
After screening several different concentrations and thiazole oran-
ge:RNA ratios, the combination of 50 nM thiazole orange and
100 nM RNA A was chosen for the displacement experiments.
Briefly, in the displacement assays, the RNA–thiazole orange com-
plex was pre-formed and added to increasing HTP concentrations.
The solutions were then transferred to a 96-well plate and scanned
for fluorescence changes. As described above, binding of thiazole
orange to the stem-loop RNA structure enhances the fluorescence
Figure. 1. (A) Structure of thiazole orange; (B) secondary structures of RNA A and B with putative intercalation site boxed and control RNA C used for the displacement assay,
nucleotides highlighted in outlined text are present in the in vitro selected aptamer (RNA A) and human pre-miRNA 23b (RNA B); (C) fold stimulation of thiazole orange
(2.6 lM) fluorescence with RNA A, B, and C at 2 lM concentration each; and (D) titration of RNA A (5 lM) with thiazole orange.

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
of thiazole orange (Fig. 2A). Addition of HTP to this complex results
in the displacement of the bound thiazole orange causing a de-
crease in the fluorescence. The extent of fluorescence decrease
can be correlated to the binding affinity of the peptide. The IC50
for the decrease in the fluorescence of thiazole orange was calcu-
lated by plotting the relative fluorescence decrease as a function
of HTP concentration. In each case, background fluorescence of thi-
azole orange alone was subtracted. These IC50 values were con-
(Fig. 3). We found that the trends in the dissociation constants
from the FID assay correlated well with those obtained from quan-
titative footprinting experiments carried out previously with larger
deviations observed for a tight binder (HTP1) and a weaker binding
compound (HTP6) (Table 1).20,21 This indicated that the FID assay
can be used reliably for screening HTPs to determine RNA-binding
affinities without the investment in time and effort necessary for
the footprinting experiments.
verted to apparent dissociation constants (KD) assuming
competitive binding mode and using a thiazole orange KD for
a
In order to further test its scope, we performed the assay with
aminoglycosides neomycin (7) and kanamycin (8) (Fig. 3). Interest-
ingly, complete displacement was not observed with these com-
pounds even at high micromolar concentrations, conditions likely
to cause them to bind these RNAs non-specifically (Table 1, Supple-
mentary Figs. 2 and 3). The more efficient displacement by HTPs
may be the result of a shared binding mode with thiazole orange
(intercalation). Groove binding by the aminoglycosides could be
compatible with the thiazole orange–RNA complex and result in
poor displacement.
Since macrocyclic scaffolds provide the possibility of orienting
different functional groups on the cyclic backbone in specific posi-
tions in space, we were interested in studying the structure activity
relationships of macrocyclic HTPs and their binding affinity to tar-
get RNA using this assay. Encouraged by the results from the dis-
placement assay, we synthesized a small macrocyclic HTP library
containing a 2-phenylquinoline intercalating unit, in order to test
RNA A of 3.1 1.8 lM as determined by quantitative RNase V1
footprinting (see Section 4 and Supplementary Information).30 It
should be noted that given the amount of RNA required to observe
the thiazole orange fluorescence enhancement, accurately measur-
ing KD’s for very tight binders (low nM or better) may not be pos-
sible with this approach.
Shown in Figure 2 is the thiazole orange displacement for a rep-
resentative macrocyclic HTP. At 50 nM thiazole orange and 100 nM
RNA concentrations, binding of thiazole orange to RNA A resulted
in an approximate 20-fold stimulation of fluorescence signal. Titra-
tion of macrocyclic HTP 2 (Fig. 2C) resulted in 96% reduction of this
initial fluorescence at the highest concentration of HTP as a result
of HTP binding and displacement of thiazole orange (Fig. 2B).
We initially tested this assay for a small set of HTPs that had
been previously shown to bind RNA A using RNase V1 footprinting
Figure. 2. (A) Schematic representation of thiazole orange displacement assay; (B) binding of HTP 2 to RNA A causes displacement of thiazole orange resulting in decrease in
fluorescence; and (C) structure of macrocyclic HTP 2.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
8917
Figure. 3. Chemical structures of HTPs and aminoglycosides analyzed by thiazole orange displacement assay. The KD values obtained from the displacement assay are
indicated below the structures for each ligand.
Table 1
the effect of changing the structure of amino acid side chains in the
peptide, the a-amino acid stereochemistry, backbone cyclization as
Comparison of dissociation constants for HTPs measured in two different assays
well as ring size (Fig. 4). In the design of our new library, our goal
was to reduce non-selective RNA binding in the HTPs as much as
possible. Since highly positively charged compounds are capable
of non-selective interactions with the negatively charged RNA
backbone, we chose to alter the core HTP structure to reduce the
number of positive charges by replacing the 4-aminobenzylamine
unit with aniline. We therefore synthesized a new 2-phenylquino-
line carboxylic acid with anilino substitution at the C4 position.
The carboxylic acid required for synthesis of the library members
was generated using a short and efficient scheme (Scheme 1).
2-Phenylquinolone 10 was synthesized from Meldrum’s acid
HTP Dissociation constants (footprinting)
M)a
Dissociation constants (FID)
(l
M)b
(
l
1
2
3
4
5
6
7
8
0.05 0.01
0.27 0.11
0.33 0.06
8.6 4.3
2.3 0.9
3.3 1.8
nd
0.22 0.06
0.6 0.1
0.71 0.04
4.4 1.3
2.7 0.6
13.2 3.1c
>60
nd
>1000
a
Conditions: 50 mM Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM MgCl2, and 10
lg/
mL of yeast tRNAPhe, 25 °C, 15 min equilibration prior to digestion with ribonu-
clease V1.
derivative,
5-(p-methyl-a-thiomethoxybenzylidene)-2,2-di-
b
Conditions: 50 mM Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM MgCl2, 50 nM
methyl-1,3-dioxane-4,6-dione (9) using microwave conditions in
very good yields. As compared to our previously reported synthesis
of 10 using standard heating conditions, we observed that the reac-
tion time decreased considerably by using microwave conditions
thiazole orange and 100 nM RNA A, 25 °C. Dissociation constants are reported as the
average of three independent measurements standard deviation. nd, not
determined.
c
Average of two independent measurements.

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
and resulted in greater yields of the quinolone.21 Amination with a
4-chloro-2-p-allylphenylquinoline derivative reported earlier by
us, using aniline in the presence of tin tetrachloride gave ester 11
in good yield.21 This methyl ester was hydrolyzed to obtain carbox-
ylic acid 12 in overall good yield. The acid was used for solid-phase
peptide synthesis of the macrocyclic HTP library as previously de-
scribed.21 The library represented members that differed in the
structure of amino acid side chains, a-carbon stereochemistry, cyc-
lic versus linear HTP as well as macrocycle ring size (Fig. 4). Disso-
ciation constants for the binding to RNA A determined using the
Figure. 4. Chemical structures of macrocyclic HTPs used in the displacement assay. The KD values obtained from the displacement assay are indicated below the structures
for each HTP.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
8919
Scheme 1. Synthesis of 2-phenylquinoline carboxylic acid 12. Reagents and conditions: (a) methyl anthranilate, Ph2O, MW, 240 °C, 20 min, 83%; (b) i—POCl3, reflux,1 h, 87%;
ii—NBS, AIBN, cyclohexane, reflux, 50 min, 77%; iii—vinyl tributyltin, Pd(PPh3)4, DMF, 45 °C, 2 h, 73%; iv—aniline, SnCl4, CH3CN, rt, 1 h and 86%; (c) LiOHꢀH2O, THF–H2O, rt,
24 h, 87%.
FID assay as discussed above are shown below each HTP structure
in Figure 4.
prepared for this purpose. As observed with the other macrocycles
in this study, ring-closing metathesis (RCM) was efficient with HTP
20 yielding the cyclic compound as the major reaction product.
However, a complex mixture of products was observed for RCM
reactions to generate the smaller HTP 21, requiring extensive HPLC
purification and mass spectrometry to identify 21 as a minor com-
ponent. Interestingly, 20 and 21 bound RNA with nearly the same
affinity and only slightly reduced from pentapeptide 13 bearing the
same overall charge, suggesting that ring size is not a major deter-
minant for binding to RNA A when comparing these tri-, tetra-, and
pentapeptides. However, the poor cyclization reaction for the tri-
peptide suggests preparing libraries of macrocyclic HTPs of this
size will be problematic.
To test the effect of changing amino acid side chain structure,
we decided to focus initially on two amino acids present in the
macrocyclic pentapeptides (positions X and Y, Fig. 4). Comparing
asparagines at these positions to leucines would allow us to deter-
mine if a preference for hydrophilic or hydrophobic side chains ex-
isted, since the asparagine and leucine side chains are of similar
size and steric differences would be minimized. RNA A was chosen
as the target for these initial studies as a representative HTP-bind-
ing RNA. It is clear from our results that asparagine is preferred at
these positions when L amino acids are used. For instance, in HTP
13, replacement of asparagine residues at X and Y with leucine
(HTP 14) dramatically reduced the binding affinity (KD = 1.4
lM
vs KD > 30 M). We also found that the position of the asparagine
l
3. Conclusion
residue in the ring is important as can be seen comparing HTP 13
to 15 or 16. Mutation of residue X in 13 from asparagine to leucine
to give HTP 15, resulted in a 4-fold loss in affinity. However, muta-
tion of residue Y in HTP 13 to give 16 was more detrimental result-
ing in a 13-fold reduction in binding affinity. Although complete
understanding of the affinity changes observed will require high-
resolution structural data, we can speculate that asparagines en-
gage in productive interactions with RNA functional groups around
the intercalation site not possible with leucines. Our previous work
with structurally related linear peptides indicated that the quino-
line 8-carboxamide substitutent is localized in the major groove
of the RNA duplex.20,22 Therefore, asparagine side chains at X and
Y may be hydrogen bonding within the major groove at the bind-
ing site. Regardless of the basis for this effect, these results indicate
that the structure of the amino acid side chain at the X and Y posi-
tions of the macrocyclic pentapeptides controls RNA affinity, signi-
fying that libraries of these molecules where X and Y are varied
contain compounds with distinct RNA recognition properties.
To determine if the observed effect arose simply from a general
preference for the more polar side chain of asparagine, we
In summary, we have adapted the fluorescent intercalator dis-
placement assay using thiazole orange to the study of RNA-binding
helix-threading peptides. This assay is highly sensitive and pro-
vides binding affinity data in good agreement with other methods.
This assay was used to define structure activity relationships for a
small library of compounds. Results of these studies indicate their
RNA binding is dependent on peptide sequence, a-amino acid ste-
reochemistry, and cyclization (vs linear peptides), but independent
of macrocyclic ring size. These studies indicate that libraries of
macrocyclic, helix-threading peptides with different amino acids
at variable positions contain members with distinct RNA-binding
properties.
4. Experimental
4.1. Preparation of 8-(methoxycarbonyl)-2-(40-methylphenyl)-
4(1H)-quinolone (10)
To a solution of 9 (0.150 g, 0.514 mmol) in phenyl ether
(3.5 mL) was added methyl anthranilate (0.1 mL, 0.770 mmol) in
a microwave vial. The vial was capped and subjected to MW irra-
diation at 240 °C for 20 min. The reaction mixture was cooled to
room temperature. Purification by silica gel column chromatogra-
phy (initially 100% hexanes to remove phenyl ether, 10–40%
EtOAc/hexanes to remove impurities, 70–80% EtOAc/hexanes to
elute product) afforded 0.121 g (83%) of the product as a white so-
lid. The characterization of the product by NMR and mass spectros-
copy has been reported previously.21
switched the
positions. The stereochemistry at the
affinity as well, as seen by comparisons to HTPs 17 and 18, which
a
-amino acid stereochemistry to D at the variable
a-carbon affects the binding
have
with
D
-amino acids at positions X and Y. Replacing the
L-asparagine
D-asparagine at both positions reduces binding affinity by 10-
fold (compare HTP 13 and HTP 18). Surprisingly, replacing the two
-leucine residues (HTP 14) with -leucine residues (HTP 17)
L
D
causes an improvement in binding affinity. The overall effect is that
with D stereochemistry at the X and Y positions, macrocycle bind-
ing is largely independent of side chain structure, since 17 and 18
bind with nearly the same affinity.
Importantly, comparison of macrocyclic HTPs 13 and 17 with
their linear precursors HTPs 22 and 23, respectively, indicated that
cyclization is beneficial for both the sequences. This result is sim-
ilar to that observed previously when comparing linear versus cyc-
lic pentapeptide HTPs binding to the binding site found on RNA
A.21 We also explored the effect of reducing the macrocycle ring
size. Macrocyclic tetrapeptide HTP 20 and tripeptide HTP 21 were
4.2. Preparation of methyl-2-(40-allylphenyl)-4-
(anilino)quinoline-8-carboxylate (11)
To a solution of methyl-2-(40-allylphenyl)-4-chloro-quinoline-
8-carboxylate (0.060 g, 0.179 mmol) (synthesized according to
our previously reported procedure) in CH3CN (5 mL) was added ani-
line (0.048 mL, 0.358 mmol) and SnCl4 (0.021 mL, 0.179 mmol).21

8920
M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
The resulting solution was stirred at room temperature for 1 h. The
reaction mixture was quenched with saturated NaHCO3, passed
through a celite pad and extracted with dichloromethane. The or-
ganic layer was dried over anhydrous Na2SO4, and the solvent
was removed under reduced pressure. Purification by silica gel col-
umn chromatography (initially 1% MeOH/CHCl3 followed by 2–3%
MeOH/CHCl3) afforded 0.060 g (86%) of the product as a yellow so-
lid. 1 H NMR (600 MHz, CDCl3) d (ppm): 7.84 (br s, 4H), 7.35–7.03
(m, 8H), 6.79 (br s, 1H), 5.87 (m, 1H), 5.01 (d, J = 0.9 Hz, 1H), 5.00
(d, J = 0.9 Hz, 1H), 3.94 (s, 3H), 3.32 (d, J = 6.5 Hz, 2H) 13C NMR
(100 MHz, CDCl3) d (ppm): 169.37 142.11, 137.19, 129.82, 129.31,
128.72, 127.30, 123.96, 123.43, 122.20, 116.39, 100.03, 52.72,
40.18, 29.94, 21.59. High-resolution CI-MS calculated mass for
CH2Cl2 (1ꢁ 2 mL) and MeOH (3ꢁ 2 mL) and lyophilized for 12 h.
The dry resin was incubated with TFA/TIS/PhOH/H2O (88:5:5:2,
3 mL) and shaken for 3.5 h to effect cleavage from the resin and
amino acid side chain deprotection. The resulting solution was col-
lected and concentrated under reduced pressure. Ether precipita-
tion of the residue, followed by extraction with water, gave the
crude product. After the standard work-up procedures, the linear
and cyclic peptides were purified using reverse-phase HPLC
(Waters Corporation) equipped with an auto sampler unit (2767
Sample Manager, Waters). The crude peptides were analyzed using
a reverse-phase analytical C-18 column (4.6 ꢁ 50 mm, SunFire,
Waters). The purification was carried out using a reverse-phase
preparative C-18 column (19 ꢁ 50 mm, SunFire, Waters). For ana-
lytical runs, a flow-rate of 1.5 mL/min was used and for the pre-
parative runs, a flow-rate of 25 mL/min was used. Typically, 15–
C
26H22N2O2: 394.1676, found [M+H]+ 394.1680 m/z.
4.3. Preparation 2-(40-allylphenyl)-4-(anilino)–quinoline-8-
carboxylic acid (12)
20
l
L of the sample was injected as an aqueous solution for analyt-
L of sample was injected when the pre-
ical runs, while 500–1000
l
parative column was used. HTPs 13–21 were injected as 5% AcOH
solutions. HTP 22 was injected as a solution in 50% ACN and HTP 23
was injected as a 50% MeOH solution to address solubility issues.
ESI-MS analyzed and calculated mass for the macrocyclic peptides
are given in Supplementary Table 1. For macrocyclic pentapeptides
13–19, olefin stereochemistry is assumed to be trans based on the
structures of HTPs 1 and 2 prepared under these conditions.21 The
olefin stereochemistry for macrocyclic tetrapeptide HTP 20 or tri-
peptide HTP 21 was not determined.
To a solution of 11 (0.250 g, 0.638 mmol) in THF/H2O (13.5 mL,
2:1) was added lithium hydroxide monohydrate (0.059 g,
1.423 mmol) and the resulting solution stirred at room tempera-
ture for 24 h. The reaction mixture was quenched with saturated
NH4Cl, when a yellow precipitate formed. The precipitate was fil-
tered, washed with water and dried under vacuum to afford
0.209 g (87%) of the product as a yellow solid. 1H NMR (500 MHz,
CDCl3/CD3OD) d (ppm): 8.42 (d, J = 7.3 Hz, 1H), 8.37 (dd, J = 8.5,
1.2 Hz, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.47–7.42 (m, 2H), 7.40–7.37
(m, 3H), 7.32–7.31 (m, 3H), 7.26–7.23 (m, 2H), 7.15 (d, J = 7.9 Hz,
3H), 6.87 (s, 1H), 5.79–5.72 (m, 1H), 4.95–4.91 (m, 2H), 3.27 (d,
4.6. Preparation of RNA for experiments
J = 6.5 Hz, 2H). 13C NMR (150 MHz, CDCl3/CD3OD)
d
(ppm):
RNA A was purchased from Dharmacon RNA Technologies and
used directly for the FID experiments. RNA B and C were purchased
from Dharmacon RNA Technologies as 20-bis(2-acetoxyeth-
oxy)methyl orthoester (20-ACE)Ò protected RNA. RNA was prepared
for the experiments by deprotecting the 20-ACE group according to
170.44, 155.12, 151.45, 145.10, 143.17, 140.33, 137.53, 136.39,
136.09, 130.51, 130.18, 130.06, 127.62, 127.20, 126.99, 126.19,
125.33, 125.29, 124.75, 117.06, 116.78, 97.02, 39.88, 21.31. High-
resolution FAB calculated mass for C25H20N2O2: 380.1519, found
[M+H]+ 380.1535 m/z.
the manufacturer’s protocol. Briefly, 400 l
L of 20-deprotection buf-
fer (100 mM acetic acid, adjusted to pH 3.8 with TEMED) was
added to the RNA pellet and mixed well. The solution was incu-
bated at 60 °C for 30 min, following which the RNA was extracted
with phenol/chloroform, ethanol precipitated, dissolved in nano-
pure water and stored at ꢃ20 °C.
4.4. Preparation of linear HTPs 22 and 23
Linear HTPs were synthesized using 9-fluoronylmethoxycar-
bonyl (Fmoc)-protected amino acids according to standard SPPS
protocols using Novagel Rink Amide resin (NovaBiochem,
0.64 mmol/g loading, 45 mg, 0.028 mmol) as described previ-
ously.21 Following coupling of carboxylic acid 12 (40 mg,
3.7 equiv), the resin was washed and cleaved using TFA/TIS/H2O
(90:8:2, 3 mL, 3.5 h). The resulting solution was collected and con-
centrated under reduced pressure. Ether precipitation of the resi-
due, followed by extraction with water gave the crude product,
which was purified by HPLC.
4.7. Quantitative footprinting of thiazole orange with RNA A
A dissociation constant for thiazole orange on RNA A was ob-
tained using RNase V1 footprinting experiments. For the prepara-
tion of 50-32P RNA, 60 pmol of RNA A in T4 Polynucleotide Kinase
buffer (70 mM Tris–HCl, 10 mM MgCl2, 5 mM dithiothreitol, pH
7.6) was treated with T4 Polynucleotide Kinase (2.0 U/
[c-
lL),
32P]ATP (2 mCi/mL) and incubated for 45 min at 37 °C followed
4.5. Cyclization via ring-closing metathesis
by heat inactivation at 65 °C for 20 min. Labeled RNA was purified
on a 19% denaturing polyacrylamide gel, visualized by storage
phosphor autoradiography, excised from the gel and eluted over-
night via the crush and soak method. After filtration, the resulting
solution was extracted with phenol/chloroform, ethanol precipi-
tated, and stored in water. Ligand/protein–RNA complexes were
formed by incubating increasing concentrations of the ligand with
50-32P labeled RNA A (ꢂ5 nM) for 15 min in reaction buffer (50 mM
Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM MgCl2) at ambient tem-
perature. Enzymatic digestions with RNase V1 (1 ꢁ 10ꢃ6 U) were
carried out for 30 min at ambient temperature and quenched with
hot formamide loading buffer. Cleaved RNA was heat denatured
and analyzed by 19% denaturing polyacrylamide gel electrophore-
sis. The cleavage data for the RNA was converted to binding data
for the ligand, assuming that the maximum cleavage efficiency cor-
responds to 0% occupancy and the minimum cleavage efficiency
corresponds to 100% occupancy. The fraction of RNA bound by
For the synthesis of macrocyclic HTPs 13–21, the linear peptides
were first assembled as described above using Novagel Rink Amide
resin (NovaBiochem, 0.64 mmol/g loading, 55 mg, 0.035 mmol).
Following the coupling of carboxylic acid 12 (52.5 mg, 4 equiv),
the resin was washed with CH2Cl2 (3 ꢁ 2 mL), DMF (3 ꢁ 2 mL),
MeOH (3 ꢁ 2 mL) and diethyl ether (3 ꢁ 2 mL) and lyophilized
for 12 h. The dried resin was transferred to a flame-dried round-
bottomed flask and preswelled with 1,2-dichloroethane (4 mL)
for 45 min. Hoveyda-Grubbs catalyst (5.5 mg, 25 mol%) was
weighed in a flame-dried vial and to this 1,2-dichloroethane
(1 mL) was added and transferred to the flask containing the sus-
pended resin. The flask was then heated at reflux for ꢂ30 h. After
cooling to room temperature, the resin was transferred to a poly-
propylene column (Bio-Rad) and drained. It was then washed with
CH2Cl2 (3ꢁ 2 mL), 10% 1,3-bis(diphenylphosphino)propane in

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 8914–8921
8921
the ligand was plotted as a function of concentration, and the data
were fitted to the equation: fraction RNA bound = [ligand]/([li-
gand] + KD(TO)). The dissociation constant (KD(TO) = 3.1 1.8 lM) is
reported as the average and standard deviation for three different
experiments (see Supplementary Fig. 1).
plate reader (Perkin Elmer, excitation 485 nm, emission 535 nm).
The background fluorescence of the control wells at each concen-
tration was subtracted from the wells containing the RNA–HTP–thi-
azole orange complex at that concentration. In order to calculate
the IC50 of fluorescence displacement, the fluorescence data was
converted to binding data assuming that maximum fluorescence
corresponds to 0% displacement. The fluorescence of the other wells
was normalized to the well with 0% displacement (100% fluores-
cence) to obtain the relative fluorescence decrease. Using Kaleida-
graph 4.0 software, the above data were plotted as a function of
HTP concentration and fitted to the equation: Percentage decrease
in fluorescence = F/(1 + IC50/[ligand]) where F = Normalized relative
fluorescence of RNA–thiazole orange complex at each HTP concen-
tration subtracted from background. With aminoglycosides 7 and 8,
there was no displacement of thiazole orange up to the highest con-
centrations (see Supplementary Fig. 2). The apparent dissociation
constants of the HTPs (KHTP) were calculated using a competitive
4.8. RNA–thiazole orange stoichiometry determination
RNA stock solutions were prepared in nanopure water and thia-
zole orange stock solutions were prepared in DMSO. A reaction mix-
ture (98
Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM MgCl2) was added to
L of increasing concentration of thiazole orange (0.1–2.5 equiv)
lL) consisting of RNA A (5 lM) in reaction buffer (50 mM
2
l
in a 96-well black OptiPlate (Perkin Elmer) at 25 °C. The mixture
was thoroughly mixed with a pipet and incubated for ꢂ15 min at
25 °C. For the control wells, 98 lL of a reaction mixture consisting
of reaction buffer (50 mM Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl,
10 mM MgCl2) was added to 2 L of increasing concentration of thi-
azole orange (0.1–2.5 equiv). The 96-well plate was then read in
Victor fluorescent plate reader (Perkin Elmer, excitation
l
model using the KD of thiazole orange with RNA
A
(KTO = 3.1 1.8 lM) as determined by RNase V1 footprinting. The
3
equation, KHTP = IC50/(1 + ([TO]/KTO))30, was used to determine the
apparent dissociation constants, where [TO] = 50 nM and is the
concentration of thiazole orange in the FID assay. The KHTP values
are reported in Tables 1 and 2.
485 nm, emission 535 nm). The background fluorescence of the
control wells at each concentration was subtracted from the wells
containing the RNA–thiazole orange complex at that concentration.
The fluorescence change was plotted as a function of equivalents of
thiazole orange added. The stoichiometry was determined using
Microsoft Excel by simultaneously solving the equations represent-
ing the pre- and post-saturation regions of the curve.
Acknowledgment
P.A.B. acknowledges NIH for financial support GM061115.
4.9. Thiazole orange displacement assay
Supplementary data
RNA stock solutions were prepared in nanopure water and thia-
zole orange stock solutions were prepared in DMSO. HTP stocks
were usually prepared in water except for HTPs 17 and 23, which
were prepared in DMSO. RNA–thiazole orange complex was formed
by adding RNA (100 nM) to a solution of thiazole orange (50 nM) in
reaction buffer (50 mM Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.08.066.
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l
l
l
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Table 2
Dissociation constants of HTPs from thiazole orange displacement
HTP
Dissociation constants (FID) (l
M)[a]
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13
14
15
16
17b
18
19
20
21
22
23
24
1.4 0.3
>30
6.2 0.6
18.9 4.1
10.1 1.8
13.4 4.2
0.35 0.08
3.9 0.3
3.6 1.9
14.2 1.6
>30
>30
a
Conditions: 50 mM Bis-TrisꢀHCl, pH 7.0, 100 mM NaCl, 10 mM MgCl2, 50 nM
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average of three independent measurements standard deviation.
b
Average of two independent measurements.