V. Marchꢂn et al.
B) RNA free exchange experiments: Each building block (24 nmol; in
0.1% TFA in H2O) was combined in an Eppendorf tube and freeze dried
or evaporated in a Speed-Vac. Buffer (240 mL; 50 mm Tris-HCl (pH 7.7),
100 mm NaCl, and 0.1 mm Na2EDTA) was then added, and the mixture
was shaken gently. The DCL mixtures were left to stand at room temper-
ature under air without stirring. After the desired time, an aliquot was
taken (approximately one third), the disulfide exchange was stopped by
addition of a 0.1% TFA solution in water (100 mL; pHꢀ2–3), and the
mixture was subjected to UV–MS–HPLC analysis.
0.58CminÀ1 and measuring the absorbance at 260 nm as a function of
temperature. The reverse denaturation curve (20 to 908C) was then re-
corded. This cooling/heating experiment ensures that the initial state cor-
responds to a thermodynamic equilibrium. In all cases, the two curves
were superimposable, which indicated that the transition was kinetically
reversible. Unless otherwise indicated, the solutions were at a concentra-
tion of 1 mm of both RNA and ligands, in 10 mm sodium phosphate buffer
(pH 6.8), 100 mm NaCl, and 0.1 mm Na2EDTA. The corresponding melt-
ing temperature values (Tm) were determined by using the baseline
method. All experiments were repeated at least three times until coinci-
dent Tm values were obtained. The error in Tm values was Æ0.28C.
B) Circular dichroism: Samples were prepared as described in the UV-
monitored melting experiment section (3 mm of both RNA and ligands, in
10 mm sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm
Na2EDTA). Spectra were recorded on a Jasco J-720 spectropolarimeter
with a thermoregulated cell holder and interfaced with a Neslab RP-100
water bath, at 208C. All CD spectra were baseline subtracted with a sep-
arately acquired buffer spectrum.
In some DCC experiments, the building blocks were incorporated as the
corresponding disulfide homodimers. In those cases, 12 nmol were used
in order to keep the same final concentration of monomer.
C) RNA-templated exchange experiments: Biotinylated RNA (6 nmol; wt
or +3-mutated) was annealed in buffer (240 mL; 50 mm Tris-HCl
(pH 7.7), 100 mm NaCl, and 0.1 mm Na2EDTA) by heating to 908C for
5 min and then slowly cooling to room temperature. After overnight in-
cubation at room temperature, the solutions were stored at 48C. The an-
nealed biotinylated RNA was then added to the Eppendorf tube contain-
ing the evaporated building blocks, and the resulting mixture left to stand
for four days at room temperature under air without stirring. After the
desired time, an aliquot was withdrawn (approximately one third), and
the disulfide exchange was stopped by addition of 0.1% TFA solution in
water (45–70 mL; pHꢀ5–6).
C) UV/Vis titration experiments: A 45–55 mm solution of the ligand (Acr1-
Nea, Acr2-Nea/Nea2, or Azq-Nea) and the corresponding amount of wt
RNA (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, or 2 equivalents) was prepared in
10 mm sodium phosphate buffer (pH 6.8) containing 100 mm NaCl and
0.1 mm Na2EDTA. The mixture was heated for 5 min to 908C and left to
cool slowly to room temperature. The absorption spectra were recorded
at room temperature. The percentage of absorption quenching was deter-
mined at the following absorption bands: 360 nm (31%) for Acr1-Nea
and 423 (40%) and 444 nm (35%) for Acr2-Nea.
Streptavidin-coated magnetic beads (Biomag Streptavidin, 5 mgmLÀ1 sus-
pension, Qiagen) were used to isolate the biotinylated RNA and the
binding ligands. In all washing procedures, a magnet was used to retain
the beads in the tube while the supernatant was pipetted off. First, the
beads (500 mL of suspension for each DCL aliquot) were separated from
the commercial buffer solution and washed with an acidic buffer (3ꢇ
500 mL of 50 mm Tris-HCl (pH 5.8), 100 mm NaCl, and 0.1 mm
Na2EDTA). DCL aliquots were added to the washed beads and incubat-
ed at room temperature. After 20 min, the beads were retained in the
vessel by using the magnet and the supernatant solution was pipetted off
again. The beads were then treated to remove the noninteracting ligands
and building blocks (3ꢇ200 mL of 50 mm Tris-HCl (pH 5.8), 100 mm
NaCl, and 0.1 mm Na2EDTA). Finally, the beads were washed with a hot
solution of 0.1% TFA in H2O in order to liberate RNA-binding ligands
(3ꢇ200 mL, incubation at 908C for 10 min). The solutions were combined
and evaporated in a Speed-Vac. The final residue was dissolved in 0.1%
TFA in H2O and subjected to UV–MS–HPLC analysis.
D) Fluorescence titration experiments: A solution of the ligand (Acr1-Nea
or Acr2-Nea) and the corresponding amount of wt RNA was prepared in
10 mm sodium phosphate buffer (pH 6.8) containing 100 mm NaCl and
0.1 mm Na2EDTA. The mixture was heated for 5 min to 908C and left to
cool slowly to room temperature. The fluorescence emission spectra were
recorded at room temperature.
E) Fluorescence binding assays: Fluorescence measurements were per-
formed in 1 cm pathlength quartz cells on a QuantaMaster fluorometer
(PTI) at 208C, with an excitation slit width of 4.0 nm and an emission slit
width of 5.2 nm. Upon excitation at 490 nm, the emission spectrum was
recorded over a range between 500 and 550 nm until no changes in the
fluorescence intensity were detected. All binding assays were performed
in 10 mm sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm
Na2EDTA.
D) UV–MS–HPLC analysis: UV–MS–HPLC analysis of DCC libraries
was performed by using a Micromass ZQ mass spectrometer equipped
with an electrospray source and a single quadrupole detector coupled to
a Waters 2695 HPLC instrument (photodiode array detector). The detec-
tion wavelength was set to 260 nm. Elution was performed on a Grace-
Smart C18 column (150ꢇ2.1 mm, 5 mm, flow rate: 0.25 mLminÀ1) with
linear gradients of H2O and ACN; both solvents contained either 0.1%
formic acid or 0.1% formic acid and 0.01% TFA. Typical gradient: 0 to
35% B in 15 min and from 35% to 80% B in 10 min. In some cases,
UV–MS–HPLC analysis was carried out with both elution conditions to
avoid the overlapping of some peaks in order to allow a more accurate
integration.
For each experiment, the fluorescence spectrum of buffer solution
(120 mL) without RNA or ligand was first taken, to be used as the base-
line. After this buffer blank, the spectrum of a 0.25 mm solution of refold-
ed RNA containing fluorescein (120 mL) was recorded, and the baseline
blank was subtracted. Subsequent aliquots of an aqueous ligand solution
(1 mL; increasing in concentration from 0 to 0.75 mm, 0.0005–3000 equiv-
alents, depending on the ligand affinity) were added to the solution con-
taining RNA, and the fluorescence spectrum was recorded after addition
of each aliquot until the fluorescein fluorescence signal at 517 nm
reached saturation (typically 5–10 min). Over the entire range of ligand
concentrations, the emission maxima varied less than 1 nm. The total
volume of the sample never changed more than 20%. The full titration
was repeated in the absence of labeled RNA to correct for the presence
of the ligandꢆs fluorescence. These spectra were subtracted from each
corresponding point of the labeled RNA titrations, and the resulting fluo-
rescence intensity was corrected for dilution (FꢇV/V0).
All peak areas of the HPLC traces were integrated and normalized by
taking into account the extinction coefficient of each compound at the
detection wavelength: e260: Acr1 37090, Acr2 13334, Azq 2930, TyrP 596,
and TrpP 3484mÀ1 cmÀ1. Histograms showing the change in the mol per-
centage for all DCL members at different times were generated in order
to verify that thermodynamic equilibrium had been reached. In addition,
histograms showing the mol percentage changes in each species of the
equilibrium mixture (amplification%) upon introduction of the target
RNA were also generated.
The emission fluorescence at 517 nm was normalized by dividing the dif-
ference between the observed fluorescence, F, and the initial fluores-
cence, F0, by the difference between the final fluorescence, Ff, and the in-
itial fluorescence, F0. This normalized fluorescence intensity was plotted
as a function of the logarithm of the total ligand concentration. Finally,
nonlinear regression with a sigmoidal dose-response curve was performed
with the software package GraphPad Prism 4 (GraphPad Software, San
Diego, CA) to calculate the EC50 values. Experimental errors were less
than or equal to Æ25% of each value.
Evaluation of the interaction between RNA and ligands
A) UV-monitored melting experiments: The impact of the ligands on the
thermal stability of wt and mutated (+3 and +14) stem-loop RNA struc-
tures was estimated by cooling/heating experiments. Samples were placed
in 1 cm pathlength quartz cuvettes in a Jasco V-550 spectrophotometer
equipped with a thermoregulated cell holder. Melting curves were re-
corded by cooling the samples from 90 to 208C at a constant rate of
For competitive experiments, a tRNA from bakerꢆs yeast (Saccharomyces
cerevisiae) was purchased from Sigma. Stock solutions of tRNAmix were
1952
ꢅ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 1946 – 1953