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Published online 12 May 2008 Nucleic Acids Research, 2008, Vol. 36, No. 10 e61
doi:10.1093/nar/gkn246
Gel-based oligonucleotide microarray approach
to analyze protein–ssDNA binding specificity
Olga A. Zasedateleva*, Andrey L. Mikheikin, Alexander Y. Turygin,
Dmitry V. Prokopenko, Alexander V. Chudinov, Elena E. Belobritskaya,
Vladimir R. Chechetkin and Alexander S. Zasedatelev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow,
Russian Federation
Received August 6, 2007; Revised March 7, 2008; Accepted April 16, 2008
ABSTRACT
Gel-based oligonucleotide microarray approach was
developed for quantitative profiling of binding affinity
of a protein to single-stranded DNA (ssDNA). To
demonstrate additional capabilities of this method,
we analyzed the binding specificity of ribonuclease
(RNase) binase from Bacillus intermedius (EC
3.1.27.3) to ssDNA using generic hexamer oligo-
deoxyribonucleotide microchip. Single-stranded
octamer oligonucleotides were immobilized within
3D hemispherical gel pads. The octanucleotides in
individual pads 5’-{N}N
1
N
2
N
3
N
4
N
5
N
6
{N}-3’ consisted
of a fixed hexamer motif N
1
N
2
N
3
N
4
N
5
N
6
in the middle
and variable parts {N} at the ends, where {N}
represent A, C, G and T in equal proportions. The
chip has 4096 pads with a complete set of hexamer
sequences. The affinity was determined by measur-
ing dissociation of the RNase–ssDNA complexes
with the temperature increasing from 08Cto508C
in quasi-equilibrium conditions. RNase binase
showed the highest sequence-specificity of binding
to motifs 5’-NNG(A/T/C)GNN-3’ with the order of
preference: GAG > GTG > GCG. High specificity
towards G(A/T/C)G triplets was also confirmed by
measuring fluorescent anisotropy of complexes of
binase with selected oligodeoxyribonucleotides in
solution. The affinity of RNase binase to other 3-nt
sequences was also ranked. These results demon-
strate the applicability of the method and provide the
ground for further investigations of nonenzymatic
functions of RNases.
INTRODUCTION
Different types of DNA microarray-based approaches
have been developed to identify sequence specificity for
regulatory proteins (1–4). Among these are the ChIP-on-
chip method, which enables identification of specific bind-
ing regions of a protein in vivo, and protein-binding DNA
microarrays for in vitro profiling of DNA-binding sites.
Universal protein-binding DNA microarrays containing
all possible single-stranded (ss) or double-stranded (ds)
DNA sequences of a given length allow to determine
in vitro the relative affinity of a protein to these sequences
and remains the powerful tool in protein–DNA binding
studies. Recently, microarrays containing all possible 8
and 10 bp DNA duplexes were used to define ds DNA
specific sites for transcription factors and low-molecular-
weight ligands (5,6). Morgan et al. (7) applied microarray
containing all possible 6 nt single-stranded sequences to
identify single-stranded motifs for a cold-shock protein
binding.
We present an alternative microarray method for
quantification of binding affinity of a protein to ssDNA.
In contrast to most oligonucleotide microchip techniques
based on 2D surface immobilization, we immobilize
single-stranded octamer oligonucleotides within 3D hemi-
spherical hydrogel pads (8–12). Immobilization of oligo-
nucleotides within the pads allows us to quantify the
affinity of a protein by measuring the complete set of
4
6
¼ 4096 dissociation curves for protein–oligonucleotide
complexes.
Guanyl-specific bacterial ribonuclease binase produced
by Bacillus intermedius (EC 3.1.27.3) was used to develop
further this approach. The earlier generation of 3D
microarrays proved to be quite efficient for quantitative
assessment of sequence specificity of low-molecular-weight
compounds, ligands and proteins in their interactions with
ssDNA, as well as dsDNA (8–11). Specific motifs found
using these oligonucleotide microchips were also con-
firmed by alternative methods (10).
The RNase binase from B. intermedius is a member of a
family of guanyl-specific enzymes that regulate cellular
metabolism by catalyzing ssRNA degradation (13–18).
The size of the substrate segment bound at the active site
*To whom correspondence should be addressed. Tel: þ7 495 1359980; Fax: þ7 495 1351405; Email: lana@biochip.ru
ß 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
of guanyl-specific RNases, particularly for barnase, the
closest homologue of RNase binase, was estimated to be
three ribonucleotides in length (19,20). The mechanism of
binase endoribonuclease activity towards ssRNA is similar
to mammalian RNase A (14,15).
Molecular structures and molecular mechanisms of
RNA cleavage are well established for many RNases, the
affinity towards different substrate sequences for many
of them is known in a broad general sense only. The
comparative analysis of substrate sequence specificity is
hampered not only by the need of massive combinatoric
turnover of RNA sequences at the active site, but also by
the essential cleavage activity of RNases. The latter
difficulty may be partially resolved by using DNA sub-
strate instead of RNA. Such replacement is commonly
performed in X-ray studies of RNase–substrate complexes
(21–23). Using NMR spectroscopy, it has been shown that
the functional dynamics at the active site of ribonuclease
binase, the mode of complexation and dissociation
constant did not change significantly after DNA substrate
substitution for RNA (24). There is additional interest in
such a substitution, taking into account the possibility of
additional functions of RNases, besides their primary
enzymatic activity (25–27).
We analyzed the enzyme-binding affinity to all possible
6-nt DNA sequences by measuring the dissociation of the
binase–DNA complexes under temperature increase in
quasi-equilibrium conditions. The enzyme was covalently
labeled with Texas Red fluorescent dye. To avoid possible
influence of the dye on the active site, we developed a
special protocol for binase labeling in the presence of
10-mer oligodeoxyribonucleotide 5
0
-GAGAGAGAGA-
NH
2
-3
0
, which was subsequently removed.
Using our microarray-based approach we demonstrate
that RNase binase shows sequence-specificity of binding
to 3-nt motifs 5
0
-G(A/T/C)G-3
0
with the following order
of preference: GAG>GTG>GCG. The affinity of RNase
binase to other 3-nt sequences was also ranked. These
results provide the ground for the further exploration of
nonenzymatic functions of RNases.
MATERIALS AND METHODS
Oligonucleotides
Oligonucleotides were synthesized at 1 mmol scale using
ABI 394 DNA/RNA Synthesizer (Applied Biosystems,
Foster City, CA, USA) and standard phosphoramidite
chemistry; 3
0
-C(7) amino modifier was purchased from
Glen Research (Sterling, VA, USA). The octadeoxyribo-
nucleotides used for the immobilization in the gel pads of
generic microchip have the structure 5
0
-{N}N
1
N
2
N
3
N
4
N
5
N
6
{N}-NH
2
-3
0
, where N
1
N
2
N
3
N
4
N
5
N
6
is one of
4
6
¼ 4096 possible 6-nt sequences and {N} corresponds to
equimolar mixture of four nucleotides A, T, G and C.
Thus, the flanking positions {N} are degenerate.
Decadeoxyribonucleotide 5
0
-GAGAGAGAGA-NH
2
-3
0
was labeled with amine-reactive fluorescent Dye-2
(N-hydroxysuccinimide ester of 4,4-difluoro-5,7-dimethyl-
4-bora-3a,4a-diaza-s-indacene-3-yl propionic acid) pro-
vided by Biochip-IMB (Moscow, Russia). Dye-2 is similar
to Bodipy dyes (Invitrogen, Carlsbad, CA, USA) and
has absorption/emission wavelengths
abs
¼ 500 nm
em
¼ 512 nm. Heptadeoxyribonucleotides 5
0
-TTGAGTT-
NH
2
-3
0
,5
0
-TTGTGTT-NH
2
-3
0
,5
0
-TTGCGTT-NH
2
-3
0
and
5
0
-TTTTTTT-NH
2
-3
0
were labeled with amine-reactive
fluorescent Dye-3 [N-hydroxysuccinimide ester of
3-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
indacene-2-yl) propionic acid] provided by Biochip-IMB.
Dye-3 is also similar to Bodipy dyes (Invitrogen) and
has absorption/emission wavelengths
abs
¼ 517 nm and
em
¼ 522 nm. To perform labeling, HPLC-purified oligo-
nucleotides were dissolved in H
2
O and cooled to 0–58C.
Dye-2 or Dye-3 dissolved in dry pyridine and cooled to
08C were added to oligonucleotide water solution (molar
ratio dye:oligonucleotide 5:1, volume ratio of solutions
1:1) and incubated at 0–58C overnight. The mixture was
diluted with 0.1 M TEAA buffer (pH 7.0), the excess of the
dyes extracted with n-butanol, and the oligonucleotides
were purified using HPLC.
The purity of the octadeoxyribonucleotides was
controlled using MALDI-TOF mass-spectrometer
COMPACT MALDI 4 (Kratos Analytical, Chestnut
Ridge, NY, USA). The concentrations of the oligodeox-
yribonucleotides were measured with spectrophotometer
Jasco V-550 (Jasco, Japan) using extinction coefficient
derived from nearest-neighbor model (28).
Generic microchip manufacturing
Generic microchips were manufactured according to the
IMAGE chip technology (12,29).
The generic microchip consisted of an array of 3D
hydrogel pads of hemispherical shape automatically
spotted on hydrophobic glass slide (3 in. by 1 in.; Cell
Associate, Inc., The Sea Ranch, CA, USA) by a pin robot
QArray (Genetix). Octamers with embedded 4096 possible
6-nt sequences extended with degenerated single nucleo-
tides at both ends were covalently immobilized in the gel
pads. Thus, each 3D hydrogel pad of the generic microchip
contained octadeoxyribonucleotides with a unique inner
6-nt sequence. The pads were 0.2 nl in volume and 80 mmin
diameter. The distance between neighbouring pads was
250 mm. The total area of the array was 17.8 15.9 mm
2
.
Since the manufacturing of IMAGE generic microchips
involved a combined step of photo-induced gel polymer-
ization and DNA immobilization, immobilized oligo-
nucleotides were uniformly distributed within the volume
of each hydrogel pad (12). The efficiency of oligonucleo-
tide immobilization inside the gel pads was 50% in
accordance with earlier results (12), while the final
concentration of immobilized oligonucleotides within the
gel pads was about 25 mM.
Covalent labeling of RNase binase
Wild-type binase was expressed in pMT416 bacterial
expression vector (Novagen, Madison, WI, USA) in
Escherichia coli strain BL21(DE3)[pLysS] cells (Novagen)
and purified by phosphocellulose chromatography as
described earlier (30,31).
To detect the RNase binase interaction with oligonu-
cleotides immobilized in the gel pads of generic microchip,
e61
Nucleic Acids Research, 2008, Vol. 36, No. 10 PAGE 2 OF 11
the enzyme was covalently labeled with Texas Red (TR)
sulfonyl chloride fluorescent dye (Molecular Probes,
Eugene, OR, USA) (absorption/emission wavelengths
abs
¼ 588 nm and
em
¼ 601 nm) (32). To prevent covalent
binding of the amine-reactive dye TR to amino acids in
the active center, the labeling of binase was performed in
the complex with fluorescently labeled 10-mer 5
0
-GAGA
GAGAGA-3
0
. This 10-mer was labeled with another
fluorophore, Dye-2, similar to Bodipy dyes (absorption/
emission wavelengths
abs
¼ 500,
em
¼ 512) to control the
completeness of removal of the 10-mer from TR-labeled
enzyme.
To perform covalent labeling, binase was dissolved
in 100 ml of 0.1 M NaHCO
3
, pH 8.5, to 10
4
M (32).
Concentrated water solution of fluorescently labeled
10-mer was added to RNase binase solution (molar ratio
oligonucleotide:enzyme 5:1). The mixture was incubated
at 08C for 10 min for binding. Solution of TR sulfonyl
chloride in dimethylformamide (20 mg/ml) cooled to 08 C
was added to binase–oligonucleotide complex (molar ratio
dye:enzyme 100:1). The enzyme was labeled at 08C for 4 h
in the dark. The labeled enzyme was purified from
oligonucleotide and the excess of unreacted molecules of
TR fluorophore by gel filtration using QAE Sephadex
A-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden).
According to absorbance measured from 700 to 190 nm,
fluorescently labeled 10-mer was removed completely. The
concentration of the enzyme was measured by absorption
at 280 nm assuming that the extinction coefficient equals
27 411 M
1
cm
1
(31).
The extent of protein modification was assessed by
MALDI-TOF mass-spectrometry and absorption spec-
trum at 595 and 280 nm and found to be 0.5 dye residue
per enzyme molecule.
Measurements of dissociation curves of complexes between
TR-labeled binase and oligonucleotides of generic microchip
All measurements on generic microchips were performed
in real time by an automated research custom-made
fluorescent microscope (Biochip-IMB) with 10 10 mm
2
field, mercury lamp as the excitation source and the filter
set for TR dye (
ex
¼ 580 nm and
em
¼ 630 nm). The
microscope was equipped with a CCD camera (SenSys,
Roper Scientific, Tucson, AZ, USA), a Peltier thermotable
with temperature controller (Melcor, Trenton, NJ, USA),
and a scanning system consisting of a two-coordinate
table, step-wise motor, and motion controller (Newport,
Irvine, CA, USA).
The generic microchip consisted of four fields. Two
fields comprised 972 gel pads each, including 36 gel pads
containing immobilized markers and four empty gel pads
in each field. Fluorescently labeled oligonucleotide
gel-5
0
-NH-TTTTTTTT-NH-TR immobilized within cor-
responding gel pads at final concentration 6 mM was used
as the marker. Two other fields consisted of 1152 gel pads
each, including 36 gel pads per field containing immobil-
ized markers. The microchip was scanned field by field at
each temperature point. Each field was exposed for 7 s,
and the whole microchip (four fields) was scanned in 55 s.
The microscope was equipped with a computer and
Imagel Research software developed in our laboratory
(33) to operate the experiments and record fluorescent
signals and images of the whole generic microchip at each
temperature point. Recording of the whole image at each
temperature enabled us to recalculate the whole set of
dissociation curves if needed.
Binding of TR-labeled binase with 4096 octadeoxy-
ribonucleotides immobilized in gel pads of the generic
microchip was performed at 08C for 4 h. Binding and
subsequent dissociation of the formed complexes were
carried out in 50 ml chamber. Both processes were
performed in Buffer A (0.1 M NaCl, 50 mM Tris–HCl,
pH 6.5, 1 mM EDTA) containing 2 10
5
M of TR-
labeled binase. The dissociation curves were recorded for
all pads of the microchip with the temperature increase
from 08Cto658C at the rate of 18C/30 min. According to
the kinetic curves of binding at 08C between TR-labeled
binase and oligonucleotides of generic microchip, the time
of about 30 min was sufficient for fluorescent signals to
reach 90% saturation. Therefore, the whole curve of the
dependence of fluorescence intensity on temperature was
obtained in quasi-equilibrium conditions reached at every
temperature step.
Data processing
Fluorescence signals from individual pads were processed
using ImaGel Research software. Fluorescent image of
each gel pad of the microchip was surrounded by inner
and outer circular frames: inner frame enclosed the gel pad
itself, while outer frame enclosed the background around
it. Intensity of fluorescence, J, from a microchip gel pad
was defined according to the equation:
J ¼
In Out
Out Dc
1
8
X
8
i¼1
In Out
Out Dc
ref
i
1
where In is the average intensity of fluorescence inside the
inner frame occupied by the gel pad, Out is the average
intensity of fluorescence in the space between the inner
and outer frames occupied by the outer region of the gel
pad, Dc is the average noise signal inside the inner frame
produced by dark current at zero illumination intensity,
while the counterpart expression with subscript ‘ref’
corresponds to the signals from empty gel pads without
immobilized oligonucleotide probes.
All experiments on the measurements of dissociation
curves for binase-ssDNA complexes were repeated twice
on two different generic microchips. The subsets of 3963
and 3891 melting curves were selected for subsequent
analysis according to the criteria developed earlier (10):
the pads whose initial signals were below 20% of the
average initial signals for the whole set were excluded from
further consideration, and they constituted up to 5% of all
curves. The filtered curves were approximated by least
squares method using the following fitting equation
described earlier (10) to obtain the set of dissociation
temperatures, T
D
:
JðTÞ¼A þ
B
1 þ
T
T
D
N
2
PAGE 3 OF 11 Nucleic Acids Research, 2008, Vol. 36, No. 10 e61
where J(T) is the measured intensity of fluorescence, T is
temperature (K), T
D
is dissociation temperature (K),
A þ B is initial intensity, A is final intensity, N is fitting
parameter. After fitting procedure, the resulting set of T
D
values was converted to centigrees. Dissociation tempera-
ture, T
D
, for the complexes between RNase binase and
oligonucleotides was defined as a temperature at which
half of the complexes in a microchip gel pad are in
nondissociated state in equilibrium thermodynamic con-
ditions. In our conditions, it may also be approximately
assessed by the temperature at which the fluorescence
intensity is half of the intensity at 08C.
In our microarray experiments, the enzyme (1 nmol)
was taken in excess with respect to the total amount
of immobilized oligonucleotides (2 10
2
nmol), or
C
p
V
chamber
>> C
imm
V
pad
n, where C
p
is the concentra-
tion of protein in applied solution, C
imm
is the concentra-
tion of immobilized oligonucleotides, V
chamber
and V
pad
are the volumes of the microchip chamber and a single
microchip gel pad correspondingly, n is total amount of
gel pads. Thus, the concentration of free protein can be
assumed to be constant and does not depend on
concentration of protein–ssDNA complexes at each
temperature point.
Taking into account this condition and in accordance
with the mass action law the equilibrium binding constant,
K(T), for the formation of protein–ssDNA complexes
inside a microchip gel pad can be expressed as follows:
KðT Þ¼
C
complex
ðT Þ
C
p
ðC
imm
C
complex
ðT ÞÞ
3
where T is temperature (8K), C
imm
is the concentration of
immobilized oligonucleotides, C
p
is the concentration of
binase in applied solution, and C
complex
(T ) is the
concentration of binase–oligonucleotide complexes.
Dissociation temperature, T
D
, for the complexes
between RNase binase and oligonucleotides is defined as
the temperature at which half of the complexes in a
microchip gel pad are in nondissociated state in equilib-
rium thermodynamic conditions, or C
complex
¼ Q
imm
/2.
Thus, at dissociation temperature T ¼ T
D
, the equilibrium
binding constant from Equation (3) can be expressed as
follows:
K
j
T¼T
D
¼
1
C
p
4
According to van’t Hoff equation,
KðT Þ¼exp
FðT Þ
RT
5
where F is the change in binding free energy and R is
universal gas constant. Taking into account the relation-
ship (4), we obtain the following relationship between
the change in binding free energy and dissociation
temperature:
F
j
T¼T
D
¼ R T
D
ln C
p
6
This relationship shows that in the process of equilibrium
dissociation the higher the dissociation temperature, T
D
,
the higher the change in binding free energy. Due to this
relationship, the binding constants keep the same value,
1/C
p
, at dissociation temperatures T ¼ T
D
, for all com-
plexes between the protein and oligonucleotides of generic
microchip [Equation (4)]. The relationship (6) also shows
that T
D
can be used for quantitative assessment of binding
affinity.
Fluorescence anisotropy measurements
Measurements of intensities of polarized fluorescence, I
||
and I
?
, were performed using a Cary Eclipse fluorescence
spectrophotometer (Varian Australia) equipped with a
manual polarizer and single cell holder with temperature
control. The excitation and emission wavelengths were 510
and 530 nm, respectively, with 10 nm band-pass and an
integration time 30 s. Target heptadeoxiribonucleotides
were labeled with Dye-3, which is similar to Bodipy dyes
(absorption/emission wavelengths
abs
¼ 517,
em
¼ 522,
molecular mass of the dye residue of a modified
oligonucleotide is 303.13 Da). Each Dye-3 labeled
heptamer was titrated with increasing concentration of
protein, with the total [protein] >> [oligonucleotide] at
each titration point. The titration was carried out at 208C,
at equilibrium conditions. After each addition, the cuvette
(1 cm 1 cm) with sample was rotated gently, equilibrated
at the required temperature for 5 min and polarized
intensities of fluorescence were measured. All titrations
were performed in buffer A. Initial concentration of
oligonucleotide was 10 nM, volume 1 ml. In total, 60 mlof
protein solution was added. The decrease in oligonucleo-
tide concentration during the titration as well as protein
absorption were taken into account in the analysis of the
data and computation of the values of anisotropy of
fluorescence.
Fluorescence anisotropy data analysis
Equilibrium binding curves obtained using fluorescence
anisotropy were fit to a standard single-site isotherm
usable when [protein] >> [oligonucleotide] (34):
AðC
p
Þ¼A
0
þ
ðA
max
A
0
ÞC
p
K
1 þ C
p
K
where A is fluorescence anisotropy, A
0
is fluorescence
anisotropy of oligonucleotide in the absence of the protein
in solution, A
max
is the value of fluorescence anisotropy
when all oligonucleotides are in bound state with the
enzyme, C
p
is the total binase concentration at each point
in the titration, and K is the association constant for
binase–DNA binding. Fitting was performed using the
program Origine 6.1 (OrigineLab Corp., Northampton,
MA, USA). Fitting parameters were K, A
0
and A
max
.
RESULTS
The affinity of binase binding to all possible 6 nt ssDNA
sequences was quantitatively assessed via measurements of
fluorescence signals from the complexes formed between
ssDNA and TR-labeled binase. To avoid the inactivation
of binase during modification, its active site was protected
e61
Nucleic Acids Research, 2008, Vol. 36, No. 10 PAGE 4 OF 11
by temporary binding of 10-mer oligodeoxyribonucleotide
5
0
-GAGAGAGAGA-NH
2
-3
0
, which was removed after
the reaction (see Materials and methods section).
Figure 1A shows the image of generic microchip
recorded after 4 h of incubation at 08C with TR-labeled
binase. Most gel pads have visible fluorescence demon-
strating strong binding of TR-labeled binase to corre-
sponding immobilized oligonucleotides. The fluorescence
intensities differ depending on their sequences.
Figure 1B shows examples of dissociation curves for
eight complexes between the enzyme and oligonucleotides
of generic microchip. Because of the variations of the
affinity of binase to different sequences, the corresponding
complexes showed different stability. Dissociation tem-
peratures vary from 26.58C for octadeoxiribonucleotide
containing inner TCTGCC 6-nt sequence to 48.38C for
octadeoxiribonucleotide containing inner AGTGTG 6-nt
sequence.
Figure 1C shows the distribution of dissociation
temperatures of binase–oligonucleotide complexes.
Dissociation of the complexes occurs below 508C, while
the mean value of dissociation temperature for all binase–
oligonucleotide complexes is 40.28C. It should be noted
that the denaturation temperature of binase in similar
physico-chemical conditions is 558C, and the conforma-
tion of the enzyme remains stable up to 508C (31,35).
Thus, the dissociation temperatures determined in this
work characterize the binding affinity of RNase binase in
its native form.
All measurements of dissociation curves for binase–
ssDNA complexes were repeated twice and the results
were reproducible. The data presented below correspond
to averages of two experiments. Table 1 (Supplementary
Material) contains the values of the dissociation tempera-
tures and the intensities of fluorescence at 408C for the
complexes between TR-labeled binase and oligonucleo-
tides of the generic microchip.
Figure 2A shows a computer representation of the
dissociation temperatures measured for the complexes
between TR-labeled binase and octadeoxyribonucleotides
of generic microchip. The most stable binase–oligonucleo-
tide complexes are found in six rectangular regions. Three
of these regions arranged in columns correspond to
common motif 5
0
-NG(A/T/C)GNN-3
0
, where N is one
of the four nucleotides. The other three rectangular
regions arranged in rows correspond to the same 5
0
-
NNG(A/T/C)GN-3
0
common motif shifted by one nucleo-
tide to the right.
The effective dissociation temperatures for shorter
motifs were obtained as the mean values over all hexamer
sequences containing a given shorter motif and summar-
ized in Figure 2B–F. G was found to be the best
mononucleotide motif, GA, AG, GG as well as GT,
TG, GC and CG were found to be the best dinucleotide
motifs, GAG, GTG and GCG the best 3-nt motifs,
NG(A/T/C)G, G(A/T/C)GN the best 4-nt motifs and
NG(A/T/C)GN G(A/T/C)GNN and NNG(A/T/C)G the
best 5-nt motifs. These results demonstrate that starting
from the length of four bases the most specific motifs are
organized by addition of degenerated nucleotides to the
ends of 5
0
-G(A/T/C)G-3
0
motifs, thus revealing the
Figure 1. Interaction of TR-labeled RNase binase with octadeoxy-
ribonucleotides immobilized on generic microchip. (A) Overview of
generic hexamer deoxyribonucleotide microchip after binding with
TR-labeled RNase binase from B. intermedius. Each octadeoxyribo-
nucleotide immobilized in a given gel pad contains a fixed hexamer
N
1
N
2
N
3
N
4
N
5
N
6
in the middle of 5
0
-{N}N
1
N
2
N
3
N
4
N
5
N
6
{N}-3
0
sequence, where variable nucleotides {N} at 5
0
- and 3
0
-ends are A, C,
G and T in equal proportions. (B) Normalized dissociation curves for
the complexes between TR-labeled RNase binase and eight octadeoxy-
ribonucleotides immobilized on generic microchip. The complexes are
characterized by different binding affinity. Dotted lines illustrate the
approximate assessment of T
D
value for 5
0
-NTCTGCCN-3
0
octadeoxy-
ribonucleotide. Sequences of the inner 6-nt long sequences of the
immobilized octadeoxyribonucleotides are shown in the upper right
corner. (C) Distribution of the number of binase–oligonucleotide
complexes plotted against their dissociation temperatures.
PAGE 5 OF 11 Nucleic Acids Research, 2008, Vol. 36, No. 10 e61
Figure 2. Computer representation of dissociation temperatures, T
D
, for complexes between TR-labeled RNase binase and octadeoxyribonucleotides
of the generic microchip. Each hexanucleotide sequence should be read by combining its 5
0
-half in the column with 3
0
-half in the row. Set (A) refers
to the initial data measured with generic hexamer microchip and averaged over two experiments. (B–F) represent effective dissociation temperatures
for all possible sequences of the length 5, 4, 3, 2 and 1 nt, correspondingly. The effective dissociation temperatures for sequences shorter than 6 nt
were obtained as the mean values over T
D
values for all 6-nt sequences containing a given shorter motif. For instance, the value for the sixth data
point in the upper row in (B) was obtained as the mean value for all hexamers from (A) containing sequence 5
0
-CCAAA-3
0
; the value for the second
element in the bottom row of (D) is the mean for all hexanucleotides containing sequence 5
0
-CTT-3
0
, etc. The re-arrangement of figure (A) into (B–F)
was carried out using VirtualChip software developed in our laboratory. Color scales of T
D
values are shown next to corresponding matrices.
e61 Nucleic Acids Research, 2008, Vol. 36, No. 10 PAGE 6 OF 11
distinct sequence-specificity of the binase towards these
motifs. This is in agreement with earlier observations
(14,19,20), which demonstrated that RNase binase is
guanyl-specific and that the length of the substrate frag-
ment in the active site is 3-nt, similarly to other guanyl-
specific ribonucleases. Our data also provide additional
information on the sequence-specificity of binase–ssDNA
binding.
Similar results were obtained by analyzing the values of
fluorescent intensities measured at 408C for TR-labeled
binase–ssDNA complexes formed in microchip gel pads
(Figure 3A–F). Figure 3A shows the same six rectangular
regions corresponding to 5
0
-NG(A/T/C)GNN-3
0
and
NNG(A/T/C)GN-3
0
as the common specific motifs shifted
by one nucleotide within the hexamer. Figure 3B–F helps
to visualize the 5
0
-G(A/T/C)G-3
0
sequence as the binase-
specific 3-nt binding motif. The results obtained at 408C
are in good agreement with the results obtained using the
dissociation temperatures and strongly suggest that
RNase binase did not undergo denaturation within the
temperature range used in the melting experiments, from
08Cto508C. These results also demonstrate the possibility
to study the binding specificity of proteins to oligonucleo-
tides immobilized on generic microchip at a constant tem-
perature. It has to be noted that in this case it is necessary
to have preliminary data on the average dissociation tem-
perature of the complexes.
The impact of the flanking nucleotides on the affinity
of RNase binase to 5
0
-G(A/T/C)G-3
0
motif is shown in
Figure 4. The mean dissociation temperatures for hex-
anucleotides containing G(A/T/C)G motifs at their
ends (i.e. one nucleotide from the end of the immobil-
ized octamer) is 1–28C lower then for those containing
G(A/T/C)G motifs in the middle. It should be noted that
mean dissociation temperatures for hexanucleotides with
G(A/T/C)G motifs near their 3
0
-ends is lower then for
those containing G(A/T/C)G motifs near 5
0
-ends. This
could be explained by the presence of amino-linker at the
5
0
-end of immobilized octadeoxyribonucleotides. Thus,
the specificity of RNase binase towards its consensus
sequence reaches its maximum when the G(A/T/C)G is
located two nucleotides from the end of immobilized
octamer.
Figure 5 shows the affinity of RNase binase to all
possible 3-nt sequences flanked by at least two nucleotides
within octanucleotide sequence and ranked in decreasing
order according to the values of effective dissociation
temperatures. Effective T
D
values for GAG, GTG and
GCG sequences are at least two degrees higher than for
other triplets. In general, RNase binase shows higher
affinity towards purine-rich 3-nt sequences as compared
with pyrimidine-rich 3-nt sequences. The affinity of RNase
binase to 3-nt homonucleotide sequences ranks in the
order GGG > AAA > CCC TTT.
To validate our results obtained using the microarray
approach, the binding affinities of unlabeled RNase binase
towards TTG(A/T/C)GTT 7-nt sequences were measured
using fluorescence anisotropy method and compared with
the binding affinity of the enzyme towards TTTTTTT 7-nt
homonucleotide sequence. We used oligonucleotides
labeled at their 3
0
ends with an analog of Bodipy dye.
According to the equilibrium titration curves shown in
Figure 6, the estimated values of association constants for
TTGAGTT, TTGTGTT and TTGCGTT oligodeoxy-
ribonucleotides were found to be (54 000 8000) M
1
,
(56 000 13 000) M
1
and (47 000 7000) M
1
, corre-
spondingly, which are identical within the limits of error.
For comparison, the association constant measured earlier
for d(GCAG) was found to be between 20 000 and
10 000 M
1
(24). Figure 6 also shows that the enzyme
exhibited significantly lower extent of affinity towards
TTTTTTT sequence than towards TTG(A/T/C)GTT
sequences. Table 2 (Supplementary Material) contains
the data for titration curves. As shown using the
microarray approach, the enzyme exhibits the highest
sequence-specificity in interaction with G(A/T/C)G 3-mers
flanked by 2 nt at both ends and one of the lowest affinity
towards TTT 3-mer flanked by 2 nt from both ends (see
Figure 5 for comparison). Thus, the difference in binding
affinities of RNase binase towards highly specific triplets
and a nonspecific one was confirmed by direct measure-
ments of their interaction with unlabeled protein in
solution.
DISCUSSION
Our microarray-based analysis proves that in the tempera-
ture range 0–508C corresponding to the native form of
RNase binase the enzyme binds with the highest affinity
to ssDNA motifs 5
0
-G(A/T/C)G-3
0
incorporated within
the longer strands with degenerate nucleotides in their
5
0
- and 3
0
-flanking regions. The order of preference is
GAG > GTG > GCG. Measurement of the affinity of
RNase binase to all possible 3-nt sequences indicates that
generally it displays the highest affinity towards purine-rich
3-nt sequences and the lowest towards pyrimidine-rich 3-nt
sequences. Its affinity towards 3-nt long homonucleotide
sequences is GGG > AAA > CCC TTT. These results on
sequence-specificity of binase towards 3-nt ssDNA
sequences correlate with the published data on the rate of
enzymatic cleavage of Np–Np bonds in ssRNA directly
related to the type of nucleoside at the 5
0
-end of the
phosphodiester bond to be split. As shown by Bulgakova
et al. (15), the enzyme preferentially splits Gp–Np and
Ap–Np bonds in ssRNA, with the order of preference
Gp–Np > Ap–Np, displaying significantly lower activity
towards Pyrp–Np bonds. In a later review, sequence-
specificity of RNase binase towards ss RNA was summar-
ized as G > A > pyrimidines (14).
The affinity of RNase binase towards 3-nt long
homodeoxyribonucleotides determined in this work also
correlates well with the published data on the rate of
cleavage of homo-polyribonucleotides by the enzyme.
Yakovlev et al. (36) demonstrated that the rate of hydro-
lysis of purine polyribonucleotides is 3–4 order of magni-
tude higher than that of pyrimidine ones.
The high-affinity motif found in this work is also in
agreement with the previous observations on 3-nt length
of the substrate segment bound at the active site. These
data were obtained during the study of catalytic activity of
microbial ribonucleases towards ssRNA (19,20).
PAGE 7 OF 11 Nucleic Acids Research, 2008, Vol. 36, No. 10 e61
Figure 3. Computer representation of fluorescence intensities, J,at408C, for complexes of TR-labeled RNase binase with octadeoxyribonucleotides
of the generic microchip. Each hexanucleotide sequence should be read by combining its 5
0
-halve in the column with 3
0
-halve in the row. Set ( A)
refers to the initial data measured with generic hexamer microchip and averaged over two experiments. (B–F) represent effective values of
fluorescence intensities for all possible sequences of the length 5, 4, 3, 2 and 1 nt, correspondingly. (B–F) were obtained from the data shown in (A) as
described in the legend to Figure 2.
e61 Nucleic Acids Research, 2008, Vol. 36, No. 10 PAGE 8 OF 11
Using fluorescence anisotropy method, we determined
the binding affinities of unlabeled enzyme towards highly
specific sequences TTG(A/T/C)GTT, which were found to
be approximately the same and significantly higher than
that for nonspecific oligodeoxyribonucleotide TTTTTTT.
In our microarray analysis, we used fluorescently
labeled enzyme. To avoid possible influence of the dye
on the active site of the enzyme, we developed a special
protocol for labeling: the enzyme was labeled in the
presence of sequence-specific oligodeoxyribonucleotide,
which was removed after the reaction. According to the
mass-spectrum, the majority of modified molecules con-
tained only one covalently attached fluorophore (data not
shown).
The specificity of hydrolytic activity of nucleases vary
widely, with some of them being strictly specific to DNA
or RNA substrates and to single-stranded or double-
stranded nucleic acids. The activity of other enzymes is
rather broad and can be directed against both RNA and
DNA, as well as single-stranded and double-stranded
nucleic acids (37). The strong binding of an enzyme to
single-stranded DNA or RNA often implies that it can
destabilize double-stranded DNA or RNA upon binding
[for discussion and further references see ref. (10)]. Indeed,
our preliminary data on melting of gel-immobilized
duplexes in the presence of binase indicate the destabiliza-
tion of dsDNA by the protein (unpublished data).
Besides the catalysis of RNA degradation, ribonu-
cleases display other biological activities, some of which
seem to be independent of their ribonucleolytic action.
Many of them exhibit cytotoxic action, triggering apop-
totic events, and therefore offer therapeutic opportunities
for cancer treatment (25–27,38–41). Thus, onconase, an
amphibian homolog of mammalian RNase A, selectively
target tumor cells and is currently in phase III of human
clinical trials as a chemotherapy treatment (25). Bacillus
intermedius RNase (binase) was shown to kill preferen-
tially mammalian cells expressing ras-oncogene (40). The
complete network of molecular interactions responsible
for RNases cytotoxic activity is not known yet and
presumably includes pathways involving both specific and
nonspecific interactions of cytotoxic RNases with cellular
components. This in turn suggests the existence of
additional targets besides the elements of protein synthesis
machinery (25,27). The study of specific and nonspecific
binding of binase to ssDNA as well as to dsDNA may
Figure 4. Dissociation temperatures of binase–oligonucleotide com-
plexes plotted against different positions of GAG, GTG or GCG motifs
within inner hexanucleotide sequences of immobilized octadeoxyribo-
nucleotides. In each of two experiments, the data sets for 63–64
hexanucleotide sequences were used to calculate mean T
D
value for the
each position. The histogram indicates the calculated mean T
D
values
averaged over two experiments. The bars indicate the margin of error
of T
D
. All sequence data refer to the inner hexanucleotide sequences of
immobilized octadeoxyribonucleotides.
Figure 5. Affinity of RNase binase to all possible 3-nt sequences
flanked by at least 2 nt from each end. The effective T
D
values were
obtained as the mean values over experimentally measured T
D
values
for the complexes between binase and immobilized 8-mers with inner
hexanucleotide sequences of the latter containing a given 3-nt sequence
in one of the middle positions. The effective T
D
values obtained in two
different experiments are shown using filled and open circles,
correspondingly. The histogram indicates the values of effective T
D
averaged over two experiments. The 3-nt motifs were ranked
corresponding to the averaged effective T
D
.
PAGE 9 OF 11 Nucleic Acids Research, 2008, Vol. 36, No. 10 e61
shed additional light on the mechanism of cytotoxic action
of bacterial ribonucleases.
In this study, we used microchip with oligonucleotides
immobilized within 3D hydrogel pads. As it was shown
earlier for IMAGE microchips (12,42), immobilized
oligonucleotides are evenly distributed within the gel pad
volume, are easily accessible because of large pores, are in
homogeneous water-like surrounding, and their intermo-
lecular interactions, as well as contacts with solid surface,
are negligible. Therefore, the interaction of immobilized
short oligonucleotides with the analyzed protein occurs in
conditions close to those in solution. This allows to
register multiple temperature dissociation curves for
protein–oligonucleotide complexes in parallel in equilib-
rium conditions and to define the affinity of a protein by
comparing dissociation temperatures: the higher the
dissociation temperature of a given complex, the higher
its binding affinity. The only limitation of this approach is
the conformational stability of the analyzed protein at
high temperature.
In most microarray approaches, the assessment of
binding affinities is carried out mostly using the analysis
of fluorescence intensities from protein–oligonucleotide
complexes formed on microarray at a particular tempera-
ture (3–7). In the present study, the analysis of dissocia-
tion curves allowed determining an optimal temperature,
at which comparison of fluorescence intensities from
protein–oligonucleotide complexes on the microchip
under protein solution can provide the same assessment
of binding specificity as the analysis of dissociation
temperatures.
The generic hexamer oligonucleotide platform used in
this work is primarily aimed at the study of sequence-
specificity of ligands and proteins binding to ssDNA,
particularly of proteins whose active sites bind relatively
short stretches of DNA. Such microchips may be used for
the ranking of complete combinatorial turnover of
binding sequences in protein–DNA complexes and infer-
ence of specific binding motifs.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
The authors are very thankful to Dr Yakovlev G.I. for
RNase binase kindly provided for this investigation and
helpful discussions. We wish to thank Dr I. Udalova,
Kennedy Institute of Rheumatology, Imperial College
(London) for fruitful discussions and valuable comments.
The assistance of Health Front Line, Ltd. (Champaign,
IL) in the preparation of this article for publication is
appreciated. This research was supported by International
Association for the Promotion of Cooperation with
Scientists from the New Independent States of the
former Soviet Union (INTAS YSF 04-83-3841). Funding
to pay the Open Access publication charges for this article
was provided by INTAS.
Conflict of interest statement. None declared.
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