Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 3. Optimization of miRNA hybridization conditions. (A) Effect
of ionic strength adjusted by NaCl on hybridization efficiency (number
of hybrid counts). (B) Performance of LNA, DNA, and RNA probe on
hybridization with complementary miR-21 and negative control of miR-
214. (C) Effect of incubation time on hybridization efficiency (number
of hybrid counts). The data depict the averages of three experiments,
and the error bars are the standard error of mean of the three trials.
as a consequence, the number of counts drops. Here, we adjusted
the ionic strength by varying the concentration of NaCl in 1× Tris-
NaCl-EDTA (TNE) buffer. Figure 3A shows the molecule counts
as a function of the concentration of NaCl in the hybridization
buffers for probes of locked nucleic acid (LNA), DNA, and RNA,
6916 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
respectively, with the same concentration of the target miR-21.
The experimental condition was considered optimum when the
highest number of molecule counts was obtained; indicating that
the largest number of individual hybrids was formed. A gradual
increase in the number of hybrids was observed from buffer
containing 0-250 mM NaCl and a decrease after the salt
concentration exceeds. We analyzed the pixel size of each
fluorescent spots of LNA/miRNA hybrids prepared in TE buffer
with 250 and 500 mM NaCl, respectively, as shown in Figure S3.
It is obvious that higher percentages of molecules with larger pixel
sizes (size >4 pixels) were found in the series of 500 mM NaCl
compared to that of 250 mM. This implies that increase in ionic
strength would induce aggregation among hybrids and cause
underestimation on the count of molecules in single molecule
level. Thus, it is concluded that the optimal NaCl concentration
was 250, 150, and 150 mM with respect to probes of LNA, DNA,
and RNA.
Oligonucleotide probe is commonly used as a reporter in
hybridization-based nucleic acid detection. Conventional miRNA
detection assays use DNA oligonucleotides as the capturing probe,
although the stability of DNA-miRNA duplex is not high.
Recently, several groups demonstrated the potential of LNAs as
an alternative to DNA probes. 8,9,50,51 LNA is a nucleic acid
analogue known as a mimic of RNA that has high binding affinity
to RNA molecules. The thermostability of the LNA-miRNA
duplex is significantly higher than that of unmodified DNA
probes. 52-55 Consequently, both the detection sensitivity and
hybridization discrimination efficiency are enhanced due to the
increase in binding affinity and stability of LNA-miRNA complex.
To assess the performance of different nucleic acid analogues of
probes in the detection of miRNA, the hybridization affinities of
LNA, DNA, and RNA probes toward (i) complementary miR-21
and (ii) negative control miR-214 (sequence showed in Experimental
Section) in free solution were investigated. As shown in
Figure 3B, LNA probe-based hybridization yields a 1.5-fold
enhancement in the molecule counts compared with those of DNA
and RNA probes, while the binding of LNA probes to negative
control of miR-214 was maintained at low level. This indicates that
LNA probe not only promotes higher capturing efficiency, but also
displays a good mismatch discrimination capability.
Incubation time also plays a role on the overall hybridization
efficiency. Counts of hybrids obtained after incubation for 15, 30,
60, and 180 min were shown in Figure 3C. It was observed that
the number of counts increased and reached maximum in the
first 60 min incubation and then reduced after 3hofincubation.
The reduction in counts after prolonged incubation may be due
to the structural conformation change of LNA strands as proven
by MALDI-TOF mass spectrometry analysis (see Supporting
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