Nucleic Acids Research, 2003, Vol. 31, No. 15 e79
© 2003 Oxford University Press
Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry
Angelika Gabler*,
Stefan Krebs,
Doris Seichter and
Martin Förster
Lehrstuhl für Tierzucht und Allgemeine Landwirtschaftslehre, Ludwig-Maximilians-Universität München, Veterinärstrasse 13, D-80539 Munich, Germany
*To whom correspondence should be addressed. Tel: +49 89 21802577; Fax: +49 89 344925; Email: angelika.gabler{at}gen.vetmed.uni-muenchen.de
Received March 5, 2003; Revised May 22, 2003; Accepted June 3, 2003
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ABSTRACT
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Alteration of gene expression by use of antisense oligonucleotides
has considerable potential for therapeutic purposes and scientific
studies. Although applied for almost 25 years, this technique
is still associated with difficulties in finding antisense-effective
regions along the target mRNA. This is mainly due to strong
secondary structures preventing binding of antisense oligonucleotides
and RNase H, playing a major role in antisense-mediated degradation
of the mRNA. These difficulties make empirical testing of a
large number of sequences complementary to various sites in
the target mRNA a very lengthy and troublesome procedure. To
overcome this problem, more recent strategies to find efficient
antisense sites are based on secondary structure prediction
and RNase H-dependent mechanisms. We were the first who directly
combined these two strategies; antisense oligonucleotides complementary
to predicted unpaired target mRNA regions were designed and
hybridized to the corresponding RNAs. Incubation with RNase
H led to cleavage of the RNA at the respective hybridization
sites. Analysis of the RNA fragments by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,
which has not been used in this context before, allowed exact
determination of the cleavage site. Thus the technique described
here is very promising when searching for effective antisense
sites.
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INTRODUCTION
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Since its introduction by Zamecnik and Stephenson more than
two decades ago (
1), antisense technology has been widely used
to specifically generate gene knockouts. The underlying strategy
is conceptually elegant. Taking advantage of knowledge of the
unique sequence of the mRNA transcribed from the gene of interest,
small oligonucleotides of complementary sequences are synthesized,
which enables them to interact by Watson–Crick base pairing
with the corresponding mRNA. Although a number of RNA oligonucleotide
derivatives and peptide nucleic acids are currently under investigation,
the most common antisense compounds are chemically modified
oligodeoxynucleotides (ODNs). One of the modes of antisense
ODN action discussed is inhibition of protein synthesis via
translational blockade; antisense ODNs that produce DNA–RNA
hybrids can interfere in the initiation process and/or progression
of the ribosomal apparatus and so induce translational arrest
(
2). The main reason for antisense ODN-mediated alteration of
gene expression, however, is supposed to be degradation of the
mRNA by endogenous RNase H (
3,
4). The ubiquitously expressed
enzyme RNase H cleaves the RNA strand of DNA–RNA hybrids,
and thus the target mRNA is no longer available as a template
for protein synthesis. This mechanism of antisense ODN-mediated
inhibition of gene expression is especially important in proliferative
tissues as RNase H activity clearly correlates with the rate
of DNA synthesis (
5), and as few as 5–6 bp are often sufficient
to be recognized as a DNA–RNA substrate by RNase H (
6).
Unlike other RNases, RNase H does not cleave RNA in a sequence-specific
manner, and scission may occur at more than one point along
the target RNA (
7).
One of the most critical steps in antisense technology is the design of effective antisense ODNs, because intramolecular base pairing of the mRNA into stable secondary and tertiary structures makes large parts of the molecule inaccessible to interacting with complementary nucleic acids, whereas sequences free from intramolecular interactions may favor interaction with other nucleic acids (8,9). Furthermore, a linear correlation was found between the percentage of translation inhibition by RNase H and the percentage of unpaired target RNA (10); taken together, these observations clearly indicate that mRNA regions free from intramolecular base pairing are ideally suited as targets for antisense ODNs. Therefore, many researchers use computer programs which make predictions of the secondary structure of the target RNA. One of the most frequently used programs is ‘mfold’ [http://www.bioinfo.rpi.edu/applications/mfold/old/rna/ (11,12)]; this program calculates the global free energy minimum of an RNA molecule, thus leading to the prediction of single-stranded and double-stranded structures. However, several reports call the reliability of prediction of accessible and inaccessible sites on mRNAs by ‘mfold’ into question (13,14); thus computer prediction methods alone seem not to be sufficient for designing effective antisense ODNs. In order to circumvent these difficulties, we constructed antisense ODNs according to computational secondary structure predictions, and tested their ability to provoke RNase H-mediated degradation of the RNA in vitro. The resulting RNA fragments were subsequently analyzed not only by gel electrophoresis, but also by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF mass spectrometry), thus enabling us to determine the exact cleavage site.
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MATERIALS AND METHODS
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Generation of in vitro transcripts
Total RNA was isolated from pig ileum with Trizol (Life Technologies)
according to the manufacturer’s protocol. cDNA synthesis
was performed with random primers and M-MLV reverse transcriptase
(Promega). Using gene-specific primers, PCR products of the
FUT2 coding sequence were generated (primer sequences: 5'-TAA
TAC GAC TCA CTA TAG GGA GAT GCT CAG CAT GCA GGC ATC T-3' and
5'-CCC TCT AGA TCA GTG CTT AAG GAG TGG GGA-3') and cloned into
pCR2.1TOPO (Invitrogen), serving as template for subsequent
PCR where an SP6 promoter was inserted at the 5' end for
in vitro transcription (primer sequences: 5'-ATT TAG GTG ACA CTA
TAG AAG ATG CTC AGC ATG CAG GCA TCT-3' and 5'-AAT TAA CCC TCA
CTA AAG GGA GAT CAG TGC TTA AGG AGT GGG GAC AG-3'; the generated
PCR product has a length of 1067 bp). A 25 µl aliquot
of this PCR product was used as template for a 50 µl
in vitro transcription of
FUT2, using the RiboMAX Large Scale RNA
Production System-SP6 (Promega).
Design of antisense ODNs
The coding sequence of the porcine FUT2 gene (GenBank accession no. AF136895
[GenBank]
) was analyzed by the computer program ‘mfold’ (11,12). The obtained data about predicted single-stranded regions along the FUT2 mRNA were chosen for design of antisense ODNs. These were unmodified phosphodiester 18mers (MWG Biotech).
RNase H digestion and subsequent analysis of ODN–RNA duplexes
A 5 µg aliquot of RNA from the in vitro transcription reactions was incubated with 1 U of RNase H (Promega) and antisense ODNs in varying concentrations for 1 h at 37°C in 1x reaction buffer [40 mM HEPES-KOH pH 8.0, 1 mM dithiothreitol (DTT), 10 mM MgCl2, 100 mM KCl, according to Matveeva et al. (15)]. Samples were electrophoresed through a denaturing polyacrylamide gel [7 M urea, 4.5% total acrylamide (ratio acrylamide:bisacrylamide = 19:1)] and visualized under UV light after staining with ethidium bromide to check the efficacy of RNase H cleavage caused by the various ODNs.
MALDI-TOF mass spectrometry
RNase H reaction products and control samples were desalted on a membrane filtration plate (Multiscreen Seq, Millipore, Bedford, MA) by washing twice with 20 µl of water and twice with 20 µl of 10 mM diammonium citrate. The samples were resuspended in a MALDI-compatible buffer (16) and further digested with 10 U of RNase T1 (LaRoche) at 37°C for 1 h in order to cleave the RNA fragments after each guanine. Then they were spotted on dried matrix droplets (saturated 3-hydroxy picolinic acid in 50% acetonitrile, 10 mg/ml ammonium citrate) on a Scout 384 stainless steel target plate (Bruker Daltonics) and analyzed on a linear Bruker Biflex III delayed extraction MALDI mass spectrometer (record in negative ion mode, 19 kV acceleration voltage, 17.45 kV IS/2 potential, 400 ns extraction delay, no. of shots 120 on average).
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RESULTS
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Design of antisense ODNs
To obtain secondary structure predictions of the porcine
FUT2 mRNA, we applied the computer program ‘mfold’, version
3.1 (
11,
12). We made an input of the
FUT2 coding sequence (1023
bases) and obtained 38 different folding structures calculated
for linear RNA at 37°C, based on free energy minimization.
We looked for sequence segments of eight or more consecutive
single-stranded bases found in parallel in at least 30 of the
38 calculated secondary structures in order to enhance the likelihood
of finding true single-stranded regions. Of the eight sequence
segments which matched these criteria, we selected four by chance,
and designed six antisense ODNs whose sequences were complementary
to the selected regions (Table
1).
Test of RNase H activity induction by specific ODNs
Each antisense ODN was incubated with the
FUT2 in vitro transcript
and RNase H as described in Materials and Methods; the concentration
of the oligonucleotides was 25 µM. After gel electrophoresis
of the samples, no more intact
in vitro transcript was visible
in any of the samples, except a small amount in the case of
ODN1 (Fig.
1). In contrast to ODNs 1, 2, 3 and 5, where distinct
cleavage patterns of the RNA were visible, ODNs 4 and 6 led
to a more complete degradation of the RNA, leaving only a smear
of small RNA fragments. We therefore selected these two antisense
ODNs for further investigation; moreover, we designed sense
control and mismatch control ODNs for both antisense ODNs (Table
2). Hence ODNs 4, 6 and control oligos were again incubated
with
FUT2 RNA and RNase H, and their concentrations were scaled
down in order to reach the point where maximum stringency in
hybridization was obtained as well as activation of RNase H-mediated
cleavage of the transcript. For ODN4, this was the case at a
concentration of 0.025 µM, and for ODN6 at 0.05 µM,
respectively, where the
in vitro transcripts were cut only once
(Fig.
2). How can the generation of these RNA fragments be explained?
Given the hybridization site of ODN4 between bases 460 and 477
of the
FUT2 mRNA (i.e. bases 357–374 of the coding sequence),
and taking into account the additional bases, linked by the
PCR primers, at both ends of the
in vitro transcript, one would
expect two RNA fragments of
380 and 690 bases, assuming that
RNase H cleaved the
FUT2 RNA only once within the hybridization
site of ODN4. In the case of ODN6, there should be two RNA fragments
of
830 and 240 bases. These assumptions are confirmed by the
results shown in Figure
2; however, the exact positions of RNase
H-mediated cleavage of the RNA are still unclear.
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Figure 1. Electrophoretic separation of FUT2 RNA after incubation with RNase H and ODNs (25 µM each). Lane M: 0.15–1.77 kb RNA ladder (Life Technologies). Lanes 1–6: ODN1, 2, 3, 4, 5 and 6, respectively. Lane C: control reaction without RNase H. The weak signal at 400 bases that appears in each lane is due to residual PCR product which was the template for the in vitro transcription.
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Determination of the exact cleavage site of RNase H by MALDI-TOF mass spectrometry
To determine the exact cleavage site of RNase H, the generated
RNA fragments were further analyzed by MALDI-TOF mass spectrometry.
With this technique, resolution is best for short RNA fragments
[up to
100 bases (
17)], and we therefore carried out further
fragmentation of the RNA in a base-specific manner by the use
of RNase T1. RNase T1 cleaves unpaired guanines, thus each specific
RNA sequence will result in a unique fragment pattern. We therefore
digested untreated
FUT2 RNA with RNase T1 in order to generate
a
FUT2-specific fragment pattern. When measured by MALDI-TOF
mass spectrometry, a characteristic peak pattern could be detected.
A change of this peak pattern after additional pre-digestion
of the
FUT2 in vitro transcript by RNase H in the presence of
ODN4 and ODN6, respectively, and subsequent digestion by RNase
T1, could easily be detected by comparison of these mass spectra
(Fig.
3).
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Figure 3. MALDI-TOF mass spectra of FUT2 RNA fragments generated after incubation with RNase H and ODN with subsequent RNase T1 digestion. Reactions were done using ODN4 and ODN6, respectively, as well as ODN4 sense and ODN6 sense; the control reaction was lacking in RNase H. Peak 2099.5 only appeared with ODN4, whereas peak 2421.2 could only be detected with ODN6.
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The main peak pattern characteristic for the
FUT2 in vitro transcript
digested by RNase T1 also appeared in reactions where the transcript
additionally has been pre-incubated with specific ODNs and RNase
H. However, in contrast to the control reaction and reactions
with sense control ODNs, the samples that have been pre-treated
with RNase H in combination with ODN4 and ODN6, respectively,
showed new mass peaks, at
2099.5 Da in the case of ODN4, and
at
2421.2 Da when ODN6 was used. Thus the RNA fragments underlying
these two mass peaks must arise from RNase H-mediated digestion.
From gel electrophoresis of the RNase H digestion products,
we could already estimate that the RNase H cleavage sites are
located nearly at, or even within, the hybridization site of
the respective antisense ODN (Fig.
2). Furthermore, it is described
that RNase H cuts the RNA portion of a DNA–RNA hybrid
within, or immediately adjacent to, the heteroduplex region
(
18); therefore, RNase H cleavage in ODN4 and ODN6 reactions
is supposed to occur within the respective hybridization site.
In order to define RNase H cleavage sites exactly, the
FUT2 RNA fragment pattern resulting from RNase T1 digestion was calculated
by RnaseCut 1.01 software available at our homepage (
http://www.vetmed.uni-muenchen.de/gen/forschung.html)
(
19). RNase T1-mediated digestion generates a 2'–3' cyclic
phosphate intermediate at the 3' end of each digestion product,
which is then transformed into a 2' (or 3') ‘linear’
phosphate; the conversion into the linear phosphate can be forced
by using more enzyme or by prolonged digestion time (16 h),
whereas almost exclusively cyclic phosphate products can be
observed when using the reverse conditions (
16). In order to
save time, we wanted to generate cyclic phosphate products;
therefore, with the conditions we applied, the observed RNA
fragments were 18 Da less in mass because of the guanine with
a cyclic phosphate having a mass of 327.2 Da, instead of 345.2
Da in the case of guanine with a linear phosphate. We therefore
calculated the RNase T1 digestion products with respect to the
detected cyclic phosphate intermediates. Figure
4 compares in
detail the resulting fragments of the
FUT2 RNA after RNase H/RNase
T1 double digestion versus RNase T1 digestion only.
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Figure 4. Cleavage of the FUT2 transcript by RNase H and RNase T1 in comparison with cleavage by RNase T1 only. RNase H hydrolyzes the phosphodiester bonds of RNA hybridized to DNA to produce 5'-P- and 3'-OH-terminated products; RNase T1 cleaves RNA after each guanine, leaving 5'-OH- and 3'-P-terminated products (shown in detail only at relevant cleavage sites). Hybridization sites of respective ODNs are in italics. (A) Generation of a 2100.4 Da RNA fragment after ODN4-induced RNase H cleavage and subsequent RNase T1 cleavage. (B) Resulting RNA fragment of 2420.6 Da when ODN6 was used.
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Figure
4A shows a calculated RNA fragment of 2507.6 Da that
results from digestion of the
FUT2 in vitro transcript by RNase
T1 only. When subtracting the last base and phosphate group
from this fragment, the corresponding mass is reduced to 2100.4
Da. The mass peak that we detected by MALDI-TOF mass spectrometry
in the RNase H reaction after application of ODN4 was 2099.5
Da (Fig.
3), that is why we conclude that incubation with RNase
H and ODN4 leads to cleavage of the
FUT2 in vitro transcript
between bases U464 and G465, leaving a 3'-OH terminated product.
In Figure
4B, the origin of a calculated RNA fragment of 2482.6
Da after RNase T1 digestion of the
FUT2 RNA is shown. If RNase
H cleaves the
FUT2 in vitro transcript in advance between bases
G908 and C909 due to the presence of ODN6, then the 3'-terminal
base G is hydroxylated (instead of being phosphorylated in the
case of RNase T1 digestion), and therefore the mass of this
RNA fragment after subsequent RNase T1 digestion is reduced
to 2420.6 Da (measured: 2421.2 Da, Fig.
3). Thus the sites of
RNase H cleavage induced by ODN4 and ODN6, respectively, could
be analyzed. Supposing that the
FUT2 in vitro transcript was
completely digested by RNase H in the presence of ODN4 and ODN6,
respectively, then the above-mentioned fragments of 2507.6 and
2482.6 Da generated in the control reactions should not be detectable
in the respective ODN MALDI-TOF mass spectra. This is not the
case; by chance, there exist other sequence segments along the
FUT2 RNA that lead to these masses when digested by RNase T1.
In Table
3, the RNA fragments generated by RNase H cleavage
and RNase T1 cleavage are listed within the mass range of interest.
Again the products resulting from RNase T1 digestion have been
calculated by using RNaseCut 1.01 software with respect to the
cyclic phosphate intermediates.
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Table 3. Calculated FUT2 in vitro transcript fragment pattern after RNase H and RNase T1 digestion, sorted according to fragment masses
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DISCUSSION
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Data on the reliability of RNA secondary structure predictions
by computer folding programs are contradictory; some researchers
report problems in finding antisense ODN-accessible sites by
the use of ‘mfold’ (
13,
14), whereas Zuker and Jacobson
(
20) could demonstrate high degrees of correspondence between
structures predicted by this program and known structures under
certain circumstances. They calculated by ‘mfold’
the secondary structures of different rRNAs with well-known
secondary structures, and the identity between predicted and
known structures was in the range of 27–70%. However,
the degree of correspondence improved to 81% when the analysis
was limited to structural domains that were present not only
in the optimal predicted secondary structure, but also in all
suboptimal structures. Thus one should avoid selecting structural
domains with large differences in the calculated suboptimal
structures when searching for reliable secondary structure predictions.
This is why we only considered
FUT2 sequence segments that were
predicted to be single stranded in at least 30 of the 38 different
foldings calculated by ‘mfold’. Single-stranded
loops of hairpins on predicted RNA secondary structures were
shown to be good target sites for antisense ODNs (
15,
21,
22);
that is why we selected only single-stranded regions for the
design of complementary antisense ODNs. As could be demonstrated,
all of the four predicted single-stranded sequence segments
that we selected have been shown to be good target sites for
antisense ODN-mediated RNase H cleavage (see Fig.
1). Thus when
intensifying selection criteria, the use of ‘mfold’
can be very helpful in finding antisense-accessible sites on
a given RNA sequence. The length of the antisense ODNs was fixed
to 18 bases, as this size seems to be a compromise where specificity
and cellular uptake are both satisfactory (
23). As represented
in Figure
1, application of the antisense ODNs at a concentration
of 25 µM led to a more or less complete degradation of
the RNA. Thus at this high concentration, the respective antisense
ODNs hybridized to not only the complementary site, but also
to many other sites of the
FUT2 in vitro transcript, leading
to multiple cleavage events by RNase H. When ODNs 4 and 6 were
scaled down to 0.025 and 0.05 µM, respectively, an optimum
was found where the
FUT2 in vitro transcript was cut at the
appropriate sites only, which was not the case when applying
control ODNs (see Fig.
2). Hence, at these concentrations, maximum
specificity could be reached.
In order to determine the exact cleavage sites produced by RNase H, we decided to use MALDI-TOF mass spectrometry. Knowledge of the cleavage site is of great importance when RNase H activity is to be maintained after chemical modification of the antisense ODN, which is a necessary step when in vivo application is desired. Normal phosphodiester oligonucleotides are readily degraded by nucleases when introduced into a biological system. The most commonly used modified oligonucleotides are phosphorothioate oligonucleotides; they are resistant to nucleolytic attack and support RNase H activity, but they have been shown to produce non-specific toxicity in both primates and rodents (24) and do not bind their target mRNA as tightly as unmodified oligonucleotides do (25), and thus one might choose other chemical modifications. Among these are new generation chemistries such as morpholino phosphorodiamidates (MFs) and 2'-O-methoxyethylribonucleotides (MOEs) that are resistant to nucleases and have a high affinity to hybridize to their target mRNA, but do not support RNase H-mediated cleavage of the DNA–RNA duplex (26,27). It is therefore advisable to develop gapped or chimeric oligonucleotides that contain a mixture of chemistries that allow for high affinity hybridization, resistance to nucleolytic attack and support of RNase H activity [as illustrated in Golden et al. (28)].
A common strategy to analyze the site of RNase H-mediated cleavage is to reverse-transcribe the resulting RNA fragments in a primer extension reaction with a labeled primer and resolve the newly synthesized fragments via electrophoresis (15,29,30). This is a troublesome and to some extent misleading method; strong RNA secondary structures can cause premature stops of reverse transcription reactions, thus generating artifactual fragments that do not result from RNase-mediated digestion. Hansen et al. (31) directly compared the applicability of primer extensions and MALDI-TOF mass spectrometry for the analysis of RNA. They searched for post-transcriptional modifications of 23S rRNA of diverse organisms. The detection of modifications by primer extension was of limited reliability because of premature termination of reverse transcription, probably due to very stable RNA secondary structures. In contrast, analysis of RNase-generated RNA fragments by MALDI-TOF mass spectrometry reliably detected almost all types of modifications, regardless of secondary structure. Thus they could demonstrate that MALDI-TOF mass spectrometry was much more suitable than primer extension for analyzing RNA. The unambiguous and easily practicable determination of RNase-generated RNA fragments by applying MALDI-TOF mass spectrometry has already also been validated by others (16,32–34). After RNase-mediated cleavage, the generated RNA fragments are directly analyzed; therefore, there is no fault-prone need to copy them. Moreover, analysis by MALDI-TOF mass spectrometry is more exact than by electrophoresis, as mass accuracy is high enough not only to determine the length of the sequence segment but also to draw conclusions about the sequence composition as the four bases differ in their masses (16). When applying MALDI-TOF mass spectrometry, most investigators use sequence-specific RNases for the degradation of the RNA (16,31,33,34). However, Hansen et al. (31) applied the non-sequence-specific RNase H in order to generate RNA subfragments of a smaller size suitable for subsequent digestion by sequence-specific RNases; they report the appearance of some unexpected peaks due to heterogeneity of the RNase H cleavage, indicating that their DNA oligonucleotides inducing RNase H-mediated cleavage have not been optimized in sequence and concentration. Optimization was not necessary because determination of RNase H-induced cleavage sites was not the purpose of their study. In contrast, Polo and Limbach (32) wanted to evaluate the efficiency and specificity of cleavage using RNase H. Therefore, they produced an oligoribonucleotide hairpin of 19 nt length and incubated it with RNase H as well as two different chimeric oligonucleotides [5'-r(NNN)d(NNNN) r(NNN)-3']. The design of the chimeric oligonucleotides allows for a maximum number of four possible cleavage sites as RNase H recognizes only DNA–RNA hybrids; this restriction and the small size of the hairpin enable analysis of expected RNA fragments by MALDI-TOF mass spectrometry without the need of further treatment. However, in the context of searching for antisense ODN-accessible sites along a given RNA, the technique described by Polo and Limbach is not sufficient. In order to detect antisense sequences that can induce RNase H-mediated cleavage in vivo, the transcript must be synthesized in full length to form the same secondary structure as under physiological conditions. This is why we perform cleavage of the full-length transcript by RNase H under native conditions, and thereafter degrade the transcript in a sequence-specific manner by RNase T1, thus producing small RNA subfragments that can easily and unambiguously be analyzed by MALDI-TOF mass spectrometry. Therefore, with the technique presented by us, the analysis of RNA sites that are accessible to antisense ODN and RNase H in vivo by using MALDI-TOF mass spectrometry succeeded for the first time. Given the convincing results, this approach should become an attractive tool for other users of antisense technology.
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ACKNOWLEDGEMENT
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The authors thank Tierzuchtforschung e.V. München for supporting
this project.
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