A biotin-conjugated pyridine-based isatoic anhydride, a selective room temperature RNA-acylating agent for the nucleic acid separation

Article abstract of DOI:10.1039/c4ob02636e

Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic acid separation. 2′-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-coated magnetic beads and disulfide chemica

Full text of DOI:10.1039/c4ob02636e

Organic &
Biomolecular Chemistry
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PAPER
A biotin-conjugated pyridine-based isatoic
anhydride, a selective room temperature
RNA-acylating agent for the nucleic acid separation†
Cite this: Org. Biomol. Chem., 2015,
3, 3625
1
a,b
a,b
c
c
a,b
a,b
S. Ursuegui, R. Yougnia, S. Moutin, A. Burr, C. Fossey, T. Cailly,
c
c
a,b
A. Laayoun, A. Laurent* and F. Fabis*
Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic
acid separation. 2’-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-
coated magnetic beads and disulfide chemical cleavage led to isolation of highly enriched RNA samples
from an initial 9/1 DNA–RNA mixture. Starting from the parent compound N-methylisatoic anhydride A
which was used at 65 °C, we improved the extraction process by designing a new generation of isatoic
anhydrides that are able to react under smoother conditions. Among them, a pyridine-based isatoic
anhydride derivative 15f was found to be reactive at room temperature, leading to enhance the efficiency
and selectivity of the extraction process by significantly reducing DNA side extraction. The extracted and
purified RNAs can then be detected by RT-PCR.
Received 19th December 2014,
Accepted 26th January 2015
DOI: 10.1039/c4ob02636e
www.rsc.org/obc
isolate RNAs from biological samples. The primary procedure
used in molecular biology laboratories is based on an extrac-
Introduction
Extraction and purification of nucleic acids from a biological tion process using a guanidinium thiocyanate–phenol–chloro-
4
a,b
sample, called Sample Prep, is a crucial step in molecular diag- form mixture.
nostics. This process is essential to remove interfering sub- to add a DNase step to remove residual genomic DNA before
The use of organic solvents and the necessity
1
a,b
5a,b
stances and isolate nucleic acids of satisfactory quality.
The analysis by RT-PCR
makes the method time-consuming
majority of commercial methods involve the use of silica and very difficult to automate. New methods to isolate pure
2
matrices, referred as “Boom chemistry”. With high chaotropic RNA from biological samples have thus recently been develo-
salt concentration, nucleic acids can bind silica and thus be ped. For example, liquid chromatography, and particularly
separated from other cellular components. This technique arginine matrices, have recently been applied to RNA isolation
6
which led to the automation of the Sample Prep step does not from eukaryotic cells. Besides separation processes based on
allow selective extraction of each nucleic acid target. Regarding physical properties of nucleic acids, chemical methods based
RNA, the contamination by predominant genomic DNA is the on selective nucleic acid functionalization could be an alter-
cause of many issues for RNA detection and analysis. Purity native. Reversible boronic ester formation between boronic acids
and integrity of RNA are two critical parameters for many and the cis-diol group of ribose has been used for ribonucleo-
3
applications based on RNA detection by RT-PCR.
side, oligoribonucleotide and RNA purification. Boronic acids,
7
The close chemical and physical properties of the two and particularly phenyl boronic acid-modified cellulose and
nucleic acid targets make the specific RNA extraction an intri- polyacrylamide
8
a,b
have been developed for this application.
cate process, and several methods have been developed to However, this method was not evaluated for the nucleic acid
9
separation from biological samples.
We have recently described a biotin-conjugated N-methyl
isatoic anhydride A (NMIA), designed to purify RNAs by selec-
tive 2′-OH acylation of ribose, capture by streptavidin-coated
a
Normandie Univ, France. E-mail: frederic.fabis@unicaen.fr
b
Université de Caen Basse-Normandie, Centre d’Etudes et de Recherche sur le
Médicament de Normandie (EA 4258 - FR CNRS 3038 INC3M - SF 4206 ICORE) UFR
des Sciences Pharmaceutiques, Bd Becquerel, F-14032 Caen, France
magnetic beads and release through a disulfide bond cleavage
1
0
(Fig. 1). This reagent allowed the selective extraction of RNA
from a DNA–RNA mixture, and the extracted RNA can be suc-
cessfully amplified by RT-PCR. However, the conditions of acy-
lation used to capture significant amounts of RNAs (65 °C at
1.5 mM of A) led to the presence of residual DNA. In order to
c
BioMérieux SA, Centre Christophe Mérieux, Parc Polytech, 5 rue des berges,
38024 Grenoble, France. E-mail: alain.laurent@biomerieux.com
†
Electronic supplementary information (ESI) available: Detailed experimental
procedures and characterization of new compounds. See DOI: 10.1039/
c4ob02636e
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Scheme 1 Synthesis of isatoic anhydride 3b.
monoester 1 which was subsequently treated with phosgene
and N-methylated with iodomethane to give 3b in 58% overall
yield (Scheme 1).
Fig. 1 Biotin-isatoic anhydride conjugated designed for RNA selective
extraction.
IA derivatives 3b–3i were then tested toward acylation of
short synthetic 27-nt RNAs and the results were compared to
the parent NMIA 3a. According to our previously reported
methodology, an RNA oligomer was incubated with isatoic
anhydride derivatives 3a–3i in a DMSO–TEAAc mixture (25/75,
pH7) at 65 °C for 1 h. After removing the tag excess, RNA oligo-
mers were subjected to an enzymatic hydrolysis and the result-
ing mixture of nucleosides was analyzed by HPLC. The ratios
of tagged nucleosides vs. non-tagged were determined and the
efficiency of the acylation process was compared for each IA
derivative. Results are depicted in Table 1.
improve the selectivity of the acylation step, and hence
decrease the ratio of extracted DNA, we were interested in the
development of more reactive isatoic anhydride (IA) derivatives
which could be able to react in smoother conditions to avoid
side reactions with DNA, while maintaining or improving the
efficiency for RNA extraction.
The enhancement of reactivity of IA derivatives toward
2
′-OH RNA acylation being the result of the higher electrophilic
1
1
character of the anhydride carbonyl group, we were able to
design more reactive RNA-acylating agents after a primary
screening of IA derivatives. Among them, we found that a pyri-
dine-based isatoic anhydride 15f was able to react with RNA at
room temperature, leading to a better selectivity in the extrac-
tion protocol compared to the parent compound A.
As expected, the introduction of electron donating groups
(
3i) or the π-excessive thiophene ring (3h) led to a decrease of
the acylation ratio, whereas the IA derivatives containing an
electron withdrawing group at position 7 (3b–d) led to an
increase of this ratio compared to 3a. When electron withdraw-
ing groups were placed at position 6 (3e–f), the reactivity was
similar to that of compound 3a. All these results are in accord-
ance with the electronic effects of substituents in aromatic
Results and discussion
IA derivatives bearing electron withdrawing groups (3b–3f) series. The electrophilic character of the reactive carbonyl
were synthesized in order to improve RNA selective acylation group of IA is indeed increased when strong electron with-
and were compared to the parent NMIA (3a) and to an isatoic drawing groups are placed at the para position. Interestingly
anhydride containing electron donating groups (3i). Hetero- the introduction of a pyridine (compound 3g) led to acylation
cyclic analogues containing a π-deficient pyridine (3g) or a ratios similar to those obtained for the 7-carboxy derivative 3b,
π-excessive thiophene ring (3h) were synthesized for comparison. respectively 9.7 and 9.4%. For these two compounds, we inves-
Except for compound 3b bearing a 7-carboxy group, all the tigated the acylation process at room temperature. Under these
anhydrides 3c–i were obtained using literature methods conditions 8-aza-NMIA 3g remained reactive, leading to a
1
0–15
(Fig. 2).
The synthesis of anhydride 3b was achieved slight decrease of the acylation ratio (7%) whereas the reactiv-
through a three-step sequence starting from 2-aminotereph- ity of the parent NMIA 3a dramatically decreased to only 1.3%
thalic acid. The selective esterification of carboxylic acid at
position 4 using chlorotrimethylsilane in methanol led to the
Table 1 Synthetic 27-nt RNA tagging with 3a–i
a
a
Tagged nucleosides
(%) at 65 °C
Tagged nucleosides
(%) at 25 °C
Tag
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
6.1
9.7
8.1
7.1
6.1
6.6
9.4
2.0
2.0
0.7
1.3
b
nd
b
nd
b
nd
b
nd
7.0
b
nd
b
nd
a
a
b
Fig. 2 Selected isatoic anhydride 3a–i. NMIA (N-MethylIsatoic-
Ratio determined by LC/MS, see experimental data for details. nd:
Anhydride) is commercially available.
non-determined.
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Fig. 3 7-Carboxy and 8-azaisatoic anhydride derivatives 15a–f functionalized for RNA selective extraction.
of acylated nucleosides. Despite the higher reactivity at 65 °C reaction with methylacrylate, using a combination of Pd(OAc)
2
of 7-carboxyNMIA 3b, this compound was not more efficient and PPh . The catalytic hydrogenation of the alkene moiety fol-
3
than NMIA at room temperature with only 1.3% of acylated lowed by the selective hydrolysis of the methylester with
nucleosides. In these experiments at room temperature, we lithium hydroxide led to the azaisatoic precursor 11 in a 41%
observed that compound 3b remained poorly soluble in the overall yield over 6 steps (Scheme 3).
reaction mixture, whereas the pyridine derivative 3g showed
The disulfide linker was introduced through a cystamine
good solubility. These results highlighted the great importance for the pyridine and the 7-carboxamidoisatoic derivatives.
of water solubility for an efficient RNA acylation. We then Cystamine was first protected as its mono-N-Boc derivative I
1
6
selected the pyridine and the 7-carboxy derivatives and syn- according to the procedure described by Niu J. et al. For the
thesized the biotin-peg -disulfide-conjugated compounds to ester of 7-carboxy derivative, we prepared a dissymmetric di-
4
further study their use in the RNA extraction and purification. sulfide linker II bearing a hydroxyl group from 2-mercaptoethyl-
The lower reactivity of the 7-carboxy derivative should be com- amine according to the procedure described by Nemmani K.V.S.
1
7
pensated by the introduction of a hydrophilic peg linker on et al. The two carboxylic acids 5 and 11 were activated
the structure leading to more water-soluble compounds.
The chemical strategy to introduce the biotin-peg
using EDC and HOBt in DCM. The addition of the disulfide
-disulfide linkers I and II led to the formation of the corresponding
4
moiety was the same as previously described for the parent amides 12b–c and ester 12a which were deprotected with TFA
NMIA A. For the 7-carboxy derivative, the biotin conjugate was in DCM to give the free amines 13a, 13b and 13c (Scheme 4).
directly coupled to the carboxylic acid group through either an The amines 13a–c were then conjugated to a biotin activated
4
amide or an ester bond whereas the pyridine ring was pre- as N-hydroxysuccinimide ester and a biotin-peg activated as
viously functionalized with a propanoyl chain. The chemical a pentafluorophenyl ester. The resulting biotinylated deriva-
synthesis required the preparation of the tert-butylaminoester tives 14a–f were obtained in yields ranging between 70 and
precursors 5 and 11 (Fig. 3). Each conjugated anhydride was 90%. The final anhydride derivatives 15a–d were obtained by
prepared with and without the peg linker in order to compare treatment with phosgene in DCM from the corresponding
4
the influence of hydrosolubility on the RNA acylation reaction. 7-carboxy aminoesters 14a–d in 50–85% yields. For the pyridine
Starting from 7-carboxyNMIA derivative 3b, tert-butylaminoe- anhydrides 15e–f, DCM was replaced by diethyl ether as the
ster 4 was obtained by opening the oxazinone ring with solvent, and the addition of triethylamine was necessary to
sodium tert-butoxide (Scheme 2). The latter compound was avoid protonation of the basic 2-aminopyridine ring. With this
then subjected to selective hydrolysis by the methyl ester procedure, the non-pegylated anhydride 15e was isolated in
group using lithium hydroxide at room temperature leading to 50% yield. Starting from the pegylated aminoester 14f, the
the 7-carboxy aminoester precursor 5 in a good overall yield.
2
formation of non-soluble aggregates in Et O hampered the
The synthesis of 8-aza aminoester precursor 11 began with cyclization reaction. The anhydride 15g was obtained in
the protection of the acid as its tert-butyl ester followed by a 25% yield after previous absorption of 14f on C -RP-silica
18
SNAr with methylamine to give 7 in good yield. The propionyl before treatment with phosgene in the presence of TEA
chain was then introduced following a selective electrophilic (Scheme 5). Starting from 2-aminoterephthalic acid, 7-carboxy
iodination at position 5 and a subsequent Heck cross-coupling anhydrides 15a–d were successfully obtained in 9 steps with
overall yields of 24, 28, 18 and 38%, respectively. In a similar
manner, azaisatoic anhydrides 15e–f were obtained in 10 steps
starting from 2-chloronicotinic acid in 11 and 10% yields
respectively.
We first evaluated the IA derivatives 15a–15f with synthetic
nucleic acids, a 27-nt oligoribonucleotide and a 27-nt oligo-
Scheme 2 Synthesis of t-butylaminoester 5 (isolated yields).
desoxyribonucleotide, using the same procedure described above
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Scheme 3 Synthesis of t-butylaminoester 11 (isolated yields).
Scheme 4 Synthesis of cystamine derivatives 13a–c (isolated yields).
Scheme 5 Synthesis of RNA tags 15a–f (isolated yields).
for the screening of compounds 3a–i. The percentages of tages ranging between 8.7 and 14.1%; the best results being
tagged nucleosides were determined respectively at 65 °C and obtained with aza compounds 15e and 15f with respective
at room temperature and compared to the results obtained for percentages of tagged nucleosides of 14.1 and 13.5%. These
the compound of first generation A. The selectivity of the acyla- observations are in accordance with results previously
tion reaction was determined as the ratio of tagged nucleo- obtained in the screening. Esters 15a–15b and amides 15c–15d
sides with both oligonucleotides (Fig. 4). At 65 °C, the exhibited the same reactivity, showing that the nature of the
percentage of tagged nucleosides for A was calculated to be linkage on the carbonyl group has no influence. When com-
7
1
.5% with RNA and 0.7% for DNA, giving a selectivity ratio of paring the results between pegylated and non-pegylated com-
0.7. The 7-carboxy and 8-azaisatoic anhydrides 15a–f were pounds no significant differences were observed in the
found to be more reactive toward RNA with a tagging percen- acylation ratios. These results can be explained by the good
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result from the combination of two effects. First, the lower reac-
tivity of the 3′-OH group compared to the 2′-OH group as demon-
strated by Weeks et al. with the 2′-deoxyATP being 10-fold less
18
reactive than 3′-deoxyATP would lead to a kinetic selectivity
favoring thus acylation of the more reactive 2′-OH group of RNA.
Secondly, the higher susceptibility of the more electrophilic
11
isatoic derivatives to hydrolysis in the reaction medium would
lead to the rapid inactivation of their acylating properties, limit-
ing thus the acylation of the less reactive 3′-OH of DNA.
We then selected the more reactive azaisatoic anhydride 15f
for further evaluation in the RNA extraction process using HIV
RNA transcripts and calf genomic DNA as biological nucleic
acid models.
The RNA extraction process was first applied to HIV RNA
transcript and compared to calf genomic DNA. The separation
efficiency was then studied using a mixture of the two nucleic
acids. Nucleic acids were incubated with A or 15f at two con-
centrations (1.5 and 30 mM). After removing the tag excess
using magnetic silica particles, biotinylated nucleic acids were
captured with streptavidin-coated magnetic beads and eluted
after disulfide cleavage with dithiothreitol (DTT). The eluted
nucleic acids were then quantified by fluorescence and extrac-
Fig. 4 Synthetic 27-nt RNA and 27-nt DNA (8 nmol) tagging with tion yields were determined for both RNA and DNA. The
a
3
0 mM of isatoic anhydride derivatives 15a–f at 65 °C and 25 °C. Ratio selectivity of the capture was evaluated by calculating the ratio
determined by LC/MS, see experimental data for details.†
of RNA/DNA extraction yields (Fig. 5). The control experiment
performed with HIV RNA transcripts and calf genomic DNA
solubility of all these compounds at 65 °C. No difference was without tag led to extracted yields below the detection limit.
observed in the selectivity between RNA and DNA acylation Non-specific interactions between streptavidin magnetic beads
with the parent compound A. Indeed all the ratios were found and nucleic acids appeared to be negligible. The compound of
to be between 10 and 12. We then turned our attention to the first generation A was evaluated at room temperature and gave
acylation reaction at room temperature. Compound A exhibited significant extraction yields only using high concentration of
a very low reactivity with only 1.0% of RNA nucleosides acyl- tag (22% of extracted RNA at 30 mM), whereas using 1.5 mM
ated. For the carboxy derivatives, the esters and amides were only led to very low extraction yields (<5%). These results are
found equally reactive, with the non-pegylated compounds 15a consistent with the low reactivity observed for A toward an
and 15c acylating 1% of RNA nucleosides, whereas the pegy- RNA oligomer at room temperature. At 65 °C this derivative
lated derivatives 15b and 15d were found to be quite more was more efficient allowing RNA extraction with the lower tag
reactive than the parent compound A, underlining that both concentration (27% of extracted RNA at 1.5 mM). The use of
the intrinsic reactivity and the aqueous solubility of IA deriva- higher tag concentration led to an increase in the yield (66% at
tives are two critical parameters to perform an efficient acyla- 30 mM) but in this case a six-fold decrease of selectivity was
tion of RNA nucleosides. These results were confirmed with observed (extracted RNA/DNA ratio: 57 at 1.5 mM vs. 10 at
aza-compounds 15e and 15f which were able to acylate RNA 30 mM). With the 8-azaisatoic derivative 15f RNA extraction
nucleosides in high ratios of 4.8 and 7.8% respectively. The yield of 24% was obtained at the lower concentration of
non-pegylated aza-derivative 15e was more reactive than the 1.5 mM. At this low concentration, 15f appeared to be as
non-pegylated carboxy-compounds and as effective as the pegy- efficient at room temperature as A at 65 °C. Additionally, a
lated ones 15c and 15e. The pegylated aza-anhydride 15f clear increase of selectivity was observed when using azaisatoic
exhibited the best acylating properties, matching with its 15f at room temperature (extracted RNA/DNA ratio of 158 at
higher water solubility. The acylation ratio between RNA and 1.5 mM) compared to A at 65 °C (extracted RNA/DNA ratio of
DNA nucleosides was calculated to be around 15.5 for the 57 at 1.5 mM) with similar RNA extraction yields. Interestingly
three pegylated compounds 15b, 15d and 15f, which rep- increasing the temperature of the acylation step at 65 °C with
resents an increase of selectivity of 50% compared to all experi- 15f at the lower concentration of 1.5 mM did not result in
ments performed at 65 °C.
improved RNA extraction yields (21% at 65 °C vs. 24% at
These preliminary results with oligonucleotides validated 25 °C), but only to a two-fold decrease of the selectivity ratio
the interest in developing room-temperature RNA acylating (extracted RNA/DNA ratio of 79 at 65 °C vs. 158 at 25 °C). At
reagents to gain a higher selectivity. It could be postulated that higher concentration of 15f (30 mM), the extracted RNA yield
this higher selectivity for RNA vs. DNA at room temperature was significantly increased at both room temperature and
with more electrophilic isatoic anhydride derivatives would 65 °C (62% and 57%, respectively).
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a
Fig. 5 Extraction of biological NA using A and 15f. Extraction yields determined using fluorescence spectroscopy, see experimental data
for details.†
Unfortunately as previously observed with A, a decrease in acids with a resulting mixture containing 86% of RNA after the
the selectivity ratio was observed (42 at 25 °C, 9 at 65 °C). extraction process leading to a selectivity of 5. Performing the
Taken together these results validate the hypothesis that sec- acylation step at room temperature with 8-azaisatoic derivative
ondary reactions with DNA can be minimized using acylating 15f increased the selectivity of the process to 28 by lowering
agents able to react under milder conditions.
the quantity of extracted DNA. A strong RNA enrichment was
Compound 15f was further evaluated for its ability to separ- achieved with 15f and the RNA/DNA ratio reached 95/5 starting
ate the two nucleic acids starting from a mixture of RNA–DNA from an initial mixture containing 90% of DNA. Nevertheless,
containing 10% of RNA and 90% of DNA in order to consider when we compared the selectivity obtained for the mixture of
the predominance of DNA in biological samples. The acylation nucleic acids with the selectivity obtained when RNA and DNA
step was performed with the lower tag concentration of are extracted separately, a strong decrease of selectivity was
1
.5 mM at room temperature and compared to the parent com- observed (28 vs. 158). This decreasing selectivity was probably
pound A at 65 °C. The selectivity of the separation process was due to interactions between the captured RNA and non acyl-
evaluated by spectrofluorescence quantification of the two ated genomic DNA but not to enhancement of the DNA
nucleic acids after capture and elution (Table 2). At 65 °C, the acylation.
use of isatoic anhydride A reversed the ratio of the nucleic
To prove this hypothesis, the RNA HIV transcript was acyl-
ated using derivative 15f at 30 mM. After removing the tag
Table 2 RNA extraction from biological RNA–DNA mixture using A
and 15f
Table 3 Co-extraction of non-acylated DNA and captured RNA
Tag at
.5 mM
Temperature
(°C)
RNA/DNA a
initial ratio
RNA/DNA
final ratio
RNA/DNA
Selectivity
a
b
1
Tag at
0 mM
Temperature
(°C)
% of RNA
extracted
% of DNAa
recovered
a
3
A
65
25
10/90
10/90
86/14 ± 1
95/5 ± 1
5 ± 0.9
28 ± 1.7
15f
15f
25
42.0
2.1
a
a
Determined using fluorescence spectroscopy (n = 2), see experimental
Determined using fluorescence spectroscopy, see experimental data
for details.
b
data for details. Ratio determined from RNA and DNA extraction yields.
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Fig. 6 RT-PCR of transcript HIV extracted with azaisatoic anhydride derivative 15f (for control experiments using non-acylated RNA and electro-
phoresis of RT-PCR amplicons, see ESI†).
excess, genomic DNA was added to the mixture before the tron withdrawing groups or using a pyridine analog of isatoic
capture step with streptavidin magnetic beads. After DTT treat- anhydride, led to an increase in the reactivity of these com-
ment and elution, RNA and DNA were quantified by fluo- pounds toward RNA and allow the efficient capture and recov-
rescence. As we had previously checked the absence of non- ery of RNA at room temperature, when the compound of first
specific interaction between magnetic beads and nucleic acid, generation A was only efficient at elevated temperatures. Due
no significant amounts of DNA should be detected. In con- to its higher water solubility, the pyridine derivative 15f was
trast, 2.1% of the initial quantity of DNA was recovered after selected and was able to extract RNA starting from a RNA–DNA
the extraction process, demonstrating clearly that the presence mixture with a higher selectivity than the previous compound
of residual DNA was due to co-elution of non-acylated DNA A. The residual quantity of DNA detected after the extraction
with captured RNA and not to DNA acylation (Table 3).
process, was mainly due to interaction of non-acylated DNA
As the amplification of RNA by RT-PCR remains a prerequi- with captured RNA and not to DNA acylation. Moreover, we
site for detection of low RNA concentrations in molecular have confirmed the compatibility of this extraction technique
biology and notably for diagnostic applications, we had pre- with the amplification of RNA for detection applications.
viously shown that RNA captured by our method with com- Besides its use for the selective extraction of RNA, this new
pound A could be amplified even with low concentration of room-temperature efficient RNA-acylating agent could find
1
0
extracted RNA (50 copies). We then evaluated the RT-PCR wider applications in the field of bioconjugate chemistry or
amplification of RNA obtained with compound 15f. RNA HIV chemical biology.
transcripts were first acylated by 15f at 15 mM, and after
capture and elution, RNAs were diluted to respectively 50, 100
and 1000 copies before performing the RT-PCR amplification
Acknowledgements
(Fig. 6a). In accordance with previous results obtained with
compound A, RNA amplification, after capture with 15f, led to
the positive HIV transcript detection, even with the lower con-
centration of 50 copies. In order to evaluate the limit of the
extraction method, we performed the same experiment using
This work was supported by the French public agency OSEO
Advanced Diagnostics for New Therapeutic Approaches, a
French government-funded program dedicated to personalized
medicine), bioMérieux Laboratories and the “Conseil Régional
de Basse-Normandie”.
(
1
5f at a high concentration of 180 mM. In this case, a positive
detection was only obtained with high RNA concentrations of
000 copies (Fig. 6b). It can be postulated that using a high
1
concentration of tag increased the probability of acylation
inside the RT-target sequence, stopping then the reverse tran-
scriptase on 2′-O-adducts as reported by Weeks in the SHAPE
protocol. Nevertheless, in the extraction process, we used a
low concentration of tag (1.5 mM) at which no inhibition of
the amplification process should be observed.
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1
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