Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates

Article abstract of DOI:10.1039/c8ob01498a

We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.

Full text of DOI:10.1039/c8ob01498a

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: N. Milisavljevic, P.
Perlikova, R. Pohl and M. Hocek, Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB01498A.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Page 1 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and
comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside
triphosphates as substrates
Nemanja Milisavljevič,1,2 Pavla Perlíková,1 Radek Pohl,1 Michal Hocek1,2*
1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610, Prague 6, Czech Republic. E-
mail: hocek@uochb.cas.cz; Tel: +420 220183324.
2Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8,
CZ-12843 Prague 2, Czech Republic.
Abstract:
We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-
deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or
dibenzofuryl groups through cross-coupling reactions of nucleosides followed by
triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside
triphosphates. We systematically studied the influence of the modification on the efficiency of T7
RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and
CTP analogues bearing smaller modifications were good substrates and building blocks for the
RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky
dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In case
of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator
needed to be used to get efficient RNA synthesis. The T7 RNA polymerase synthesis of modified
RNA can be very efficiently used for synthesis of modified RNA but the method has constraints
in sequence of the first three nucleotides of the transcript, which must contain a non-modified G
in the +1 position.
1

Organic & Biomolecular Chemistry
Page 2 of 19
View Article Online
DOI: 10.1039/C8OB01498A
TOC graphics
Synthesis and systematic study of T7 RNA polymerase incorporation of base-modified
ribonucleotides for the enzymatic synthesis of modified RNA.
2

Page 3 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
Introduction
Studies toward detailed understanding of the structure and many diverse biological functions of
nucleic acids require the use of chemically modified DNA or RNA probes.1 In addition to
classical chemical synthesis of oligonucleotides on solid support,2,3 enzymatic methods for
synthesis nucleic acids play an important role.4-7 The polymerase catalyzed synthesis of DNA
using modified 2'-deoxyribonucleoside triphosphates (dNTPs) is very well established4-6 and
there are many commercially available DNA polymerases capable of incorporation of modified
nucleotides as well as many diverse methodologies for construction of single- or double-stranded
DNA bearing one, several or many modifications.6 On the other hand, both chemical and
enzymatic synthesis of modified RNA is more problematic and less well established,7-9 despite
the extensive current efforts in the use of modified RNA probes for imaging, chemical biology
and therapeutic applications.6,9,10-16 The enzymatic production of modified RNAs can be based
either on the RNA polymerase catalyzed synthesis using modified ribonucleoside triphosphates
(NTPs) or on enzymatic posttranscriptional modifications of RNA (e.g. alkylation by
methyltransferases etc.).6 Posttranscriptional chemical modifications through bioorthogonal
chemistry is another alternative approach to modified RNA.17,18
The enzymatic synthesis of modified RNA almost exclusively relies on the use of
bacteriophage T7 RNA polymerase to avoid the need of complex transcription factors. The T7
RNA polymerase requires specific promoters19-21 and presence of guanosines in the +1 and/or +2
positions to ensure efficient transcription initiation.22 The known examples of polymerase
synthesis of modified RNAs include the incorporation of useful functional groups, i.e. biotin for
affinity probes,23,24 alkyne or azido groups for click reactions,25,26 5-vinylU for further chemical
modifications,27 5-iodoU for posttranscriptional cross-coupling modification,28 amino acid-like
side chains for selection of aptamers,29-31 diazirine for cross-linking32 or fluorophores.33-37 In vast
majority of cases, the modification was attached at position 5 of uridine triphosphate (UTP)
because of easy synthetic access and because the modification at this position are not much
perturbing the structures of RNA duplexes. Only scattered few examples were published on
incorporation of modified cytidines.38-41 Modified adenine NTPs were never studied
systematically and just a few examples of successful incorporation of 7-aryl-42 and 7-ethynyl-7-
deazaadenine43 or -8-aza-7-deazaadenine44 nucleotides were reported. In addition, fluorescent
analogues of NTPs (including GTP)45 and even formylpyrrole46-48 or thioisocarbostyryl49,50
3

Organic & Biomolecular Chemistry
Page 4 of 19
View Article Online
DOI: 10.1039/C8OB01498A
nucleobase surrogates, extending the genetic alphabet, were successfully incorporated by T7
RNA polymerase. Apart from base-modified NTPs, also several examples of 2'-sugar-modified
NTPs were reported51 to be substrates for T7 RNA polymerase. Despite those many excellent
works dealing with incorporation of specific modified nucleotides (mostly 5-substituted uracils or
non-natural bases), there was no systematic study and comparison of substrate activity of NTPs
bearing different modifications at pyrimidine or 7-deazapurine bases. Since the enzymatic
synthesis of base-modified RNA is an important and underexplored area, such systematic study
may facilitate further use of this approach. Therefore we synthesized a small library of NTPs
(derived from all four bases) bearing modifications of different bulkiness and systematically
studied them as substrates for T7 RNA polymerase.
Results and Discussion
Chemistry
In order to study the influence of substituents on incorporation of modified nucleotides by T7 RNA
polymerase, we designed a small library of substituted NTPs (NRTP). The 7-deazapurine NTPs
were substituted at position 7 whereas the pyrimidine NTPs at position 5. The substituents were
chosen with increased bulkiness: methyl (for deazapurines only), ethynyl, phenyl, 2-benzofuryl
and dibenzo[b,d]furan-4-yl. The synthesis (Scheme 1) was based either on preparation of the
corresponding modified nucleosides followed by triphosphorylation,52 or on the direct Suzuki-
Miyaura cross-coupling modifications of iodinated NTPs.53 The 7-methylation of 7-iodoadenosine
(unprotected AI) and -guanosine (N2-pivaloyl-2',3',5'-tribenzoyl protected GI) was performed
through Pd-catalyzed reactions with trimethylaluminum (Scheme 1A). The Sonogashira cross-
coupling reactions of iodinated nucleosides with TMS-acetylene followed by desilylation was used
to introduce ethynyl group and the Suzuki reaction for attachment of the aryl groups. The
triphosphorylation of nucleosides was performed according the literature procedure52 using POCl3
in PO(OMe)3, followed by reaction with pyrophosphate and triethylammonium bicarbonate
(TEAB) to give the desired NRTPs in moderate to good yields (Scheme 1B). In case of synthesis
of GETP, the triphosphorylaton was performed with silylated nucleoside GTMSE and the
concomitant desilylation occurred during the work up. Finally, most of the aryl derivatives of ATP,
UTP and CTP were prepared by the direct aqueous Suzuki coupling of iodinated NITPs with the
4

Page 5 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
corresponding arylboronic acid using Pd catalyst and triphenylphosphine-3,3',3"-trisulfonate
(TPPTS) ligand.53 The desired NRTPs were obtained in good yields of 31-75% (Scheme 1C).
Scheme 1. A) Synthesis of modified nucleosides, B) triphosphorylation of nucleosides and C)
cross-coupling reactions on NTPs. Reagents and conditions: (i) HMDS, 40°C, AlMe3, Pd(PPh3)4,
THF, 80°C, 2 h; (ii) NaOMe/MeOH, 80°C, 16 h; iii) TMS-acetylene, Pd(PPh3)4, CuI, DMF, r.t.,
16 h; (iv) R-B(OH)2, Pd(OAc)2, TPPTS, H2O/ACN (2:1), Cs2CO3 100°C, 2 h for nucleosides or 1
h for NTPs; (v) R-B(OH)2, Na2PdCl4, TPPTS, H2O, 60°C, 1 h.
5

Organic & Biomolecular Chemistry
Page 6 of 19
View Article Online
DOI: 10.1039/C8OB01498A
Biochemistry
All the synthesized NRTPs were then systematically tested as substrates for T7 RNA polymerase.
Since all the literature examples of T7 RNA polymerase syntheses of modified RNAs use specific
promoters containing guanosines in the +1 and/or +2 positions,19-22 for the testing of incorporation
of modified ARTPs, URTPs and CRTPs, we also designed DNA templates containing the promoter
and three guanosines in the +1-+3 position.54 The 5’-terminal nucleotide in the antisense strand of
DNA templates was always a 2’-MeO ribonucleotide to minimize nontemplated nucleotide
addition.55 For each modified base, we designed three different templates encoding the
incorporation of either one, three or seven modified nucleotides (in the latter case there were also
two adjacent incorporations of modified nucleotides) (Figure 1, Table S1 in Supporting
Information). The transcription has been performed in analogy to literature procedures25-39,54 using
[α-32P]-GTP or [α-32P]-ATP and the RNA products were analyzed by PAGE and quantified using
QuantityOne software (Figure 2, means from 2-3 independent experiments). The transcription
conditions needed to be carefully optimized to avoid misincorporations, especially when ATP
analogues (ARTP) were used. Addition of detergent (Triton X-100) and fine tuning of Mg2+ ion
concentration lead to minimization of misincorporated transcript content in negative control
experiments (See Figure S1 in Supporting Information). The RNA products were also characterized
by MALDI-TOF which in most cases confirmed the correct full-length modified RNA (see Table
S2 and Figures S6-S44 in Supporting Information). In some cases, we also observed a one-
nucleotide longer RNA product containing an additional n+1 nucleotide incorporated in a non-
templated manner.
Figures 1 and 2 show that the single incorporations of a modified nucleotide, as well as
incorporations of three modified nucleotides at separate positions, worked with excellent (for
smaller substituents) to moderate (for bulky BF or DB substituents) conversions to give the desired
modified RNAs (RNA_1NR or RNA_3NR). Modified nucleotides bearing smaller substituents
(Me, E or Ph) were typically comparable or just slightly worse substrates than the natural
nucleotides in these experiments. On the other hand, in the more challenging experiments, aiming
at incorporation of seven modified nucleotides with two pairs of modified bases in adjacent
6

Page 7 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
positions, the polymerase synthesis of RNA_7NR was somewhat less efficient than the synthesis
of natural non-modified RNA. The transcriptions with bulky DB-substituted NDBTPs did not give
any significant amounts of the full-length transcripts.
Figure 1. (A) Scheme of T7 RNA polymerase synthesis of modified RNA; (B) examples of
PAGE analysis of the transcripts; (C) sequences of the modified RNA transcripts RNA_XNR.
7

Organic & Biomolecular Chemistry
Page 8 of 19
View Article Online
DOI: 10.1039/C8OB01498A
Figure 2. Quantifications of the relative conversions (compared to transcription with all four
natural NTPs: N+) of T7 RNA polymerase transcription experiments
The study of processing of modified GRTPs was more difficult because of the requirements of
the T7 RNA polymerase for the presence of guanosines in the +1,+2 and/or +3 positions of the
transcript. At first we designed sequences with three adenosines at +1-+3 positions and tried the
8

Page 9 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
transcriptions under the same conditions as above. Figure 3A shows that the transcriptions did not
work even for the positive control (using all natural NTPs). Then we designed a sequence
commencing with three guanosines and the transcription worked only when natural NTPs were
used. It indicates that the modified GRTPs are not suitable for transcription initiation. Next, we
designed a sequence with only one G at the +1 position (followed by CA). The result was
encouraging showing that the transcription worked reasonably well at least for GMeTP but still
was negligible for all other bulkier GRTPs. Apparently, the polymerase does not tolerate a bulky
modification at the +1 position. Therefore, we used the sequence GCA in the first positions of the
transcript and used guanosine monophosphate (GMP) for initiation of the transcription56 (Figure
3B). Now, the transcriptions leading to the difficult RNA_pg7GR transcripts proceeded with
acceptable efficiency for the smaller modifications (35-50% compared to natural RNA). Again,
the most bulky dibenzofuryl modified GDBTP gave only trace amounts of the transcript.
Figure 3. (A) Scheme of attempted T7 polymerase synthesis of modified RNA using modified
GRTPs; (B) synthesis of modified RNA using modified GRTPs in presence of GMP.
9

Organic & Biomolecular Chemistry
Page 10 of 19
View Article Online
DOI: 10.1039/C8OB01498A
Conclusions
We synthesized a complete set of 18 modified pyrimidine or 7-deazapurine NTPs bearing
substituents of increasing bulkiness (methyl, ethynyl, phenyl, benzofuryl and dibenzofuryl). For
alkyl or ethynyl substituted nucleotides, the best approach was the synthesis of the corresponding
nucleoside followed by triphosphorylation. For most of the aryl substituted NRTPs, the direct
Suzuki-Miyaura cross-coupling of halogenated NITPs with the corresponding arylboronic acid
gave the desired modified nucleotides in s single step.
Systematic screening of the incorporation of the modified ribonucleotides into RNA using
NRTPs as substrates in presence of T7 RNA polymerase was performed using several DNA
templates encoding for different RNA transcripts. It should be noted that the procedures had to be
optimized to minimize misincorporations. The assays revealed that the modified pyrimidine
nucleotides URTPs and CRTPs bearing smaller or middle-sized substituents (up to benzofuryl)
worked as good to very good substrates for the T7 RNA polymerase and the nucleotides were
incorporated with moderate to good efficiency even into difficult RNA sequences containing 7
modifications with two pairs of modified bases at adjacent positions. The most bulky
dibenzofuryl modification still worked reasonably well on pyrimidine NTPs in easier sequences
but not in the difficult sequences. Transcriptions with modified 7-deazaadenine ARTPs worked
also well for smaller or middle sized modifications (but not for bulky dibenzofuryl). However,
special attention had to be paid to optimize the procedure to avoid misincorporations.
Much more problematic and difficult was the polymerase synthesis of RNA containing modified
7-deazaguanosines. The synthesis of RNA commencing with guanosine-free AAA sequence did
not work and the sequence starting with GGG worked only for non-modified GTP. Using a
modified promoter encoding for RNA starting with GCA sequence we at least observed some
transcription product when using GMeTP (but not the bulkier GRTPs) indicating that the
polymerase does not tolerate bulky modification at the +1 position. The problem was solved with
using GMP for initiation of transcription. In presence of GMP, the synthesis of GR-modified
RNA worked well for all GRTPs (except for the most bulky GDBTP). The lack of transcription
with modified GRTPs in the absence of GMP could be explained by the fact that the Km for the
+1 substrate is much higher than for any other elongating position and therefore the sterically
10

Page 11 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
hindered nucleotide which can be tolerated in others positions cannot be tolerated in the +1
position.
Most of the previous studies were performed only with 5-modified UTPs25-37 and with just few
examples of 5-modified CTPs.38-41 Our current work for the first time compares the same
substituents directly linked (without any flexible tether) to different NTPs. It not only confirms
the previous findings25-39 that the pyrimidine NTPs bearing even bulkier groups at position 5 are
good substrates for T7 RNA polymerase, but also shows that the corresponding 7-substituted 7-
deazapurine NTPs (with smaller or middle-sized groups up to benzofuryl) can be reasonably
efficiently incorporated into RNA. The substituents here were not selected for specific function
but to study the influence of the size of the group on the enzymatic incorporation. Nevertheless,
at least the UTP, CTP and 7-deazaATP derivatives bearing smaller hydrophobic substituents
certainly have a potential for applications in selection of aptamers or other functional RNAs,14-16
The limitations for practical use are the first three nucleotides of the transcript (the transcript
must start with at least one non-modified guanosine) and problematic use of modified GRTPs.
The difficult sequences not accessible by T7 polymerase synthesis (e.g. any sequence starting
with non-G nucleotides) still remain a challenge for future development which in principle could
be solved by engineered RNA polymerases not dependent on specific promoter.57 Studies in this
direction are under way in our laboratory.
Experimental
Synthesis and characterization data of the following known compounds have been reported
59
previously: AE, AITP and APhTP,39 CE and UE,58 GPh, GBF and GTMSE
,
CITP,60 UITP,61 UPhTP62
and UBFTP37 were previously reported. Detailed synthetic procedures and full characterization data
for all new compounds is given in the Supporting Information. Only the most important general
procedures are given below.
General procedure A: Suzuki cross-coupling on modified nucleosides
Water/ACN mixture (2:1, 15 mL) was added through septum to an argon purged vial containing
iodinated nucleoside (0.2 mmol), corresponding boronic acid (2.4 mmol) and Cs2CO3 (399 mg, 1.2
mmol), followed by the addition of the degassed solution of Pd(OAc)2 (7 mg, 0.02 mmol) and
11

Organic & Biomolecular Chemistry
Page 12 of 19
View Article Online
DOI: 10.1039/C8OB01498A
TPPTS ligand (69 mg, 0.12 mmol) in water/ACN (2:1, 10 mL). After argon/vacuum exchange, the
reaction mixture was left stirring at 100°C overnight. The mixture was cooled to the room
temperature, coevaporated with silica gel and purified by high performance reverse phase flash
chromatography (0 100% MeOH in water) using C18RediSep column on a CombiFlash
Teledyne ISCO system.
General procedure B: phosphorylation of modified nucleosides
Modified nucleoside (1 equiv.) was dried at 60°C overnight under vacuum. It was then suspended
in PO(OMe)3, stirred at room temperature for 15 minutes, followed by cooling to 0°C and addition
of POCl3 (1.2 eqiv.). The reaction mixture was left stirring at 0°C (1.5-24 h) and then ice cold
solution of (NHBu3)2H2P2O7 (5 equiv.) and tributyl amine (5 eqiv.) in anhydrous DMF (1-5 mL)
was added. The reaction mixture was stirred at 0°C for another hour. Then aqueous solution of
TEAB (2M, 2 mL, 4 mmol) was added and the mixture was evaporated under reduced pressure.
The residue was coevaporated several times with water. The product was purified by
chromatography on DEAE Sephadex column (01.2M aq. TEAB) and then by HPLC (C-18
column, 0.1M TEAB in water to 0.1M TEAB in 50% aq. MeOH), it was coevaporated several
times with water and, where possible, converted to the sodium salt form (Dowex 50 in Na+ cycle).
General procedure C: Suzuki coupling on iodinated triphosphates
Degassed solution of Pd(OAc)2 (0.5 mg, 0.002 mmol) and TPPTS (6 mg, 0.01 mmol) in
water/ACN (2 mL, 2:1) was added to the water/ACN (5 mL, 2:1) solution of NITP (30 mg, 0.04
mmol), corresponding boronic acid (0.11 mmol) and Cs2CO3 (70 mg, 0.2 mmol). The reaction
mixture was left stirring for 30 min at 100°C. After cooling to the room temperature, the mixture
was concentrated under reduced pressure and the product was purified by HPLC (C-18 column,
0.1M TEAB in water to 0.1M TEAB in 50% aq. MeOH), coevaporated several times with water
and, where possible, converted to a sodium salt form (Dowex 50 in Na+ cycle).
In vitro transcription with modified NTPs
A solution of template oligonucleotides (100 M each) in annealing buffer [Tris (10 mM), NaCl
(50 mM), EDTA (1 mM), pH 7.8] was heated to 95°C for 5 minutes and slowly cooled to 25°C
over a period of 45 minutes. The resulting DNA (50 M) was used as a template for transcription
12

Page 13 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
reactions. In vitro transcription reactions were performed in the total volume of 20 L in 40 mM
Tris buffer (pH 7.9) containing modified NTP (2 mM), three natural NTPs (2 mM), DTT (10 mM),
MgCl2 (25 mM), Ribolock RNase inhibitor (1U/L), Triton X-100 (0.1%), dsDNA template (0.625
M), T7 RNA polymerase (2U/L, Thermoscientific) and [α-32P]-GTP (111 TBq/mmol, 370
MBq/mL, 0.4 L) or [α-32P]-ATP (111 TBq/mmol, 370 MBq/mL, 0.4 L) if transcription was done
with modified GTPs. In the negative control experiment, water was used instead of the solution of
modified NTP, and in the positive control the natural NTP (2 mM) was used instead. The
transcription reactions were performed at 37°C for 2-4 h. The samples (2 L) were mixed with
RNA loading dye (2 L, Thermoscientific), heated to 75°C for 10 minutes and cooled on ice. The
samples were then analyzed by gel electrophoresis on 12.5% denaturing polyacrylamide gel
containing 1xTBE buffer (pH 8) and urea (7 M) at 42 mA for 45 minutes. The gels were dried
(85°C, 75 minutes), autoradiographed and visualized by phosphoimager (Typhoon 9410,
Amersham Biosciences). Transcription efficiencies were deteremined densitometrically from two
or three independent experiments using QuantityOne software.
In vitro transcription with modified GTPs in the presence of GMP
A solution of template oligonucleotides (100 M) in annealing buffer [Tris (10 mM), NaCl (50
mM), EDTA (1 mM), pH 7.8] was heated to 95°C for 5 minutes and slowly cooled to 25°C over
a period of 45 minutes. The resulting DNA (50 M) was used as a template for transcription
reactions. In vitro transcription reactions were performed in the total volume of 20 mL in 40 mM
Tris buffer (pH 7.9) containing modified GTP (2 mM), GMP (30 mM), CTP (2 mM), UTP (2 mM),
ATP (2 mM), DTT (10 mM), MgCl2 (25 mM), Ribolock RNase inhibitor (1U/L), Triton X-100
(0.1%), dsDNA template (0.625 M), T7 RNA polymerase (3U/L, Thermoscientific) and [α-32P]-
ATP (111 TBq/mmol, 370 MBq/mL, 0.4 L). In the negative control experiment, water was used
instead of the solution of modified GTP, and in the positive control the natural GTP (2 mM) was
used instead. The transcription reactions were performed at 37°C for 4h. The samples (2 L) were
mixed with RNA loading dye (2 L, Thermoscientific), heated to 75°C for 10 minutes and cooled
on ice. The samples were then analyzed by gel electrophoresis on 12.5% denaturing
polyacrylamide gel containing 1xTBE buffer (pH 8) and urea (7 M) at 42 mA for 45 minutes. The
gels were dried (85°C, 75 minutes), autoradiographed and visualized by phosphoimager (Typhoon
13

Organic & Biomolecular Chemistry
Page 14 of 19
View Article Online
DOI: 10.1039/C8OB01498A
9410, Amersham Biosciences). Transcription efficiency was deteremined densitometrically from
two independent experiments using QuantityOne software.
MALDI-TOF analysis of modified RNAs
A solution of template oligonucleotides (100 M each) in annealing buffer [Tris (10 mM), NaCl
(50 mM), EDTA (1 mM), pH 7.8] was heated to 95°C for 5 minutes and slowly cooled to 25°C
over a period of 45 minutes. The resulting DNA (50 M) was used as a template for transcription
reactions. In vitro transcription reactions were performed in the total volume of 50 µL in 40 mM
Tris buffer (pH 7.9) containing modified NTP (2 mM), three natural NTPs (2 mM), DTT (10 mM),
MgCl2 (25 mM), Ribolock RNase inhibitor (1U/L), Triton X-100 (0.1%), dsDNA template (1.25
M), T7 RNA polymerase (3U/L, Thermoscientific). The transcription reactions were performed
at 37°C for 2-4h. Then, DNAse I (2U, Thermoscientific) was added and samples were incubated
for further 15 minutes. The samples were then purified on NucAway spin colums (Ambion, elution
done in DEPC-H2O) as per supplier’s protocol. Purified samples were analyzed by MALDI-TOF
mass spectrometry. The MALDI-TOF spectra were measured on a MALDI-TOF/TOF mass
spectrometer with 1 kHz smartbeam II laser. The measurements were done in reflectron mode by
droplet technique, with the mass range up to 30 kDa. The matrix consisted of 3-hydroxypicolinic
acid (HPA)/picolinic acid (PA)/ ammonium tartrate in ratio 9/1/1. The matrix (1 μL) was applied
on the target (ground steel) and dried down at room temperature. The sample (1 μL) and matrix (1
μL) were mixed and added on the top of the dried matrix preparation spot and dried at room
temperature.
14

Page 15 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
Electronic Supporting Information. Full experimental part, characterization of all compounds,
additional gels and copies of spectra.
Acknowledgement:
Funding by European Regional Development Fund; OP RDE (No.
CZ.02.1.01/0.0/0.0/16_019/0000729 to M.H.), Czech Science Foundation (206-12-G151 to
N.M.) and Czech Academy of Sciences (Praemium Academiae award to MH) is gratefully
acknowledged.
15

Organic & Biomolecular Chemistry
Page 16 of 19
View Article Online
DOI: 10.1039/C8OB01498A
References:
(1) R. R. Breaker, Nature 2004, 432, 838–845.
(2) C. B. Reese, Org. Biomol. Chem. 2005, 3, 3851–3868.
(3) R. I. Hogrefe, B. Midthune, A. Lebedev, Isr. J. Chem. 2013, 53, 326–349.
(4) M. Hollenstein, Molecules 2012, 17, 13569–13591.
(5) A. Hottin, A. Marx, Acc. Chem. Res. 2016, 49, 418–427.
(6) M. Hocek, J. Org. Chem. 2014, 79, 9914–9921.
(7) L. Anhäuser, A. Rentmeister, Curr. Opin. Biotechnol 2017, 48, 69–76.
(8) A. Somoza, Chem. Soc. Rev. 2008, 37, 2668–2675.
(9) E. Paredes, M. Evans, S. R. Das, Methods 2011, 54, 251–259.
(10) F. Wachowius, C. Höbartner, ChemBioChem 2010, 11, 469–480.
(11) A. Haller, M. F. Soulière, R. Micura, Acc. Chem. Res. 2011, 44, 1339–1348.
(12) A. Chirkova, M. Erlacher, R. Micura, N. Polacek, Curr. Org. Chem. 2010, 14, 148–161.
(13) R. Kole, A. R. Krainer, S. Altman, Nat. Rev. Drug. Discov. 2012, 11, 125–140.
(14) M. Famulok, G. Mayer, Chem. Biol. 2014, 21, 1055–1058.
(15) M. Famulok, J. S. Hartig, G. Mayer, Chem. Rev. 2007, 107, 3715–3743.
(16) D. Bumcrot, M. Manoharan, V. Koteliansky, D. W. Y. Sah, Nat. Chem. Biol. 2006, 2, 711–
719.
(17) J. T. George, S.G. Srivatsan, Methods 2017, 120, 28–38.
(18) J. M. Holstein, A. Rentmeister, Methods 2016, 98, 18−25.
(19) Y. W. Yin, T. A. Steitz, Science 2002, 298, 1387–1395.
(20) I. Kuzmine, P. A. Gottlieb, C. T. Martin, J. Biol. Chem. 2003, 278, 2819–2823.
(21) W. P. Kennedy, J. R. Momand, Y. W. Yin, J. Mol. Biol. 2007, 370, 256–268.
(22) J. F. Milligan, D. R. Groebe, G. W. Witherell, O. C. Uhlenbeck, Nucleic Acids Res. 1987,
15, 8783-8798.
(23) P. R. Langer, A. A. Waldrop, D. C. Ward, Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 6633-
6337.
(24) Y. Ito, A. Suzuki, N. Kawazoe, Y. Imanishi, Bioconjug. Chem. 2001, 12, 850–854.
(25) A. A. Sawant, P. P. Mukherjee, R. K. Jangid, S. Galande, S. G. Srivatsan, Org. Biomol.
Chem. 2016, 14, 5832–5842.
(26) A. A. Sawant, A. A. Tanpure, P. P. Mukherjee, S. Athavale, A. Kelkar, S. Galande, S. G.
Srivatsan, Nucleic Acids Res. 2016, 44, e16.
(27) J. T. George, S. G. Srivatsan, Bioconjug. Chem. 2017, 28, 1529–1536.
(28) M. B. Walunj, A. A. Tanpure, S. G. Srivatsan, Nucleic Acids Res 2018, 10.1093/nar/gky185.
(29) T. M. Dewey, A. Mundt, G. J. Crouch, M. C. Zyzniewski, B. E. Eaton, J. Am. Chem. Soc.
1995, 117, 8474–8475.
(30) N. K. Vaish, A. W. Fraley, J. W. Szostak, L. W. McLaughlin, Nucleic Acids Res. 2000, 28,
3316–3322.
(31) J. D. Vaught, T. Dewey, T. B. E. Eaton, J. Am. Chem. Soc. 2004, 126, 11231–11237.
(32) C. C. Smith, M. Hollenstein, C. J. Leumann, RSC Adv. 2014, 4, 48228–48235.
(33) A. A. Tanpure, S. G. Srivatsan, ChemBioChem 2014, 15, 1309–1316.
(34) S. G. Srivatsan, Y. Tor, J. Am. Chem. Soc 2007, 129, 2044–2053.
(35) M. G. Pawar, S. G. Srivatsan, Org. Lett. 2011, 13, 1114–1117.
16

Page 17 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
(36) S. G. Srivatsan, Y. Tor, Chem. Asian J. 2009, 4, 419–427.
(37) A. A. Tanpure, S. G. Srivatsan, Chem. Eur. J. 2011, 17, 12820–12827.
(38) M. M. Hanna, Y. Zhang, J. C. Reidling, M. J. Thomas, J. Jou, Nucleic Acids Res. 1993, 21,
2073–2079.
(39) D. A. Di Giusto, W. A. Wlassoff, S. Giesebrecht, J. J. Gooding, G. C. King, Angew. Chem.
Int. Ed. 2004, 43, 2809–2812.
(40) G. Stengel, M. Urban, B. W. Purse, R. Kuchta, Anal. Chem. 2010, 82, 1082-1089.
(41) P. N. Asare-Okai, E. Agustin, D. Fabris, M. Royzen, Chem. Commun. 2014, 50, 7844-7847.
(42) A. Bourderioux, P. Nauš, P. Perlíková, R. Pohl, I. Pichová, I. Votruba, P. Dzubák, P.
Konecný, M. Hajdúch, K. M. Stray, T. Wang, A. S. Ray, J. Y. Feng, G. Birkus, T. Cihlar, M.
Hocek, J. Med. Chem. 2011, 54, 5498–5507.
(43) P. Perlíková, G. Rylová, P. Nauš, T. Elbert, E. Tloušťová, A. Bourderioux, L. P. Slavětínská,
K. Motyka, D. Doležal, P. Znojek, A. Nová, M. Harvanová, P. Džubák, M. Šiller, J. Hlaváč, M.
Hajdúch, M. Hocek, Mol. Cancer. Ther. 2016, 15, 922–937.
(44) Y. Zheng, P. A. Beal, P, Bioorg. Med. Chem. Lett. 2017, 26, 1799-1802.
(45) L. S. McCoy, D. Shin, Y. Tor, J. Am. Chem. Soc. 2014, 136, 15176–15184.
(46) K. Moriyama, M. Kimoto, T. Mitsui, S. Yokoyama, I. Hirao, Nucleic Acids Res. 2005, 33,
e129.
(47) R. Kawai, M. Kimoto, S. Ikeda, T. Mitsui, M. Endo, S. Yokoyama, I. Hirao, J. Am. Chem.
Soc. 2005, 127, 17286–17295.
(48) M. Kimoto, M. Endo, T. Mitsui, T. Okuni, I. Hirao, S. Yokoyama, Chem. Biol. 2004, 11,
47–55.
(49) (a) Y. J. Seo, D. A. Malyshev, T. Lavergne, P. Ordoukhanian, F. E. Romesberg, J. Am.
Chem. Soc. 2011, 133, 19878–19888. (b) Y. J. Seo, S. Matsuda, F. E. Romesberg, J. Am. Chem.
Soc. 2009, 131, 5046–5047.
(50) C. Domnick, F. Eggert, S. Kath-Schorr, Chem. Commun. 2015, 51, 8253–8256.
(51) (a) Y. Masaki, H. Ito, Y. Oda, K. Yamazaki,N. Tago, K. Ohno, N. Ishii, H. Tsunoda, T.
Kanamori, A. Ohkubo, M. Sekine, K. Seios, Chem. Comm., 2016, 52, 12889-12992. (b) J. B. J,
Pavey, A. J. Lawrence, I. A. O’Neil, S. Vortler, R. Cosstic, Org. Biomol. Chem, 2004, 2, 869-
875. (c) Y. Huang, F. Eckstein, R. Padilla, R. Soussa, Biochemistry, 1997, 36, 8231-8242.
(52) J. Ludwig, Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131–133.
(53) P. Capek, H. Cahová, R. Pohl, M. Hocek, C. Gloeckner, A. Marx, Chem. Eur. J. 2007, 13,
6196–6203.
(54) H. Gruegelsiepe, A. Schön, L. Kirsebom, R. K. Hartmann in Handbook of RNA biochemistry
(Eds.: R. K. Hartmann, A. Bindereif, A. Schön, E. Westhof), WILEY‐ VCH Verlag GmbH &
Co. KGaA, Weinheim, pp. 3-95.
(55) C. Kao, M. Zheng, S. Rudisser, RNA 1999, 5, 1268-1272.
(56) C. T. Martin, J. E. Coleman, Biochemistry 1989, 28, 2760-2762.
(57) T. Chen, F. E. Romesberg, J. Am. Chem. Soc. 2017, 139, 9949–9954.
(58) D. Qu, L. Zhou, W. Wang, Z. Wang, G. Wang, W. Chi, B. Zhang, Anal. Biochem. 2013,
434, 128–135.
(59) P. Nauš, O. Caletková, P. Konečný, P. Džubák, K. Bogdanová, M. Kolář, J. Vrbková, L.
Slavětinská, E. Tloušt’ová, P. Perliková et al., J. Med. Chem. 2014, 57, 1097-1110.
17

Organic & Biomolecular Chemistry
Page 18 of 19
View Article Online
DOI: 10.1039/C8OB01498A
(60) A. Krause, A. Hertl, F. Muttach, A. Jäschke, Chem. Eur. J. 2014, 20, 16613-16619.
(61) K. R. Anilkumar, M. Shanmugasundaram, Tetrahedron Lett. 2012, 53, 2530-2532.
(62) T. Pesnot, L. M. Tedaldi, P. G. Jambrina, E. Rosta, G. K. Wagner, Org. Biomol. Chem.
2013, 11, 6357–6371.
18

Page 19 of 19
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01498A
TOC entry
Synthesis and systematic study of T7 RNA polymerase incorporation of base-modified
ribonucleotides for the enzymatic synthesis of modified RNA.