Modified nucleotides as substrates of terminal deoxynucleotidyl transferase

Article abstract of DOI:10.3390/molecules22040672

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N⁴-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3′-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3′-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.

Full text of DOI:10.3390/molecules22040672

molecules
Communication
Modified Nucleotides as Substrates of Terminal
Deoxynucleotidyl Transferase
Daiva Taurait e˙ 1,*,†, Jevgenija Jakubovska 1,†, Julija Dabužinskait e˙ , Maksim Bratchikov 2 and
1
Rolandas Meškys 1
1
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center,
Vilnius University, Sauletekio al. 7, Vilnius LT-10257, Lithuania; jevgenija.jakubovska@bchi.vu.lt (J.J.);
julija.dab@gmail.com (J.D.); rolandas.meskys@bchi.vu.lt (R.M.)
2
Department of Physiology, Biochemistry, Microbiology and Laboratory Medicine, Faculty of Medicine,
ˇ
Vilnius University, M. K. Ciurlionio g. 21, Vilnius LT-03101, Lithuania; maksim@pcr.lt
*
†
Correspondence: daiva.tauraite@bchi.vu.lt; Tel.: +370-5-223-4384
These authors contributed equally to this work.
Academic Editor: Suzanne Peyrottes
Received: 16 March 2017; Accepted: 19 April 2017; Published: 22 April 2017
Abstract: The synthesis of novel modified nucleotides and their incorporation into DNA sequences
opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore,
broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide
derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as
2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were
tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing
4
N -aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a
nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3’-terminally modified
ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried
out. Enzymatic post-synthetic 3’-modification of ONs with various photo- and chemically-reactive
groups opens novel possibilities for future applications, especially in analysis of the mechanisms of
polymerases and the development of photo-labels, sensors, and self-assembling structures.
4
Keywords: terminal deoxynucleotidyl transferase; N -amino-2’-deoxycytidine triphosphate;
4
-thio-2’-deoxyuridine triphosphate; 4-bromo-2-pyridone; 4-chloro-2-pyridone; UV cross-linking
1
. Introduction
Oligonucleotides (ONs) bearing nucleobase or carbohydrate backbone modifications have a wide
range of applications such as the development of aptamers [ ], specific labelling of nucleic acids or
proteins [ ], functionalization of surfaces [ ], scaffolding of biocatalysts [ ] or creation of biosensors [ ].
Different strategies are employed to insert functional groups into ONs. The most common are
synthesis by the standard solid-phase phosphoramidite chemistry [ ], enzymatic synthesis by DNA
or RNA polymerases using modified (2’-deoxy)ribonucleoside 5’-triphosphates (dNTPs or NTPs) as
substrates [ 11], post-synthetic modification [12 13] and DNA ligation [14]. A broad spectrum of
modified dNTPs as well as diverse polymerases have been tested and utilized for the synthesis of sugar-,
phosphate- or base-modified DNA [15 19]. A straightforward approach to enzymatically prepared
modified ONs is DNA end labelling based on terminal deoxynucleotidyl transferase (TdT) [20 21].
1,2
3
4
5
6
7
8
–
,
–
,
TdT adds random nucleotides at the 3’-OH terminus of DNA using dNTPs as substrates in a completely
template-independent manner. Beyond its natural substrate, TdT is known to use a wide variety
of nucleoside triphosphate analogues such as p-nitrophenyl triphosphate [22], 5-substituted indolyl
deoxynucleotides [23], dinucleoside 5’,5’-tetraphosphate [24], pyrene-nucleoside triphosphate [25,26]
Molecules 2017, 22, 672; doi:10.3390/molecules22040672
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Molecules 2017, 22, 672
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or benzo-expanded dNTP [27]. Several recent reports demonstrate a modified nucleotide incorporation
by TdT in order to provide nuclease resistance [28], introduce a functional group [29] or upgrade 3’-end
labelling with “click” chemistry [30]. The crystal structure of TdT [31] provides a better understanding
of reaction mechanism, however details regarding nucleotide selection remains elusive.
0
Halogenated nucleotides such as 5-bromo-2 -deoxyuridine (BrdU) and 5-iodo-2’-deoxyuridine
(IdU) have been comprehensively studied as photo-reactive chromophores [32–35]. Toxic effects
caused by UVB and UVC radiation are enhanced when BrdU or IdU is present in DNA, while these
are UVA photosensitizers only along with DNA-intercalating UVA chromophore [36]. An alternative
strategy to photoactivate DNA by UVA is to use modified nucleotides which may shift absorbance
maximum to UVA region [37]. In addition, modification of nucleobase might improve radiosensitizing
properties under hypoxic conditions as in the case of 4-bromo- and 5-bromopyridone analogues of
BrdU [38]. Despite above mentioned properties, halogen-containing ONs may be further modified
during palladium-catalysed cross-coupling [39]. Altogether, halogenated nucleotides and ONs not only
contribute to investigation of DNA lesions but also act as precursors for post-synthetic modifications.
Thio group-bearing (deoxy)ribonucleotides have been extensively used as intrinsic photolabels to
analyse the three-dimensional structure of RNA molecules, to identify contacts between nucleic acids
and proteins, to detect epitranscriptomic marks, and to manipulate individual DNA molecules [40
Similarly to halogenated nucleotides, thio-nucleotides such as 4-thio-2’-deoxyuridine (4-thiodU),
-thio-5-bromo-2’-deoxyuridine (4-thio-5-Br-dU), 6-thioguanosine (6-thioG) or 4-thiothymidine
4-thioT) operate as UVA sensitizers while the latter is easily incorporated into DNA during
replication [45–47].
Recently, much attention has been paid to the preparation of hydrazine functionality-containing
–44].
4
(
4
nucleotides and ONs [48–53]. It has been shown that N -aminocytidine induces an AT to GC transition
and can function as a mutagen [54]. Compared to well-known amino-modified ONs, hydrazines exhibit
enhanced reactivity with active esters at neutral to acidic conditions as well as generate more stable
adducts—hydrazones—reacting with aldehydes [50]. Usually, the hydrazine group is introduced
into ON post-synthetically [48,49,52,53] and only recently the protected hydrazine amidites have
been reported [50 51]. Both conjugation and immobilization of ONs continue to develop towards
,
high-throughput technologies, therefore significance of hydrazine-modification is obvious. Pyridone-,
cyano- and carboxy-functionalities are also worth mentioning since they open many ways for further
chemical or enzymatic modifications of nucleobases [55–58].
Whereas the utility of thio- or hydrazine-modified ONs is evident, the lack of convenient building
blocks as well as methods for their introduction into ONs has thus far hindered the wide use of
these functional modifications. Here we present a synthetic approach to produce triphosphates of
4-thio-2’-deoxyuridine and 2’-deoxycytidine analogue bearing hydrazine group at the 4th position.
To the best of our knowledge, the template-independent enzymatic labelling approach has not yet been
4
used for the synthesis of 3’-modified ONs with 4-thio-2’-deoxyuridine, N -amino-2’-deoxycytidine or
4
2
2
-Br(Cl)-pyridones.
. Results and Discussion
.1. Synthesis of Modified Nucleotides
4-Thio-2’-deoxyuridine (2) was prepared by adapting and modifying methods reported in the
literature [59 60]. A general synthetic approach consists of three stages: (i) protecting the hydroxyl
,
groups on the sugar by acylation; (ii) replacing the oxygen atom at 4-position with a sulphur atom using
Lawesson‘s reagent; and (iii) removing the protecting groups with sodium methylate to produce the
4
-thio-2’-deoxyuridine. The overall yield of three steps was 63%. Synthesized 4-thio-2’-deoxyuridine
was converted to the nucleoside triphosphate according to the one-pot phosphorylation method.
4
N -Amino-modified cytidine triphosphate could be prepared via bisulphite-mediated displacement
4
of the C4 amino group of cytosine, for example N -amino-2’-deoxycytidine triphosphate was

Molecules 2017, 22, 672
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synthesized from 2’-deoxycytidine triphosphate (dCTP) via microwave-mediated bisulphate-catalysed
transamination with hydrazine under strict control of reaction conditions [61]. In this research
4
we synthesized N -modified dCTP directly and simply from the prepared 4-thio-2’-deoxyuridine
triphosphate in 66% yield at room temperature (Scheme 1).
4
Scheme 1. Synthesis of 4-thio-2’-deoxyuridine triphosphate (
3
) and N -amino-2’-deoxycytidine
◦
triphosphate (
4
). Reagents and conditions: (i) Ac O, NaOAc, 90 C, 30 min; (ii) Lawesson’s reagent,
toluene, 90 C, 2 h; (iii) NaOCH , CH OH, rt, 30 min; (iv) POCl , Bu N, trimethyl phosphate, 0 C,
◦
0 min; then Bu N, (NHBu ) H P O , CH CN, 0 C, 15 min; (v) NH -NH , H O, rt, 18 h.
3 3 2 2 2 7 3 2 2 2
2
◦
◦
3
3
3
3
9
5-Cyano-2’-deoxyuridine can be synthesized by treatment of 2’-deoxy-5-iodouridine with
KCN, or by treatment of 5-trifluoromethyl-2’-deoxyuridine with concentrated NH OH [62].
4
5
-Carboxy-2’-deoxyuridine can be obtained from the oxidation of thymidine by menadione-mediated
photosensitization [63] or from the alkaline hydrolysis of 5-trifluoromethyl-2’-deoxyuridine [64]. In this
work 5-substituted 2’-deoxyuridine derivatives were obtained via glycosylation of silylated 5-cyano-
or 5-carboxy-uracil with acetylated 2-deoxyribose in the presence of tin (IV) chloride as Lewis acid
catalyst. Synthesized 5-substituted nucleosides were converted to the nucleotides according to the
one-step phosphorylation method (Scheme 2).
Scheme 2. Synthesis of 5-cyano- and 5-carboxy-dUTP. Reagents and conditions: (i) 1,1,1,3,3,3-
◦
hexamethyldisilazane (HMDS), trimethylsilyl chloride (TMSCl), 80 C, 4h; then 1,3,5-O-triacetyl-2-
deoxyribose, SnCl , rt, 5 h; (ii) NaOCH , CH OH, rt, 30 min; (iii) POCl , Bu N, trimethyl phosphate,
4
3
3
3
3
◦ ◦
–4 C, 2–3 h; then Bu N, (NHBu ) H P O , CH CN, 0 C, 10–15 min.
3 3 2 2 2 7 3
0
This method allowed accessing the triphosphate from the appropriate modified nucleoside in 20%
yield. The triphosphates were best isolated by ion exchange chromatography using a diethylaminoethyl
(DEAE) Sephadex A-25 column with a gradient of LiCl, followed by precipitation of modified
nucleotides from acetone/methanol mixtures. The one step phosphorylation procedure allowed
us to prepare the nucleoside triphosphates with >95% purity in a fast way for further studies.
2
.2. Incorporation of Modified Nucleotides by Terminal Deoxynucleotidyl Transferase
In order to determine whether these novel modified nucleotides can be incorporated into ON,
primer extension reactions (PEX) were conducted using TdT. Among all DNA polymerases TdT is
unique for its broad utilization of divalent metal ions. TdT is able to use several metal cations such
2
+
2+
2+
2+
as Mg , Zn , Co , Mn
[65]. Furthermore, each divalent metal ion contributes to the kinetics of
2+
nucleotide incorporation differently. It has been shown that in the presence of Co TdT preferentially
incorporates purine nucleotides while Mg2+ facilitates incorporation of pyrimidine nucleotides [66].

Molecules 2017, 22, 672
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Zn2+ can also improve template-independent synthesis [67]. To monitor the discrimination between
incorporation of modified nucleotides by TdT two buffer systems were used. In our studies the first
system is based on sodium glutamate and Mg2+ which is likely to be the basic enzyme cofactor in vivo
Contrary, another buffer system which is referred to as optimal buffer for TdT is absolutely artificial
.
2+
compared to in vivo conditions as it contains potassium cacodylate and Co . Moreover thinking of
2+
2+
2+
Co as a versatile metal ion cofactor capable of playing a role of both Mg and Zn , a more efficient
nucleotide incorporation using latter buffer system could be anticipated [68].
Figures 1 and 2 illustrate non-templated 3’-end elongation using 2-pyridone-based nucleoside
4
triphosphates and compounds
deoxycytidine triphosphate, N -amino-dCTP),
3
(4-thio-2’-deoxyuridine triphosphate, 4-thio-dUTP),
4
(N -amino-2’-
4
5CN
7
(5-cyano-2’-deoxyuridine triphosphate, dU
TP),
5COOH
2+
8
(5-carboxy-2’-deoxyuridine triphosphate, dU
TP) catalyzed by TdT in Mg -containing and
2+
its optimal (Co -containing) buffer system, respectively. In general, the utilization of the optimal
buffer for TdT resulted in more efficient incorporation of natural nucleotides as well as non-natural
analogues (Figure 2). It should be noted that in the case of optimal reaction conditions TdT utilizes
nearly every member of our library of non-natural nucleotides presented here whereas more than
a half of compounds tested were unsuitable substrates for TdT under milder (glutamate/Mg )
conditions. This in turn proves already established strict dependency of TdT catalytic activity and
more importantly substrate specificity on reaction buffer.
2+
It was previously demonstrated that 2-pyridone-based nucleotides are utilized by several
template-dependent DNA polymerases [69]; however mostly inefficiently since only a single
nucleotide was incorporated with no further elongation. Data provided in Figure 1 (lanes 7 and
8
) revealed that longer products (up to ten extra nucleotides) were generated using 4-chloro-2-
4Cl
pyridone-2’-deoxyriboside triphosphate (dPyr TP) and 4-bromo-2-pyridone-2’-deoxyriboside
triphosphate (dPyr4 TP) as substrates than with equivalent concentrations of dUTP and dCTP
suggesting that these compounds were better substrates for TdT. In addition, a substantial increase
in the length of the elongation products by these two compounds in optimal environment was
observed (Figure 2, lanes 7 and 8). Hence, in order to introduce varying number of monomers for the
template-independent synthesis of halogenated nucleic acids, different buffers can be applied.
Br
Figure 1. Polyacrylamide gel electrophoresis (PAGE) analysis of primer extension reactions (PEX)
2
+
with terminal deoxynucleotidyl transferase (TdT) in glutamate/Mg buffer. Lane 1, primer labelled
3
3
at the 5
'-end with a radioactive isotope of phosphorus (5’- P-labelled primer); lanes 2–12, products
of PEX using: lane 2, 2’-deoxythymidine triphosphate (dTTP); lane 3, 2’-deoxyuridine triphosphate
(
(
4
dUTP); lane 4, 2’-deoxycytidine triphosphate (dCTP); lane 5, 2-pyridone-2’-deoxyriboside triphosphate
dPyrTP); lane 6, dPyr4 TP; lane 7, dPyr TP; lane 8, dPyr TP; lane 9, dPyr5COOHTP; lane 10,
OH
4Cl
4Br
-thio-dUTP; lane 11, dU5CNTP; lane 12, dU5COOHTP; lane 13, N -amino-dCTP.
4
To the contrast, severe discrimination between two buffers was observed in the case of
-pyridone-2’-deoxyriboside triphosphate (dPyrTP) and 4-hydroxy-2-pyridone-2’-deoxyriboside
2

Molecules 2017, 22, 672
5 of 16
triphosphate (dPyr4 TP) acting as substrates for TdT. Figure 1 illustrates that utilization of both
OH
dPyrTP and dPyr4 TP by TdT was not detectable in glutamate/Mg buffer (lanes 5 and 6). However
OH
2+
2+
in the presence of Co TdT uses the former as substrate for the incorporation and subsequent
elongation while only a single incorporation of dPyr4
OH
is observed (Figure 2, lanes 5 and 6).
Recently, it was demonstrated that binding affinity of TdT to the 3’-end of ON modified with
-hydroxy-4-pyridone-based nucleotides depended on the Mg2+ concentration and caused formation
3
of unfavourable secondary structure of 3’-terminus [70]. As a consequence after incorporation of
single nucleotide further elongation was impeded. Our results support such interpretation and further
suggest that not only buffer composition but also additional nucleobase modifications may lead to
steric hindrance.
Figure 2. PAGE analysis of PEX with TdT in cacodylate/Co2+ buffer. Lane 1, 5 - P-labelled primer;
0 33
lanes 2–12, products of PEX using: lane 2, dTTP; lane 3, dUTP; lane 4, dCTP; lane 5, dPyrTP; lane 6,
4
OH
4Cl
4Br
5COOH
dPyr
TP; lane 7, dPyr TP; lane 8, dPyr TP; lane 9, dPyr
TP; lane 10, 4-thio-dUTP; lane 11,
dU5CNTP; lane 12, dU
5COOH
TP; lane 13, N -amino-dCTP.
4
4-Thiouracil nucleobase is naturally occurring in some transfer RNA species and is widely
used as a photoactivatable RNA-RNA, RNA-DNA, RNA-protein cross-linking agent [71]. Hence,
T7 RNA polymerase uses 4-thio-UTP as a substrate during transcription in vitro, while it is known that
4
-thiothymidine is readily incorporated into DNA during replication [45
,46]. The data presented here
revealed that 4-thio-dU was incorporated into ON with similar efficiency to its natural counterpart
2+
dU possibly due to its very similar structure. Despite the fact that Co -containing buffer system
enhances utilization of certain nucleotide analogues, only a slight increase in incorporation of 4-thio-dU
compared to glutamate/Mg2 buffer was observed (Figure 2, lane 10). Nevertheless, these results
suggest 4-thio-dUTP as a promising photo-affinity label for DNA.
+
4
N -Amino-dCTP appeared to be a moderate substrate for TdT in both buffer systems compared
to native nucleoside triphosphates. On the other hand, an ON bearing at least several hydrazine
modifications could be further modified or cross-linked to nucleic acid molecule, protein or
appropriately pre-treated surface.
Overall, dPyrTP, dPyr4ClTP, dPyr4BrTP, 4-thio-dUTP and N -amino-dCTP emerged as substrates
4
used by TdT for incorporation and subsequent elongation whereas dPyr4
OH
and dU
5CN
were only
is slightly
5CN
incorporated but further elongation was halted. Only a single incorporation of dU
confusing as it is known that TdT is able to incorporate modified pyrimidine nucleotides with bulky
72]. Neither dPyr5
COOH
TP nor dU
5COOH
TP were used by TdT as substrates
groups at fifth position [30
,

Molecules 2017, 22, 672
6 of 16
under both conditions tested. It might be speculated that a negative charge of both nucleotides was
detrimental to an activity of TdT.
So far, no atoms have been unambiguously identified between the amino acid residues of TdT
and the primer nucleobases that reside in sufficient proximity to one another to be considered a polar
interaction, thus indicating that binding must rely entirely upon interaction with the sugar-phosphate
backbone. It is assumed that the broad substrate specificity of TdT is based on polymerase insensitivity
to the chemical structure of an incoming dNTP although the structure and accessibility of the
3
’-terminus of ONs is crucial [26
does not appear to be influenced by
the size of modified nucleotides [11]. For example, it was shown that smaller nucleotides bearing
-fluoroindolyl- and 5-nitroindolyl- modifications were more efficiently elongated whereas their
bulkier counterparts (e.g., 5-napthylindolyl-modified), have been refractory to elongation. Hence, the
inability to elongate large, bulky, non-natural nucleotides likely results from steric constraints [11 73].
,
28]. In addition, it has been proposed that kinetics of extension
π
-electron surface area of nucleotide analogues but rather by
5
,
However, the clear difference between efficiency of incorporation of dPyr4 , dPyr (or dPyr
OH
4Cl
4Br
)
and 4-thio-dU as well as poor elongation of 5-cyano-modified dU by TdT cannot be explained in
this way. TdT requires at least three deoxynucleotide residues on the primer strand for an efficient
catalysis of the tailing reaction [31]. Subsequently, when polymerase reaches the extended section,
3
’-end modifications could block the entrance of an incoming dNTP by enhancing primer affinity to
TdT. Consequently reaction may stall explaining why only limited number of residues are appended,
e.g., in the case of 4-thio-dU. The same result would also be observed if the 3’-modified primer fails to
interact with the active center of TdT or it folds into unfavourable secondary structures as in the case of
pyrene-2’-deoxynucleotide [26] or 2-methyl-3-hydroxypyridin-4(1H)-one 2’-deoxynucleotide (dH) [74].
In our hands TdT does not extend the 4-thio-dU-elongated primer in the presence of deoxythymidine
triphosphate (dTTP) (data not shown); a similar effect has been observed with dH [70].
2
.3. UV Cross-Linking of Oligonucleotides to Terminal Deoxynucleotidyl Transferase
In order to examine the potential of thio-modified ON to serve as a cross-linking agent,
UVA-induced linking assay was conducted. Successful PEX using both 4-thio-dUTP and dUTP
a control reaction) were carried out where an average of 6 and 15 nucleotides were incorporated,
(
respectively (data not shown). The next step was to select the model protein of interest to be cross-linked
to 4-thiouridine-containing ON (4-thio-dU-ON). It was decided to cross-link the modified ON to TdT
for several reasons. First of all, it is obvious that using TdT eliminates the need to search for proteins
which specifically interact with the 3’-end of modified ONs. Secondly, it could be anticipated that,
after catalysis, thio-groups would be located adjacent to amino acid side chains in the active site of
TdT and thus revealing an actual interaction with nucleobase(s). Due to the fact that the generation
of photo-induced complexes strongly depends on the distance between a photo-reactive group and
amino acid residues it has been suggested that a successful cross-linking would be an evidence, even if
indirect, of interactions between nucleobases and TdT.
Figure 3 shows the effect of exposing PEX mixtures to 365 nm UV light. Based on the slower
mobility of the 33P label it was clear that the 4-thio-dU-ONs cross-linked to TdT (Figure 3, lanes 4–6).
Data provided in Figure 3 (lanes 4 and 5) indicate that induction of cross-links depended upon a dose
of UVA irradiation. A vast excess of TdT over ON did not improve cross-linking efficiency (Figure 3,
lane 6). Moreover, it was elucidated that no cross-linked complexes were detected during irradiation
at lower intensity (data not shown).

Molecules 2017, 22, 672
7 of 16
Figure 3. PAGE analysis of cross-linked complexes of 4-thio-dU-ONs and TdT. Lane 1, recombinant
TdT; lane 2, molecular mass marker (kDa); lane 3, 4-thio-dU-ON:TdT UV-free control; lane 4,
2
cross-linked complexes of 4-thio-dU-ON:TdT, dose of UV irradiation ~17.2 J/cm ; lane 5, cross-linked
2
complexes of 4-thio-dU-ON:TdT, dose of UV irradiation ~4.6 J/cm ; lane 6, cross-linked complexes of
2
4
-thio-dU-ON:TdT (excess of TdT), dose of UV irradiation ~17.2 J/cm ; lane 7; dU-ON:TdT control,
2
dose of UV irradiation ~17.2 J/cm .
The free TdT has a molecular mass of 45–47 kDa as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), whereas cross-linked complex run at the
position of 55 kDa marker (Figure 3, lane 1 and lanes 4–6, respectively). This suggests a 1:1 covalent
complex between TdT and 4-thio-dU-ON (molecular mass of 8–10 kDa). A second complex could be
noted with a molecular mass of ~130 kDa though exact constitution of this heavier complex could not
be ascertained easily (Figure 3, lane 4). It might be speculated that formation of more complicated
cross-links was inevitable since ON could contain up to ten 4-thiouracil nucleobases, which theoretically
might be triggered to generate at least several cross-links, whether it would be covalent bonds between
two ONs, two protein molecules or aggregates of mixed origin. No cross-linking was observed in the
absence of UV light and in the presence of dU-ONs.
TdT has a pivotal role in vivo to generate junctional diversity during V(D)J recombination by
adding random nucleotides [20]. A detailed mechanism of antibody gene recombination still needs
to be puzzled out. Although the biological role of TdT is tightly connected to its ability to utilize a
wide variety of substrates in vitro and in vivo, there is no exact explanation on substrate selectivity of
TdT up to date [75]. Data presented here slightly contradicts with the already established assumption
that a broad substrate selectivity of TdT is based on unspecific interaction with sugar-phosphate
backbone rather than specific contacts with nucleobases. Formation of UV-induced complexes between
4
-thio-dU-ONs and TdT suggests an existing juxtaposition of the 4-thiouracil base with the appropriate
amino acid side chains in the TdT.
All these observations clearly show that multiple interactions between the substrate and TdT take
place and the nature of nucleobase plays an important role with respect to nucleotide selection and
chain elongation. Therefore, further studies are needed to elucidate a substrate selectivity of TdT.

Molecules 2017, 22, 672
8 of 16
3
. Materials and Methods
3
.1. General Information
Chemicals and solvents were purchased from Sigma-Aldrich (Steinheim, Germany) and Alfa
Aesar (Karlsruhe, Germany) and used without further purification. Thin-layer chromatography
TLC) was carried out on TLC aluminium sheets coated with silica gel 60 F254 (Merck, Darmstadt,
(
Germany) and column chromatography on silica gel 60 (0.063–0.200 nm) (Merck). Reverse phase
chromatography was carried out on Grace C-18 flash cartridges (BÜCHI, Flawil, Switzerland).
Purification of nucleotides was carried out on diethylaminoethyl (DEAE) Sephadex A-25 columns (GE
Healthcare, Helsinki, Finland) with a linear (0.05–0.4 M) gradient of LiCl as a mobile phase. Melting
points were determined with a MEL-TEMP melting point apparatus (Electrothermal, Staffordshire,
UK) in capillary tubes and are not corrected. NMR spectra were recorded in DMSO-d or D O on
6
2
1
13
31
an Ascend 400: H-NMR–400 MHz, C-NMR–100 MHz and P-NMR–162 MHz (Bruker, Billerica,
MA, USA). Chemical shifts ( ) are reported in ppm relative to the solvent resonance signal as an
δ
internal standard. UV spectra were recorded on a Lambda 25 UV/VIS spectrometer (Perkin Elmer,
Singapore). High-performance liquid chromatography mass-spectrometry (HPLC-MS) analyses were
performed using a high performance liquid chromatography system, equipped with a photo diode
array detector (SPD-M20A) and a mass spectrometer (LCMS-2020, Shimadzu, Kyoto, Japan) equipped
with an electrospray ionization (ESI) source. The chromatographic separation was conducted using a
◦
YMC Pack Pro column, 3
×
150 mm (YMC, Kyoto, Japan) at 40 C and a mobile phase that consisted
of 0.1% formic acid water solution or 5 mM ammonium acetate buffer (solvent A), and acetonitrile
solvent B). Mass spectrometry data was acquired in both positive and negative ionization mode and
analyzed using the LabSolutions LCMS software (Software version 5.42 SP6, Shimadzu, Kyoto, Japan).
(
3
.2. Synthesis of 4-Thio-2'-deoxyuridine (2)
(
i) 3’,5’-Bis-O-acetyl-2’-deoxyuridine (1i). A mixture of acetic anhydride (804
µ
L, 8.52 mmol) and
◦
sodium acetate (116 mg, 1.42 mmol) was heated for 10 min at 90 C. Then 2’-deoxyuridine (1) (648 mg,
2
.84 mmol) was added to the hot solution and stirred for 30 min. After the reaction was completed
TLC), the mixture was quenched with sodium bicarbonate and extracted with chloroform. The organic
phase was dried (Na SO ) and evaporated under reduced pressure. The residue was purified by
(
2
4
column chromatography (silica gel, chloroform/methanol mixture, 10:0
→
10:1). Yield 828 mg (93%),
◦
+
white solid, melting point (mp) 85–88 C. retention factor (R ) = 0.75 (CHCl /MeOH 5:1). MS (ESI ):
f
3
+
−
1
m/z 313.00 [M+H] , 311.00 [M-H] . UV (CH OH) λmax (log ε) 262 (9.60) nm. H-NMR (DMSO-d ):
6
3
δ
= 2.06 (s, 3H, CH ), 2.07 (s, 3H, CH ), 2.28–2.34 (m, 1H, CH ), 2.43–2.46 (m, 1H, CH ), 4.16–4.19 (m,
3 3 2 2
1
1
H, CH), 4.21–4.24 (m, 2H, CH ), 5.16–5.20 (m, 1H, CH), 5.71 (d, 1H, J = 8.1 Hz, CH=CH), 6.16 (dd,
2
H, J = 6.2, 8.2 Hz, CH), 7.66 (d, 1H, J = 8.1 Hz, CH=CH), 11.40 (s, 1H, NH). C-NMR (DMSO-d6):
13
δ = 21.05, 21.23, 36.18, 64.10, 74.38, 81.65, 84.91, 102.72, 140.83, 150.83, 163.44, 170.47, 170.61.
(ii) 3’,5’-Bis-O-acetyl-4-thio-2’-deoxyuridine (1ii). To a solution of 3’,5’-bis-O-acetyl-2’-
deoxyuridine (818 mg, 2.62 mmol) in toluene (43 mL ) Lawesson’s reagent (636 mg, 1.57 mmol)
◦
was added. The reaction mixture was stirred for 2 h at 90 C. After the reaction was completed (TLC),
the mixture was quenched with sodium bicarbonate and extracted with chloroform. The organic phase
was dried (Na SO ) and evaporated under reduced pressure. The residue was purified by column
2
4
chromatography (silica gel, chloroform/methanol mixture, 10:0
→
10:1). Yield 850 mg (98%), yellowish
+
+
−
H] . UV (CH OH)
3
oil, R = 0.56 (CHCl /MeOH 9:1). MS (ESI ): m/z 329.00 [M + H] , 326.95 [M
−
f
3
1
λmax (log ε) 330 (16.50) nm. H-NMR (DMSO-d ): δ = 2.05 (s, 3H, CH ), 2.06 (s, 3H, CH ), 2.36–2.39
6 3 3
(m, 1H, CH ), 2.45–2.48 (m, 1H, CH ), 4.20–4.27 (m, 3H, CH, CH ), 5.17–5.22 (m, 1H, CH), 6.07–6.11
2 2 2
(
m, 1H, CH), 6.37 (d, 1H, J = 7.6 Hz, CH=CH), 7.57 (d, 1H, J = 7.6 Hz, CH=CH), 12.78 (s, 1H, NH).
1
3
C-NMR (DMSO-d6):
δ = 21.04, 21.22, 36.59, 64.02, 74.30, 82.10, 85.91, 113.39, 136.16, 148.08, 170.47,
1
70.60, 190.75.

Molecules 2017, 22, 672
9 of 16
(
iii) 4-Thio-2’-deoxyuridine (2). A mixture of 3’,5’-bis-O-acetyl- 4-thio-2’-deoxyuridine (840 mg,
2.55 mmol) and 1M sodium methylate (5.1 mL) was stirred for 30 min at room temperature.
The reaction was monitored with TLC. The crude reaction mixture was purified by reverse phase
column chromatography (C-18 cartridges, water/methanol mixture, 10:0
→
10:2). The solvents were
◦
removed under reduced pressure to afford yellowish solid reaction product, mp 147–150 C. Yield 438
+
−
mg (70%), R = 0.19 (CHCl /MeOH 9:1). MS (ESI ): m/z 242.95 [M − H] . UV (H O) λmax (log ε) 332
f
3
2
1
(
16.50) nm. H-NMR (DMSO-d ):
δ
= 2.03–2.06 (m, 1H, CH ), 2.11–2.15 (m, 1H, CH ), 3.56–3.61 (m,
6
2
2
2
H, CH ), 3.79–3.84 (m, 1H, CH), 4.20–4.24 (m, 1H, CH), 4.93 (bs, 1H, OH), 5.25 (bs, 1H, OH), 6.09 (t,
2
1
H, J = 6.6 Hz, CH), 6.25 (d, 1H, J = 7.4 Hz, CH=CH), 7.64 (d, 1H, J = 7.4 Hz, CH=CH), 11.79 (s, 1H,
13
NH). C-NMR (DMSO-d ):
δ
= 40.46, 49.05, 61.64, 70.70, 85.39, 88.00, 113.25, 135.39, 192.53. The NMR
6
data were consistent with those reported previously [59].
3
.3. Synthesis of 4-Thio-2'-deoxyuridine-5'-triphosphate (3)
To a suspension of synthesized 4-thio-2’-deoxyuridine (
2
, 125 mg, 0.51 mmol), tributylamine
◦
(
0
244
µL, 1.02 mmol) in trimethyl phosphate (1.5 mL) cooled to 0 C, phosphorous oxychloride (52
µL,
◦
.56 mmol) was added and the reaction mixture was stirred at 0 C for 90 min. After the reaction
L, 0.37 mmol) and 0.5 M tributylammonium pyrophosphate
solution ((NHBu ) H P O , 5.12 mL) in acetonitrile were added dropwise. After stirring for 15 min at
was completed (TLC), tributylamine (87
µ
3
2
2
2
7
◦
0
C the reaction mixture was poured into ice-water and neutralized with saturated sodium bicarbonate
solution. The reaction mixture was purified by ion exchange chromatography on DEAE-Sephadex
A25 column (30 mL) with a linear gradient (0.05–0.4 M) of LiCl as the mobile phase. The product was
eluted with 0.3 M LiCl, the solution was concentrated under reduced pressure to several mililiters
and poured into a 40 mL mixture of acetone/methanol, 4:1. The formed precipitate was collected
by centrifugation (4000 rpm, 10 min) and twice washed with a mixture of acetone/methanol, 4:1.
The nucleotide was dissolved in 2 mL of water and evaporated under reduced pressure. Slightly acidic
solution of nucleotide was neutralized with 1 M sodium hydroxide solution to pH 7.0. The synthesis
and purification afforded 0.102 mmol (20% yield) of 4-thio-2’-dUTP. Compound was quantified by
−
1
−1
cm at 332 nm. R = 0.16 (dioxane/iPrOH/H O/NH OH
f
2 4
the extinction coefficient of 16.500 M
×
+
+
−
4
:2:5:1). MS (ESI ): m/z 484.95 [M + H] , 482.90 [M-H] . UV (H O) λmax (log
ε
) 332 (16.50) nm.
2
1
H-NMR (D O):
δ
= 2.18–2.26 (m, 1H, CH ), 2.21–2.25 (m, 1H, CH ), 4.12–4.15 (m, 3H, CH , CH),
2
2
2
2
4
.50–4.54 (m, 1H, CH), 6.19 (t, 1H, J = 6.5 Hz, CH), 6.57 (d, 1H, J = 7.6 Hz, CH=CH), 7.75 (d, 1H,
J = 7.6 Hz, CH=CH). 31P-NMR (D O):
δ
= –20.39 (t, J = 18.5 Hz, P ); 10.85 (d, J = 18.6 Hz, P ); –5.19
β
−
α
2
(d, J = 18.7 Hz, Pγ).
4
3
.4. Synthesis of N -Amino-2'-deoxycytidine-5'-triphosphate (4)
To a solution of 4-thio-2’-deoxyuridine triphosphate 3)(32 mg, 0.066mmol) in water (2 mL)
hydrazine hydrate (7 mg, 0.13 mmol) was added and the reaction mixture was stirred at room
temperature for 18 h. After the reaction was completed (TLC), the reaction mixture was purified
by ion exchange chromatography on DEAE-Sephadex A25 column (20 mL) with a linear gradient
(
0.05–0.4 M) of LiCl as the mobile phase. The product was eluted with 0.25–0.3 M LiCl, the solution
was concentrated under reduced pressure to several mililiters and desalted using reverse phase
4
column C-18. The synthesis and purification afforded 0.044 mmol (66% yield) of N -amino-dCTP.
−
1
−1
Compound was quantified by the extinction coefficient of 13.700 M
×
cm at 274 nm. Rf = 0.12
+
+
−
(
dioxane/iPrOH/H O/NH OH 4:2:5:1). MS (ESI ): m/z 483.00 [M+H] , 480.95 [M
−
H] . UV (H O)
2
4
2
1
λmax (log
ε
) 274 (13.70) nm. H-NMR (D O-):
δ
= 2.60–2.68 (m, 1H, CH ), 2.73–2.81 (m, 1H, CH ),
2
2
2
4
.31–4.39 (m, 3H, CH , CH), 4.43–4.41 (m, 1H, CH), 6.38 (t, 1H, J = 6.3 Hz, CH), 6.63 (d, 1H, J = 7.9 Hz,
2
31
CH=CH), 8.09 (d, 1H, J = 7.9 Hz, CH=CH). P-NMR (D O-):
J = 15.0 Hz, P
in [61].
δ
=
−
19.09 (t, J = 14.9 Hz, P
β
);
−
10.05 (d,
2
α
);
−
5.86 (d, J = 15.0 Hz, Pγ). The NMR spectra were consistent with the data reported

Molecules 2017, 22, 672
10 of 16
0
0
3
.5. Synthesis of 5-Cyano-2 -deoxyuridine (5) and 5-Carboxy-2 -deoxyuridine (6)
To a solution of 5-cyanouracil or 5-carboxyuracil (1.32 mmol) in dichloromethane (6 mL)
hexamethyldisilazane (HMDS, 550
µ
L, 2.64 mmol) and trimethylsilyl chloride (TMSCl, 168
µ
L,
◦
1.32 mmol) were added. The mixture was heated for 4 h at 80 C. Then the mixture was cooled
to room temperature and a solution of 1,3,5-O-triacetyl-2-deoxyribose (342 mg, 1.32 mmol) in
dichloromethane (7 mL) and SnCl (309 L, 2.64 mmol) were added. The reaction mixture was
stirred for 5 h at room temperature. After the reaction was completed (TLC), the mixture was diluted
with chloroform and washed with sodium carbonate solution. The organic phase was dried (Na SO )
µ
4
2
4
and evaporated under reduced pressure. The residue was purified by column chromatography (silica
gel, chloroform/methanol mixture, 10:0 10:1). The acetylated nucleosides were de-protected with
M sodium methylate solution. The crude reaction mixtures were purified by reverse phase column
→
1
chromatography (C-18 cartridges, water/methanol mixture, 10:0→10:2). The solvents were removed
under reduced pressure to afford 5-substituted 2’-deoxyuridine derivatives 5 and 6.
◦
5
-Cyano-2’-deoxyuridine (
5
). Yield 116 mg (35%), white solid, decomposes at 220 C. R = 0.25
f
+
−
(
CHCl /MeOH 9:1). MS (ESI ): m/z 252.05 [M
−
H] . UV (CH OH) λmax (log
ε
) 277 (11.70) nm.
3
3
1
H-NMR (DMSO-d6): δ = 2.09–2.13 (m, 2H, CH ), 3.54–3.58 (m, 1H, CH ), 3.60–3.64 (m, 1H, CH),
.86–3.90 (m, 1H, CH ), 4.23–4.27 (m, 1H, CH), 6.09 (t, 1H, J = 6.3 Hz, CH), 8.64 (s, 1H, C=CH).
2
2
3
2
13
C-NMR (DMSO-d6):
δ = 40.87, 61.97, 70.81, 88.19, 88.44, 90.73, 115.19, 146.71, 149.76, 160.51.
The NMR spectra were in agreement with the data published in [62].
◦
5
-Carboxy-2-deoxyuridine ( ). Yield 140 mg (40%), white solid, decomposes at 250 C. R = 0.15
6
f
+
+
−
H] . UV (H O) λmax (log ε)
2
(CHCl /MeOH 5:1). MS (ESI ): m/z 273.00 [M + H] , 271.00 [M
−
3
1
2
75 (9.70) nm. H-NMR (DMSO-d ):
δ
= 2.28–2.35 (m, 2H, CH ), 3.78–3.82 (m, 2H, CH ), 4.19–4.23 (m,
H, CH), 4.55–4.59 (m, 1H, CH), 6.29 (t, 1H, J = 6.4 Hz, CH), 8.84 (s, 1H, C=CH). C-NMR (DMSO-d6):
= 39.17, 61.92, 70.81, 86.34, 87.73, 105.17, 148.71, 158.76, 162.53, 167.14. The H-NMR spectrum was
6
2
2
13
1
δ
1
consistent with the previously reported one [64].
3
.6. Synthesis of 5-Substituted Deoxyuridine TriphosphateDerivatives 7 and 8
Nucleoside ( or , 0.2 mmol) was dissolved in trimethylphosphate (1 mL) and cooled to 0 C.
Tributylamine (71
◦
5
6
µ
L 0.3 mmol) was added, followed by phosphorous oxychloride (30
µ
L, 0.3 mmol)
◦
5
0
min later. After several hours of stirring at 0–4 C (TLC), tributylamine (42 mL, 0.2 mmol) and
.5 M (NHBu ) H P O in acetonitrile (2 mL) were added dropwise. After stirring for 10–15 min the
3
2
2
2
7
reaction mixture was poured into water and neutralized with saturated sodium bicarbonate solution.
The crude reaction mixture was purified by ion exchange chromatography on DEAE-Sephadex A25
column (20 mL) with a linear gradient (0.05–0.4 M) of LiCl as mobile phase. The product was eluted
with 0.25–0.3 M LiCl, the solution was concentrated under reduced pressure to several mililiters
and poured into a 40 mL mixture of 4:1 acetone/methanol,. The formed precipitate was collected
by centrifugation (4000 rpm, 10 min) and washed twice with a mixture of 4:1 acetone/methanol.
The nucleotide was dissolved in water (2 mL) and evaporated under reduced pressure. Slightly acidic
solution of nucleotide was neutralized with 1 M NaOH solution to pH 7.0.
5
-Cyano-2’-deoxyuridine-5’-triphosphate (
7). The synthesis and purification afforded 0.039 mmol (19.5%
−
1
yield) of 5-cyano-2’-dUTP. Compound was quantified by the extinction coefficient of 11.700 M
cm at 277 nm. R = 0.16 (dioxane/iPrOH/H O/NH OH 4:2:5:1). MS (ESI ): m/z 491.90 [M
UV (H O) λmax (log ε) 277 (11.70) nm. H-NMR (D O): δ = 2.30–2.35 (m, 1H, CH ), 2.65–2.72 (m, 1H,
2 2
×
−
1
+
−
−
H] .
f
2
4
1
2
CH ), 4.01–4.04 (m, 2H, CH ), 4.16–4.20 (m, 1H, CH), 4.52–4.56 (m, 1H, CH), 6.12 (m, 1H, CH), 8.52
2
2
31
(
s, 1H, CH=C). P-NMR (D O):
δ
=
−
20.69 (t, J = 19.1 Hz, P
β
), –10.95 (d, J = 18.7 Hz, P
α
),
−
5.33 (d,
2
J = 19.1 Hz, Pγ).
-Carboxy-2’-deoxyuridine-5’-triphosphate (
yield) of 5-carboxy-2’-dUTP. Compound was quantified by the extinction coefficient of 9.700 M
5
8). The synthesis and purification afforded 0.041 mmol (20.5%
−
1
−1
×
cm

Molecules 2017, 22, 672
11 of 16
+
−
at 275 nm. R = 0.10 (dioxane/iPrOH/H O/NH OH 4:2:5:1). MS (ESI ): m/z 510.85 [M
−
H] . UV
f
2
4
1
(
H O) λmax (log
2
ε
) 275 (9.70) nm. H-NMR (D O, 400 MHz):
δ
= 2.31–2.37 (m, 1H, CH ), 2.65–2.72 (m,
2
2
1
H, CH ), 4.01–4.04 (m, 2H, CH ), 4.10–4.14 (m, 1H, CH), 4.49–4.53 (m, 1H, CH), 6.16 (m, 1H, CH), 8.43
2
2
31
(
s, 1H, CH=C). P-NMR (D O, 162 MHz):
δ
=
−
20.58 (t, J = 17.9 Hz, P
β),
−
10.91 (d, J = 18.3 Hz, Pα),
2
−
5.28 (d, J = 17.9 Hz, Pγ). The NMR spectra were consistent with those reported previously [76].
3
.7. Synthesis of 2-Pyridone Nucleoside Triphosphates
(
3-Hydroxy-5-(2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphosphate (dPyrTP), (3-hydroxy-
4OH
5
2
2
2
-(4-hydroxy-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphosphate (dPyr
-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr TP), (5-(4-bromo-
-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr TP), and (5-(5-carboxy-
-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr5
TP), (5-(4-chloro-
4Cl
4Br
COOH
TP) were prepared
as described previously [70].
3
.8. Incorporation of Modified Nucleotides by Terminal Deoxynucleotidyl Transferase
The DNA primer (5’-TAATACGACTCACTATAGGGAGA-3’) was purchased from Metabion
Planegg/Steinkirchen, Germany), TdT was purchased from Thermo Fisher Scientific (Vilnius,
(
33
33
−1
Lithuania). The primer sequence was 5’- P labelled by treatment with [
γ
- P]-ATP (~3000 Ci mmol ,
Perkin Elmer) using T4 polynucleotide kinase (Thermo Fisher Scientific) according to manufacturer’s
TM
recommendations. The 5’-labelled primer was desalted using Zeba Spin desalting columns (7K
MWCO, Thermo Fisher Scientific). The reaction mixtures were prepared in a total volume of 10
µ
L by
buffer contains 200 mM
potassium cacodylate, 25 mM Tris, 0.01% Triton X-100, 1 mM CoCl , pH 7.2, included in TdT kit) or 5
33
adding 5’- P-labelled primer (0.5
µL, 5 pmol), 2
µL
5 × TdT reaction buffer (1×
×
2
glutamate buffer (1
X-100, 1 mM MgCl , pH 8.2), modified dNTP (final concentration 10
distilled water. The reaction mixtures were incubated at 37 C for 5 min. The reactions were quenched
×
buffer contains 20 mM sodium glutamate, 20 mM NaCl, 10 mM DTT, 0.5% Triton
µM), TdT (0.5
µL, 50 nM) and
2
◦
with 20 µL of loading solution (95% v/v formamide, 20 mM EDTA, 0.03% w/v bromophenol blue, 0.03%
w/v xylene cyanol). Reaction products were analyzed by denaturing PAGE (15% polyacrylamide, 8 M
urea) in TBE (Tris/Borate/Ethylendiaminetetraacetic acid, EDTA) buffer (89 mM Tris, 89 mM boric
acid, 2 mM EDTA, pH 8.3) and visualized using a FLA-5100 imaging system (FUJIFILM, Tokyo, Japan).
3
.9. Formation of 4-Thio-dU-ON:Protein Cross-links
The cross-linking apparatus was constructed with slight modifications as described
previously [77]. It consisted of an ice container, 96-well plate, a sheet of parafilm and a UV light
2
source (Epileds, Tainan, Taiwan). Samples were irradiated at 365
±
5 nm (200–220 mW/cm ) 5 and
2
1
5 mm away from the surface of the light source, which provided dose of UV irradiation ~17.2 J/cm
2
and ~4.6 J/cm , respectively. A sheet of parafilm was placed over the top of 96-well plate, and taped
to the plate on all four sides. Each well was pressed to create a shallow groove. The plate was kept on
ice before and during irradiation.
33
UV cross-linking of 4-thio-dU-elongated DNA primer (5’- P-labelled) to TdT was carried out
2+
after PEX. PEX were performed in Co -containing buffer as described above. Immediately after
incubation, the reaction mixtures were chilled on ice and transferred as 10 L drops to the wells on
the parafilm tape. The ice container was placed underneath a 365 nm UV, so that the samples were
and 15 mm from the surface of the light source. Following incubation on ice the samples were
irradiated at 365 nm for 5 min. Then the samples were transferred from parafilm wells to microtubes
and quenched with 20 L of loading solution. To verify the products of elongation reactions using
-thio-dUTP and dUTP by TdT samples were examined on 15% polyacrylamide gel containing 8 M
µ
5
µ
4
urea. Alternatively, to examine potential TdT-ON cross-links generated by irradiation, the samples
◦
were supplemented with SDS loading dye, heated for 5 min at 95 C, and analyzed by electrophoresis
on a 14% w/v SDS-PAGE gel. Proteins were stained with Coomasie Briliant Blue staining solution

Molecules 2017, 22, 672
12 of 16
(
Applichem, Darmstadt, Germany). TdT and radiolabelled-ONs cross-links were visualized using an
FLA-5100 imaging system (FUJIFILM).
4
. Conclusions
We have synthesized novel modified nucleotides that can be incorporated into DNA sequences
by terminal deoxynucleotidyl transferase in the primer-extension reaction. We have succeeded
4
in the enzymatic elongation by 4-thio-2’-deoxyuridine, N -amino-2’-deoxycytidine and 2-pyridone
base-bearing nucleotides utilizing template-independent TdT, which produced the artificial DNA
0
fragments tailed with several to tens nucleotides at the 3 -end. For example, 4-thio-dU-label
has potential in studying specific proteins which interact exclusively with the DNA 3’-end,
hydrazine-bearing ONs could be successfully applied in a conjugation to improve in vivo delivery
of therapeutic ONs or immobilization, whereas halogenated-ONs seem to be irreplaceable
radiosensitizing agents. Enzymatic post-synthetic modification of ONs with various photo- and
chemically-reactive groups is in progress towards further use for the modification of DNA or RNA.
Thus, this study would open many possibilities for future applications, especially in the development
of photo-labels, sensors, and self-assembling structures.
Acknowledgments: This work was supported by the Research Council of Lithuania (project No. MIP-042/2012).
Author Contributions: R.M., J.J. and D.T. conceived and designed the experiments; D.T., J.J. and J.D. performed
the experiments; R.M., J.J., M.B., and D.T. analyzed the data; M.B. contributed reagents and materials; D.T., J.J.
and R.M. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Sample Availability: Not available.
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