Selenium-atom-modified thymidine enhances the specificity and sensitivity of DNA polymerization and detection

Article abstract of DOI:10.1039/d0cc07922g

Nucleobase mismatches can jeopardize DNA polymerization specificity, causing mutations and errors in DNA replication and detection. Herein we report the first synthesis of novel 2-Se-thymidine triphosphate (^SeTTP), describe the single-selenium

Full text of DOI:10.1039/d0cc07922g

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Selenium-atom-modified thymidine enhances
the specificity and sensitivity of DNA
polymerization and detection†
Cite this: Chem. Commun., 2021,
57, 5434
Received 10th December 2020,
Accepted 18th February 2021
a
b
Yang Li,
and Zhen Huang*ab
Yingying Zhou,a Danyan Luo,
Zhaoyi Yang,b Lillian Ruoduo Hub
DOI: 10.1039/d0cc07922g
rsc.li/chemcomm
Nucleobase mismatches can jeopardize DNA polymerization specificity, and synthesis, since they can cause mutations and reduce DNA
causing mutations and errors in DNA replication and detection. Herein polymerization specificity.19,20 This wobble pairing needs to be
we report the first synthesis of novel 2-Se-thymidine triphosphate addressed in order to facilitate highly-specific DNA polymeriza-
(
SeTTP), describe the single-selenium atom-specific modification strategy tion and accurate detection.
(SAM) against T/G mismatches, and demonstrate SAM-assisted poly-
A T/G wobble pair is formed by the alternative hydrogen-
merization and detection with much higher specificity and sensitivity. bond (Fig. 1a) through T 2-exo-oxygen, which does not partici-
SAM can effectively suppress the formation of non-specific products in pate in the T/A base pairing.21 Since the atomic radius of
DNA polymerization and detection. Thus, SAM enhances the specificity selenium (1.16 Å) is much larger than that of oxygen (0.73 Å)
of DNA synthesis by approximately 10 000 fold, and in turn, it allows the in the same family group of elements, and Se has poor ability in
detection of clinical COVID-19 viral RNA in low copy numbers (single- hydrogen-bond formation,22 we have hypothesized that by
digit copies), while the conventional RT-qPCR does not.
substituting the 2-O atom with Se, thymidine triphosphate
modified with the 2-Se atom (SeTTP) can be used to discriminate
Studying the mechanisms of nucleic acid polymerization and against the G template during DNA polymerization, significantly
replication is important in molecular and cellular biology as increasing T specificity to the A template. The incorporated Se at
well as nucleic acid detection and diagnostics. Detection of the 2-position of thymine discourages the T/G wobble pairing
specific nucleic acids is essential for pathogen identification (Fig. 1b) while keeping SeT/A the same as canonical T/A without
and epidemic control,1–4 while nucleic acids play vital roles in any significant difference, indicated by the UV-melting study.23
living systems as genetic materials.5 In addition to the enzy- This Se atom can greatly increase the electronic and steric effects,
matic synthesis, many recent studies have demonstrated the discouraging the wobble pairing, and thereby the Se-atom sub-
non-enzymatic synthesis of nucleic acids.6,7 Since the base stitution of thymine 2-oxygen enhances the SeT/A specificity.
pairs are the basis of nucleic acid recognition,8,9 they are of
Herein we report the first synthesis of SeTTP and its
great significance in studying the mechanisms of genetic recognition-and-incorporation by DNA polymerase, offering
information storage,10,11 sequence recognition, DNA and RNA
diagnosis, DNA replication,12 RNA transcription13 and protein
translation. Furthermore, the canonical pairing and stacking of
DNA nucleobases (T/A and C/G) determine the structure and
stability of DNA duplex.14,15 The non-canonical base pairs (e.g.,
U/G wobble pair) in RNAs (such as ribozymes and viral RNAs)
diversify RNA structures and enhance RNA functions,16–18 while
they (e.g., T/G wobble pair) are not desired in DNA recognition
a Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,
College of Life Sciences, Sichuan University, Chengdu 610000, Sichuan,
P. R. China
b Szostak-CDHT Large Nucleic Acids Institute & SeNA Research Institute,
Fig. 1 The base pair structures with or without the Se replacement. (a) T/A
pair and T/G wobble pair; (b) SeT/A pair without the Se disruption, while
SeT/G wobble pair with the Se disruption.
Chengdu 610000, Sichuan, P. R. China. E-mail: huang@senaresearch.org
† Electronic supplementary information (ESI) available: Experimental section,
NMR, Maldi-Tof, sequencing data. See DOI: 10.1039/d0cc07922g
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Scheme 1 Synthesis of SeTTP and Se-DNA. (a) CH3I, DIEA, toluene, DCM,
room temperature; (b) Se, NaBH4, EtOH; (c) TCA, beta-mercaptoethanol,
EtOH; (d) tributyl ammonium pyrophosphate, 2-chloro-4H-1,3,2-benzo-
dioxaphosphorin-4-one, DMF, TBA; (e) I2, H2O, DMF; (f) triethylamine,
H2O; (g) DNA polymerization.
much higher specificity and sensitivity for DNA polymerization
and detection. Our synthesis started from 50-trityl-2-thiothymidine
1 (Scheme 1), and CH3I was used to alkylate the 2-thio-
functionality in order to activate the 2-thio-moiety and offer 2.
Compound 2 was reacted with freshly prepared NaSeH in order to
incorporate the Se functionality,23 synthesizing 3 in 80% yield,
followed by detritylation with trichloracetic acid, offering
2-seleniothymidine 4 (SeT). The generated Se-nucleoside 4 was
converted to its triphosphate 5 via the one-pot and mild
synthesis.24,25 The Se-triphosphate (5, SeTTP) was purified and
characterized by NMR, MS and HPLC to confirm its integrity and
purity, followed by DNA synthesis with SeTTP and DNA poly-
merase, such as Klenow and Taq.
To investigate the SeTTP recognition by DNA polymerases,
we designed a DNA primer (10 nt, 50 FAM) and the ‘‘standing-
start’’ template (30 nt) that allow incorporation of one ‘‘T’’ on
the underlined template ‘‘A’’ (Fig. 2a). Indeed, SeTTP offered the
extension with only one ‘‘T’’, while canonical TTP allowed the
extension with up to three ‘‘T’’s (Fig. 2b and c). In addition, our
time-course experiments showed that in the presence of TTP or
SeTTP and the other three dNTPs, DNA polymerase effectively
synthesized the full-length products (Fig. 2e and f), while it
didn’t synthesize the products in the absence of TTP or SeTTP
under the same conditions. Furthermore, it was exciting to
discover that the incorporation rate with SeTTP was approxi-
mately 95% of TTP (Fig. 2b–d). Furthermore, it is worthy to note
that in the presence of SeTTP or TTP and the other three dNTPs
Fig. 2 The SeTTP recognition and Se-DNA synthesis by DNA polymerase.
The products of DNA extension reactions were analyzed by denaturing
PAGE, offering the bands of single-stranded DNAs. (a) DNA 50-FAM primer
and template used here. (b) and (c) Primer extension by Klenow in the
presence of only TTP and SeTTP, respectively; lanes 1, 2, and 3 (negative
controls) omitted template, TTP or SeTTP, and Klenow, respectively. The
time-course experiments in lanes 4–10 contained all, including the primer,
template, Klenow, TTP or SeTTP. (d) The plot of the product formation in
(b) and (c). (e) and (f) Primer extension by Klenow in the presence of the
other three dNTPs and TTP and SeTTP, respectively; the other conditions
were identical to (b) and (c). (g) The plot of the product formation in
(e) and (f).
(dATP, dCTP and dGTP), the yield of the full-length Se-DNA was extension was observed (Fig. 3b). The formation of the wobble-
approximately 90% of that of the canonical DNA (Fig. 2e–g). extended full-length products was prevented only in the presence of
Obviously, SeTTP can be recognized almost as well as the multiple ‘‘G’’ on the templates (at least 3 ‘‘G’’). However, when
canonical TTP by DNA polymerase and its incorporation speci- SeTTP was used to replace TTP (Fig. 3c), the wobble-pairing
ficity is much higher than that of canonical TTP.
extended products were not observed. This surprising result indi-
To investigate the discrimination of SeTTP on wobble pairing, cated that SeTTP discouraged the T/G wobble pairing and polymer-
we designed DNA templates with G, GG and GGG (Fig. 3a), and ization, and the quantitative PAGE analysis is presented in Fig. 3d.
included ‘‘TC’’ before the underlined template ‘‘G, GG or GGG’’ in In addition, to study the steady-state kinetics of SeTTP incorpora-
order to allow a ‘‘running-start’’ wobble-pairing extension. We tion compared to TTP, the primer and template 1 were used for the
found that in the DNA polymerization with only dATP and dGTP investigation, in the presence of dATP and dGTP. We found that
(a negative control), the full-length product via the primer exten- the wobble-pairing extension rate of SeTTP on the G-template was
sion was not observed. When canonical TTP was added to the approximately 11 000 folds slower than that of canonical TTP
polymerization with only dATP and dGTP, the T/G wobble-pairing (Fig. 3e–g), shedding light on the high specificity of SeTTP.
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Fig. 4 Inhibition of non-specific PCR DNA amplification with SeTTP. In
lane 1–4, the PCR experiments were performed with two DNA templates
by using TTP or SeTTP with other canonical dNTPs. In lane 5–8, the PCR
DNA products from lane 1–4 were diluted 50 times, followed by an
additional 10 cycles of PCR with all canonical dNTPs.
TTP or SeTTP had identical sequences. Furthermore, SeT-DNA
can be easily converted to the corresponding canonical DNA by
a few cycles of PCR with canonical dNTPs. Our discovery may
offer a new strategy for DNA amplification, gene cloning,
reverse transcription and nucleic acid detection.
Nucleic acid detection is essential in the prevention and
control of epidemics, such as the one caused by the COVID-19
virus, which has infected over 14 million people and resulted in
six hundred thousand deaths worldwide. Unfortunately, it has
been reported that nucleic acid detection of COVID-19 viral
RNA offered a high false rate (approximately 20% false positive
and negative per detection). In order to reduce the false
diagnosis rate, we have decided to explore the COVID-19
detection with our unique SAM strategy (Fig. 5). First, we
investigated its anti-background interference by including the
viral background RNA (namely, human total RNA) in the
detection. Since the typical concentration of background RNA
in clinical COVID-19 detection is 0.25 ng mLꢀ1, we set the
detection concentration of background RNA as 0.025, 0.25,
and 2.5 ng mLꢀ1. Our experiment demonstrated that the
Se-approach can successfully suppress the nonspecific ampli-
fication (Fig. 5a). No or low fluorescence signal was observed in
the presence of supplementary SeTTP, while high signals were
observed similarly with supplementary water or canonical TTP,
and the high signals could be mis-interpreted as false positives.
Furthermore, we have found that the Se-strategy can detect
viral RNA in up to single-digit 5 copies (Fig. 5b), while the
conventional kit can detect up to 100 copies, thereby indicating
the higher sensitivity of the Se-strategy (20 folds better) than the
conventional one. Furthermore, we examined clinical COVID-19
viral RNA samples (Fig. 5c) and found that our SAM methodology
detected 1000-fold-diluted viral RNA samples, while the com-
Fig. 3 Facilitation of DNA polymerization specificity by SeTTP. The pro-
ducts of DNA extension reactions were analyzed by denaturing PAGE,
offering the bands of single-stranded DNAs. (a) DNA 50-FAM primer and
templates used in (b), (c), (e) and (f). (b) Primer extension reaction by Taq
DNA polymerase with the canonical dNTPs, including TTP. Lane 1: the
reaction without Taq (negative control); template-1 was used in lane 2–4;
lane 2: the reaction with dATP and dGTP; lane 3: with all dNTPs; lane 4:
with dATP, dGTP and TTP; template-2 was used in lane 5–7; lane 5: the
reaction with dATP and dGTP; lane 6 with all dNTPs; lane 7: with dATP,
dGTP and TTP; template-3 was used in lane 8–10; lane 8: the reaction with
dATP and dGTP; lane 9 with all dNTPs; lane 10: with dATP, dGTP and TTP.
(c) Primer extension reactions with the conditions identical to (b), where
SeTTP was used instead of TTP. (d) Relative yields of the full-length
products in lanes 4, 7 and 10 of (b) and (c). (e) and (f) The time-course
experiments of DNA synthesis on T/G mispairing (template-1 for single T/G
discrimination) in the presence of TTP and SeTTP, respectively. (g) The plot
of product formation is shown in (e) and (f).
To further demonstrate the specificity value of SeTTP in DNA mercial kits (or supplementary TTP) did not. Moreover, in order
cloning and amplification, such as PCR, we designed two to investigate the non-specific product suppression, we performed
different target fragments (Fig. 4). We found that in the absence RT-qPCR and melting-temperature studies26 and discovered that
of SeTTP, the PCR nonspecific amplification was a serious in the presence of SeTTP, the non-specific products were not
problem (indicated by the smeared by-products), while in the observed, while they were when supplemented with water or
presence of SeTTP (50%), the nonspecific products were almost TTP, indicated by multiple peaks (Fig. 5d–f). Our study indicated
completely suppressed, and the product yields were as high as the severe non-specific amplification by RT-qPCR, especially when
the canonical. Further, the PCR products were analyzed by the copy number of viral RNAs was low (Fig. 5d and e). However,
sequencing to confirm their integrity. The sequencing results non-specific amplification in nucleic acid detection can be signifi-
(see ESI,† S15–S22) have indicated that the PCR products with cantly inhibited by supplementary SeTTP (Fig. 5f).
5436 | Chem. Commun., 2021, 57, 5434–5437
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be useful in nucleic acid detection, such as COVID-19 epidemic
diagnostics, control and prevention by enhancing the sensitivity
and accuracy of nucleic acid detection.
This work was supported by the National Natural Science
Foundation of China (21761132029 and 22077089), SeNA
Research Institute, and the Fundamental Research Funds for
the Central Universities.
Conflicts of interest
There are no conflicts to declare.
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