Site-specific incorporation of multiple units of functional nucleotides into DNA using a step-wise approach with polymerase and its application to monitoring DNA structural changes

Article abstract of DOI:10.1039/C8CC09444F

We have developed a new method, a step-wise approach with polymerase, for site-specific incorporation of multiple units of functional nucleotides into DNA to form hairpin secondary structures. The fluorescence of the resulting DNA incorporating the functional nucleotides varied upon transitioning from single-strand to hairpin and duplex structures.

Full text of DOI:10.1039/C8CC09444F

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Site-specific incorporation of multiple units of functional
nucleotides into DNA using a step-wise approach with
polymerase and its application to monitoring DNA structural
changes
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Binh Huy Lea, Van Thang Nguyena and Young Jun Seoa,b,*
www.rsc.org/
complementary part. Therefore, there remains room to develop more
advanced methods for direct site-specific labeling into DNA.
We have developed a new method, a step-wise approach with
polymerase, for site-specific incorporation of multiple units of
functional nucleotides into DNA to form hairpin secondary
structures. The fluorescence of the resulting DNA incorporating
the functional nucleotides varied upon transitioning from single-
strand to hairpin and duplex structures.
DNA labeling is an essential aspect of various DNA-related
studies, including diverse biotechnology applications1–4
,
DNA
and genetic
The most common methods for introducing a
structural transformation5–7
diagnosis11–13
, ,
gene expression8–10
.
fluorophore or quencher into DNA are chemical synthesis (i.e., solid
phase synthesis)14–16 and enzymatic synthesis17–19. The chemical
synthetic method is especially practical for labeling DNA for use in
DNA model studies and DNA probing systems, because it takes
advantage of convenient procedures developed for the synthesis of
oligonucleotides. Nevertheless, this method has problems when
applied to the direct labeling of naturally produced DNA20–22; in
other words, only very short lengths of DNA can be labeled this way
because of the limited yield of each synthetic cycle. In such cases,
enzymatic labeling using DNA polymerase can be a more promising
method of direct labeling and can be applied with natural DNA,
although it remains very challenging.
Scheme 1 Schematic representation of the step-wise approach using polymerase
to incorporate the functional nucleotides
For artificial base pair systems, the most common method for
enzymatic labeling involves using polymerase to introduce the
fluorophore and quencher directly into the DNA23–26. This approach
can be a very powerful one for incorporating the fluorophore and
quencher site-selectively. Another method involves applying an
artificial base that is complementary to a natural A, T, G, or C base
in DNA27–29. Unfortunately, both methods have limitations that
affect their practical applications. The use of artificial base pairs
requires a template DNA that contains the artificial base against the
opposite position of an incoming artificial nucleotide30. Furthermore,
it can be difficult to introduce multiple artificial base pairs
sequentially into DNA because of the instability of the DNA
secondary structure31. An artificial base that is complementary to a
natural A, T, G, or C base is also limited for site-specific labeling
into DNA because the artificial base might be introduced into any
In this paper we report a method for introducing functional
nucleotides into DNA using a step-wise approach with polymerase.
Our goal was to develop a new technique that would allow us to
incorporate multiple functional nucleic acids into desired DNA
positions—both sequentially and site-specifically—and to then use
this system for the detection of DNA secondary structural
transformations. DNA trinucleotide repeat (TNR) sequences,
especially CNG repeat sequences (CAG/CTG repeat sequences), are
interspersed in the human genome and have been discovered
recently in the coding and untranslated regions32,33. These hairpin
structures are involved in many hereditary neurological diseases,
including Huntington’s disease, fragile X syndrome, myotonic
dystrophy, and spinocerebellar ataxias34–37. Accordingly, several
diverse diagnostic tools have been developed for identifying these
genetic disorders (CAG/CTG repeat sequences); for example,
fluorescence-based molecular beacons, fluorescence in situ
hybridization, electrochemical sensors, and southern blotting of the
polymerase chain reaction38, 39. A drawback of each of these
methods is the need to use the indirect labeling method to monitor
the CNG repeat sequence. Accordingly, here we focused on
aDepartment of Bioactive Material Sciences, Chonbuk National University, South
Korea.
bDepartment of Chemistry, Chonbuk National University, South Korea.
* Fax: +82-63-270-3408; Tel.: +82-63-270-3417; E-mail: yseo@jbnu.ac.kr.
Electronic Supplementary Information (ESI) available: [Full experimental details;
characterization of compounds; UV absorption and circular dichroism spectra; UV
spectroscopic melting curves for oligonucleotides]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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developing an enzymatic method, based on a step-wise approach
with polymerase, for direct and site-specific labeling of fluorescent
and quenching nucleotides into DNA—in particular, into the CNG
repeat DNA sequence—and then using the system to monitor
changes from single-strand to hairpin to duplex structures. To realize
this strategy, we developed fluorescent and quenching nucleotides
that could be recognized by DNA polymerase and introduced into
DNA using a step-wise approach.
View Article Online
DOI: 10.1039/C8CC09444F
We synthesized the fluorescent (dUpyrTP) and quencher
(dUazoTP) nucleotides by introducing a pyrene fluorophore and an
azo functional group, respectively, at the 5´-position of
deoxyuridine28. Next, we synthesized the triphosphates of these
fluorescent and quencher materials, using the Yoshikawa method40,
to check their suitability for enzymatic incorporation and extension.
We examined the fluorescence and quenching properties of these
materials from their UV–Vis and fluorescence spectra (Fig. S1).
To investigate the changes in fluorescence upon changing the
structure of the DNA, we designed a CNG repeat DNA template that
formed a hairpin secondary structure (Table 1).
Fig. 1 Denaturing PAGE characterization of the proposed strategy for the
introduction of one dUazoTP unit into the P1:T1 duplex sequence. (A)
Fluorescence and (B) staining (EtBr) images of the gel. (a) Primer P1; (b) Template
T1; (c) after incorporating one dUazoTP unit; (d) after extending with dATP, dGTP,
and dCTP; e) full-length product with incorporation of dUpyrTP; (f) full-length
product after treatment with Lambda exonuclease. Primer P1 (0.1 mM) and
Template T1 (0.1 mM) were annealed to form a Primer P1:Template T1 duplex,
by heating to 95 °C and then slowly cooling to room temperature. nPfu DNA
polymerase (2 U) was then added with 2 mM dUazoTP and then the system was
incubated at 37 °C for 12 h; this sample was purified using a QIAquick nucleotide
removal kit (step 1). nPfu DNA polymerase (2 U) and 2 mM dATP, dGTP, and dCTP
were added to this sample, which was then incubated at 37 °C for 12 h and
purified using a QIAquick nucleotide removal kit (step 2). nPfu DNA polymerase (2
U) and 2 mM dUpyrTP were added into the products from step 2 and then the
system was incubated at 37 °C for 12 h and purified using a QIAquick nucleotide
removal kit (step 3). The full-length product was treated with Lambda
exonuclease (5 U, glycine/KOH buffer, 37 °C, 30 min) (step 4). All polymerase
extensions were stopped by adding twice the amount of stop buffer (10 mM
EDTA, 10 mM NaOH, 0.1% xylene cyanol, and 0.1% bromophenol blue in
Table 1: Designed Oligonucleotides
Sequence
5´–3´
Primer P1
ATC CAA GGA TGC ATT
[Phosphate] A CTG CTG CTG CTG CTG CTG
CTG CTG CTG CTG A AAT GCA TCC TTG GAT
[Phosphate] A CTG CTG CTG CTG CTG CTG
Template T1
Template T2 CTG CTG CTG CTG AA AAT GCA TCC TTG
GAT
formamide). All product solutions were loaded onto
a 20% denaturing
[Phosphate] A CTG CTG CTG CTG CTG CTG
Template T3 CTG CTG CTG CTG AAA AAT GCA TCC TTG
GAT
polyacrylamide gel.
the DNA, the primer-extended DNA product was separated again
(using the nucleotide removal kit) and then dUpyrTP was
introduced through the step-wise approach. As expected, a new band
appeared at a position higher than that of the template, with a
corresponding blue fluorescence signal at the same position,
confirming that the fluorescent dUpyrTP unit was indeed introduced
into the DNA when using DNA polymerase (Fig. 1A-B, lane e). In
the next step, we used Lambda exonuclease to remove the template
To confirm that multiple labeling was possible when using DNA
polymerase, we purchased (from Bioneer) three different
templates—the oligonucleotides T1, T2, and T3—that could be
converted into hairpin structure by the CTG repeat sequence. To
examine the multiple incorporation of functional nucleotides, we
introduced one, two, or three deoxyadenosine bases into the DNA
template that was complementary to the 3´-end of the primer, to
introduce the dUazoTP quencher units sequentially. We also
designed DNA templates that contained the deoxyadenosine units at
the 5´-end so that the pyrene-attached deoxyuridine (dUpyrTP)
units could be incorporated into the complementary positions. T1
was a sequence capable of introducing one dUazoTP unit and one
dUpyrTP unit; T2 could introduce two dUazoTP units and one
dUpyrTP unit; and T3 could introduce three dUazoTP units and
one dUpyrTP unit. With this approach, we expected to observe
different quenching effects, depending on the number of quencher
units, when monitoring the structural transformations. We used P1
as a primer; through screening, we selected nPfu special polymerase
for DNA walking. The DNA template and primer were annealed and
then DNA polymerase (nPfu special polymerase) and dUazoTP
were added for the step-wise approach using polymerase; the DNA
product was isolated using a QIAquick nucleotide removal kit.
The success of the step-wise approach using polymerase was
confirmed using gel electrophoresis (Fig. 1B). After the first step
with dUazoTP, we added dATP, dGTP, and dCTP into the product
and performed the next step. At this stage, we confirmed the success
of the step-wise approach strategy through the observation (gel
electrophoresis) of a new band at a position slightly higher than that
of the template DNA (Fig. 1B, lane d).
DNA selectively (because it featured, at the 5´-end,
a
monophosphate that could be recognized by Lambda exonuclease)41.
Indeed, when using Lambda exonuclease, only the template DNA
that contained the monophosphate at the 5´-end was removed, and
the presence of the remaining full-length product was confirmed
through gel electrophoresis (Fig. 1A-B, lane f).
In the next step, we examined the multiple incorporation of
dUazoTP into the DNA during the step-wise process. We also
performed the same step-wise approach using the T2 and T3
sequences. Fig. S2 reveals similar patterns of results for the
incorporation of dUazoTP when using the sequences T2 and T3.
When employing T2 and T3 as templates, we observed fully
extended DNA after the sequential introduction of two or three
dUazoTP quenchers and one dUpyrTP unit. These results
confirmed that DNA walking was an efficient process for the
continuous introduction of dUazoTP quencher units into DNA.
To confirm the successful introduction of the quencher dUazoTP
and the fluorophore dUpyrTP during the step-wise approach, we
radio-labeled 32P into the primer and performed the step-wise
approach using an otherwise identical process. After the introduction
of dUazoTP, we detected a new band of slower mobility (Fig. 2,
lane 2). We also observed much slower patterns of mobility upon
increasing the number of incorporated dUazoTP units (Fig. 2, lanes
5 and 8) based on the templates T1, T2, and T3. These results
confirmed that dUazoTP was indeed introduced into the DNA when
using the DNA polymerase.
We presume that the slightly slower mobility in the gel was due
to the hydrophobicity of the slightly bulky dUazoTP quencher. Our
results indicated that the dUazoTP quencher could indeed be
introduced and extended into the DNA when using the nPfu special
polymerase. To introduce the fluorescent dUpyrTP nucleotide into
2 | J. Name., 2012, 00, 1-3
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Fig. 3 (A) CD spectra of the C-T1 product. (B) Melting temperature of the C-T1
hairpin structure. All DNA hairpin state samples were prepared in 25 mM Trizma
buffer (50 mM NaCl, 10 mM MgCl2) at pH 7.2. DNA single-strand samples were
prepared in only Tris buffer.
We also examined the change in fluorescence of the pyrene
fluorophore in response to the structural transformations of the DNA.
We expected to observe quenching from the hairpin structure,
because the distance between the pyrene fluorophore and the azo
quencher would be sufficiently close for quenching. Indeed, we
observed dramatically different quenching properties during the
change from a single strand to a hairpin duplex (Fig. 4). We also
added the fully matched complementary sequence to the hairpin
structure to confirm that the change in the fluorescence signal
originated from the structural transformation. We observed a
Fig. 2 Gel electrophoresis characterization of the proposed strategy. Lane 1:
Primer P1; Lane 2: Primer P1:Template T1 + one dUazoTP; Lane 3: Primer
P1:Template T1 + one dUazoTP + dATP, dGTP, dCTP; Lane 4: Primer P1:Template
T1 + one dUazoTP + dATP, dGTP, dCTP + one dUpyrTP; Lane 5: Primer
P1:Template T1 + two dUazoTP; Lane 6: Primer P1:Template T1 + two dUazoTP +
dATP, dGTP, dCTP; Lane 7: Primer P1:Template T1 + two dUazoTP + dATP, dGTP,
dCTP + one dUpyrTP; Lane 8: Primer P1:Template T1 + three dUazoTP; Lane 9:
Primer P1:Template T1 + three dUazoTP + dATP, dGTP, dCTP; Lane 10: Primer
P1:Template T1 + three dUazoTP + dATP, dGTP, dCTP + one dUpyrTP. Primer P1
was labeled with 5´-[32P]. DNA Primer P1 (5´-labeled with 32P; 0.5 mM) and
Template T1, T2, or T3 (1 mM) were annealed to form the Primer P1:Template X
duplex, by heating to 95 °C and then slowly cooling to room temperature. nPfu dramatic recovery of fluorescence upon formation of the duplex
DNA polymerase (2 U) was added with 2 mM dUazoTP, the system was incubated
at 37 °C for 12 h, and then this sample was purified using a QIAquick nucleotide
removal kit (step 1). nPfu DNA polymerase (2 U) and 2 mM dATP, dGTP, and
dCTP were added to this sample, which was then incubated at 37 °C for 12 h
before being purified using a nucleotide QIAquick removal kit (step 2). nPfu DNA
polymerase (2 U) and 2 mM dUpyrTP were added into the product from step 2
and then incubated at 37 °C for 12 h before purifying using a DNA removal kit
(step 3). All polymerase extensions were stopped by adding twice the amount of
stop buffer (10 mM EDTA, 10 mM NaOH, 0.1% xylene cyanol, and 0.1%
bromophenol blue in formamide); the solution was then loaded onto a 20%
denaturing polyacrylamide gel.
structure (Fig. 4). Thus, monitoring DNA structural transformations
was indeed possible after introducing these fluorescent and quencher
substances using the step-wise approach.
We compared the quenching efficiencies with respect to the
number of dUAzoTP quencher units. When two or three quenchers
were present, we found—unexpectedly—that the quenching
efficiency did not increase proportionally. Indeed, we observed only
small variations in the fluorescence spectra upon varying the number
of quenchers (Fig. S5).
350
C-T1 single
C-T1 hairpin
C-T1 hairpin + target
We also examined the extension behavior using the natural
nucleotides dATP, dGTP, and dCTP (Fig. 2, lanes 3, 6, and 9); we
observed extended products from all of the templates T1, T2, and T3,
with different numbers of dUazoTP units. Interestingly, the multiple
incorporation of dUazoTP did not interfere with the full-length
DNA product. Finally, we introduced dUpyrTP and observed
slightly higher mobility compared with that of the extended product
(Fig. 2, lanes 4, 7, and 10). Thus, we confirmed that dUazoTP and
dUpyrTP had been introduced smoothly into the DNA during the
step-wise process.
Next, we examined the possibility of using these full-length
products to monitor DNA secondary structural changes. For this
experiment, we increased the amount of the full-length product (C-
T1, C-T2, C-T3) subjected to treatment with Lambda exonuclease
and then purified the products using HPLC; we then attempted to
convert the single-strand DNA to a hairpin DNA structure by
applying specific salting conditions. The conversions of these
structures were confirmed using circular dichroism (CD)
300
250
200
150
100
50
0
450
500
550
600
Wavelength (nm)
Fig. 4 Fluorescence spectra of C-T1 on single strand state, hairpin state and
duplex with complementary sequence in 25mM Trizma buffer (50mM NaCl,
19mM MgCl2, pH 7.2) at 25 oC. DNA single strand sample were prepared in only
Tris buffer.
Next, we examined the generality of this step-wise approach with
polymerase by examining the behavior after incorporating a 32-base
homogeneous continuous dA sequence and dC rich sequence, as a
potential hindrance, into the template. To apply the step-wise
approach for homogeneous continuous dA, we designed three
templates of different lengths (Template homoA-1, homoA-2,
spectroscopy (Figs. 3A and S3), which provided clear evidence for homoA-3). We incorporated dUazoTP into the first position using
template homoA-1; next, we used template homoA-2 for the
extension; finally, for incorporation of dUpyrTP, we used template
homoA-3. Figure S6 reveals that clear site-specific incorporation
occurred when using our step-wise approach with polymerase. To
apply the step-wise approach for dC rich sequence, we designed
template G4-1 and template G4-2. We also observed that our system
operate for the dC-rich sequence as shown Figure S7. This result
confirmed that our system could operate even when using
the DNA hairpin duplex structure (a strong negative band at 260 nm;
a positive band at 275 nm)42. In contrast, no specific positive or
negative band appeared in the single-strand state. Measured melting
temperatures confirmed the structure of the hairpin duplex. We
observed no melting temperature from the single strand, but
measured a melting temperature of 77.6 °C for the hairpin structure
(Figs. 3B and S4).
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7.
8.
S. Takahashi, K. T. Kim, P. Podbevsek, J. Plavec, B. H. Kim and N.
homogeneous A sequences and dC rich sequences (see Supporting
Information).
Sugimoto, Journal of the American Chemical SociVeiteyw, A2r0tic1l8e,O1nl4in0e,
DOI: 10.1039/C8CC09444F
5774.
We also measured the yield of full length products using
quantitation method by ethidium bromide fluorescence emission
(Table S1). Interestingly, we observed decreasing tendency of yield
depends on the increasing the number of dUazoTP (36.78% for C-
T1, 25.0% for C-T2, 16.1% for C-T3). We also observed lower
yield (14.4%) using template G4-1 and G4-2 than homoA1-A3 (23.9
%). Truncated products were detected from C-T2 and C-T3 and
template G4-1 and G4-2, while C-T1 and homoA1-A3 sequence did
not show any truncated product. We suspect that the low yield of
multiple dUazoTP incorporated full length product and G-C rich full
length product using template G4-1and G4-2 (G-C rich full length
product has more probability to form the non-canonical DNA
secondary structure than A-T rich full length product using homo
A1-A3), may be originated from hindrance of non-canonical DNA
secondary structure when the polymerase binds to these modified
DNA. Lower yield of homoA1-A3 (23.9%) sequence relative to the
C-T1 (36.7%) might be from multiple use of Lambda exonuclease (3
times) to remove the template in each step ( homo A1, A2, A3).
From this result, we believe that the secondary structure incorporated
by functional nucleotides and the number of step would be critical
factor to obtain the high yield of full length product using this
method.
In conclusion, we have developed a novel direct enzymatic DNA
labeling method based on step-wise approach using polymerase.
This method employs DNA polymerase to perform the step-by-step
incorporation of functional nucleotides into DNA. We applied
dUazoTP as the quencher and dUpyrTP as the fluorophore in the
step-wise process, and incorporated them, using this direct and site-
specific labeling method, into the CNG repeat sequence, which
forms a hairpin structure, to allow monitoring of the structural
transformations of DNA. The incorporation of multiple units of
dUazoTP and dUpyrTP was possible when using this approach for
direct labeling. Our direct and site-specific method for labeling DNA
with dUazoTP and dUpyrTP resulted in dramatic signal quenching
upon the transformation from single-stranded to hairpin structures,
with the fluorescence recovering after adding the perfect
complementary sequence for the formation of the duplex. This step-
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Acknowledgment
This study was supported by the Basic Science Research Program
through the National Research Foundation of Korea
(2017R1A2B4002398), funded by the Republic of Korea.
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4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C8CC09444F
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We have developed a new method, a step-wise approach with polymerase, for site-specific incorporation of
multiple units of functional nucleotides into DNA to form hairpin secondary structures. The fluorescence of the
resulting DNA incorporating the functional nucleotides varied upon transitioning from single-strand to hairpin and
duplex structures.