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University of Groningen
Modular assembly of functional DNA-based systemsSancho Oltra, Núria
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Chapter 5
An ATP sensor based on DNA-directed split
mDHFR reassembly
In this chapter the creation of an ATP sensor based on the triggering of an enzymatic
reaction by DNA hybridization is described. The approach makes use of an ATP aptamer
which liberates a DNA sequence after molecular recognition, necessary for the reassembly
of the split enzyme murine dihydrofolate reductase (mDHFR). The importance of the
stability of the folded structures is discussed and different DNA sequences are tested.
Chapter 5
98
5.1. Introduction
5.1.1. Molecular sensors
Changes in the concentration of analytes in living organisms are controlled by allosteric
proteins that respond to the binding of effector molecules. These binding events thus
result in an enhancement or reduction of protein activity depending on the composition
of the environment. The mechanism that allosteric proteins follow to reestablish the
balanced conditions involves structural changes that trigger a specific response.[1]
This
principle of translation of a binding event into a detectable signal is followed by many
biosensors. The diversity of existing biosensors is very broad and has inspired the creation
of mimics mostly for biomedical applications in the diagnosing and sensing of the
function, chemistry and structure of cells and tissues and in toxicity analysis. The current
(bio)chemical tools allow for the creation of molecular sensors with tunable features and
enhanced affinity and specificity for numerous substrates. Many examples of molecular
sensors using peptides,[2]
conjugated polymers[3]
and peptide nucleic acids[4]
have been
reported.
5.1.2. DNA in the development of biosensors
Interesting approaches in the development of biosensors involving the use of DNA have
been investigated.[5, 6]
DNA represents a particularly attractive molecule for this purpose
due to the specificity in the hybridization of the nucleobases that allows for the
construction of complex structures[7, 8]
with different functionalities[9]
in a predictable
manner. Besides the duplex conformations that DNA can present by the specific
hybridization between nucleobases, DNA is able to adopt alternative tertiary structures
capable to specifically recognize targets such as small molecules,[10]
proteins,[11]
viruses[12]
and cells.[13]
These nucleic acids are known as aptamers. Even though most of the known
aptamers are RNA-based,[14-16]
many different DNA aptamers have been identified over
the years via in vitro selection and their application as functional molecular sensors have
been broadly investigated.[17-20]
Although mostly used for biomedical purposes, some
examples in the literature showed their application in the control over the formation of
complex nucleic acid-based architectures.[21, 22]
These approaches are based in the small-
molecule-triggered liberation of a so-called toehold sequence (pale gray in I) (Figure 1).
Thus, relying on the formation of kinetically driven DNA architectures, more complex
DNA-based structures are therefore favored to assemble. Hence, the structural change
from the unbound aptamer, known as misfolded aptamer, to the folded aptamer is
induced by the binding of a ligand. In this way, the response of the system to chemical
stimuli results in the assembly of complex and functional DNA nanostructures that can be
identified by gel electrophoresis.
An ATP sensor based on DNA-directed split mDHFR reassembly
99
Figure 1. Schematic representation of small molecule-triggered DNA hybridization. I corresponds to the
initiator strand which is able to hybridize to the hairpin A after ligand recognition by liberation of the
toehold sequence (pale gray in I).
These approaches represent very elegant examples of signal amplification by triggering
the hybridization of DNA strands. However, research in which the signal amplification
involves the triggering of an enzymatic reaction using DNA as signal transducer remains
rather unexplored. To date, the research carried towards this purpose has been limited to
the use of ribozymes[23]
and deoxyribozymes.[24]
The use of enzymes, which can catalyze
reactions at very low concentrations of analytes, would allow for the development of
highly sensitive molecular sensors. This is particularly useful in toxicity analysis for
example, where the detection of analytes present in very low concentrations is required.
In this chapter, the development of a molecular sensor based on the triggering of an
enzymatic reaction by small molecule recognition using DNA as the signal transducer is
investigated.
5.2. Design
The design of the system consists of the combination of the small-molecule-triggered
DNA hybridization concept with the split-enzyme reassembly approach described in the
previous chapter (Figure 2). Once the DNA aptamer binds to the target molecule (the
analyte), a structural rearrangement is expected to take place. This structural change will
result in the liberation of the DNA sequence that was embedded in the misfolded
aptamer (in pale gray), the so-called toehold sequence, and which is necessary for the
total hybridization of both oligonucleotide-protein conjugates. By the total hybridization
of the conjugates, the correct reassembly of the split enzyme will be induced. The
reassembled enzyme will therefore catalyze the reaction, thus amplifying the signal of
molecular recognition. With this approach, differences in the reactivity of the system are
expected to be observed in the presence and absence of target molecule. Since the
Chapter 5
100
detection is signaled and amplified via an enzymatic reaction, a molecular sensor with a
high degree of sensitivity can be created.
analyte
3'5' 3'
3'
3'
5'
5'
SE1
SE2 3'
3'
5'
5'
SE1
SE2
5' 3'
substrate product
5'
5' 3'
3' 5'
inactive
Figure 2. General scheme of small molecule-triggered split enzyme reassembly. In the absence of target
molecule the hybridization of the protein-oligonucleotide conjugates to the template is not optimum
resulting in a wrong reassembly of the enzyme and hence, in a lower activity. In the presence of target
molecule the template is fully available for hybridization allowing for total hybridization and optimum
enzyme reassembly. SE1 and SE2 correspond to the split enzyme fragments.
The target molecule selected for this design is adenosine triphosphate (ATP). The aptamer
capable of recognizing ATP has been studied extensively for example for the creation of
molecular sensors.[25-27]
This aptamer has an equilibrium dissociation constant for ATP of
1-10 μM. In the present approach the aptamer sequence is appended to the sequence
complementary to both oligonucleotide-protein conjugates involved in the split protein
design. Additionally, a short DNA sequence (dark gray in Figure 2) is incorporated
between the aptamer and the template sequence which will assist in the formation of a
stem-looped structure when the target molecule is not present. As described before, part
of the DNA template sequence will be embedded in the misfolded aptamer when ATP is
not present, preventing the reassembly of the enzyme and therefore an inactive or less
active catalyst is obtained. Once the molecular recognition occurs, the toehold sequence
will be liberated, enhancing the total hybridization of the conjugates and the subsequent
enzyme reassembly.
An ATP sensor based on DNA-directed split mDHFR reassembly
101
Murine dihydrofolate reductase (mDHFR) which catalyzes the NADPH dependent
reduction of dihydrofolate to tetrahydrofolate (Scheme 1) was selected as the split
enzyme.
Scheme 1. NADPH dependent reduction of dihydrofolate to tetrahydrofolate catalyzed by mDHFR.
As described in the previous chapter, the catalytic reaction can be followed by the
consumption of NADPH monitored by UV-Vis measurements at 340 nm. This split
enzymatic system has been well studied in conjugation to oligonucleotides[28]
and as its
activity has been proven to be dependent on the DNA hybridization, it appears perfectly
suitable for the present approach.
The design of the aptamer sequences was based on previous studies involving ATP
aptamers.[22]
Several considerations had to be made in the design regarding the stability
of the misfolded and folded DNA-based structures. Highly stable misfolded aptamer
structures in the absence of ATP could prevent the system from rearranging once the ATP
is present, thus the liberation of the toehold sequence and therefore the optimal
reassembly of the enzyme is prevented. On the other hand, the misfolded aptamer
structure requires a minimum stability to prevent the liberation of the toehold sequence
in the absence of ATP. Thus, a balance between the stability of the DNA structures in the
presence and absence of ATP had to be found.
5.3. Results
The initial experiments were conducted using an aptamer sequence in which six
nucleobases of the template were embedded in the misfolded aptamer. Additionally, a
deletion in the sequence was incorporated to reduce the stability of the structure in the
absence of ATP (Figure 3). The experiments were carried out using equimolar amounts of
protein-DNA conjugates (Nterm-mDHFR and Cterm-mDHFR conjugates) and aptamer in 20
mM Tris-HCl buffer (pH 8.3, 300 mM NaCl, 5 mM MgCl2) containing dihydrofolate and
NADPH. The conditions used were based on the literature.[22]
The reassembly of the
enzyme was achieved following a rapid dilution protocol[29]
analogous to the procedure
described in the previous chapter. The course of the reaction was monitored by the
NADPH consumption at 340 nm. Three different experiments were performed; using
samples containing ATP, not containing ATP and with the addition of ATP during the
course of the reaction.
Chapter 5
102
ACCTTG C
G
AA
T
G
ACCTGGG
TGGAC
TGG
AGTATTGCGGAG
GAA
GG
CAGACATGTCAGCGTTCTCACCAGTC ACCTT
GC GAA TG
ACCTG
GG
TGGACT
GGAGTAT
TGCG
GA
GGAAGG
CAGACATGTCAGCGTTCTCACCAGTC
ATP
GACTGGTGAGAACGCTGACATGTCTGACCTTG3' 3'5' 5'
3'3' 5'5'
GACATGTCTGACCTTG3' 5'
GACTGGTGAGAACGCT3' 5'
1.
2.
ATP
ATP
Figure 3. Schematic representation of misfolded and folded aptamer structures in the absence and the
presence of ATP, respectively. Reassembly of the split enzyme by DNA hybridization is depicted when the
toehold is liberated after molecular binding.
The catalytic activity of the system was determined by comparison of the initial rates,
calculated from the slope of the kinetic curves monitored by UV/Vis spectroscopy. The
initial 15% of the reaction was discarded, as explained in the previous chapter. Although
the differences observed where not large, a noticeable difference in the slope of the
curves could be observed in the presence and absence of ATP (Table 1, entry 2; Figure 4).
This indicated that the presence of ATP had induced a structural change of the aptamer
liberating the toehold and allowing for the total hybridization of the protein-DNA
fragments with the subsequent enzyme reassembly.
Table 1. Initial rates for the reduction of dihydrofolate to tetrahydrofolate catalyzed by the split enzyme
mDHFR system in the detection of ATP using aptamers prehybridized in Milli Q water.[a]
Entry Bases in
toehold
Mismatches
in aptamer
deletions Initial rate with
ATP (· 10-9
M · s-1
)
Initial rate without
ATP (· 10-9
M · s-1
)
1[c]
- - - - 19.72 ± 0.41
2 6 - 1 18.52 ± 1.41 9.62 ± 1.29
3 13 4 1 - 9.19 ± 0.42
4 11 3 1 - 9.22 ± 2.13
5 16 5 1 - 11.2 ± 4.73
[a] All experiments were performed in duplicate using equimolar amounts of protein-DNA conjugates and
aptamers to a final concentration of 0.1 μM, 100 μM dihydrofolate, 100 μM NADPH, 2 mM ATP in buffer (20
mM Tris-HCl, pH 8.3, 300 mM NaCl, 5 mM MgCl2) at 25 ⁰C, unless noted otherwise. All aptamers where
prehybridized in Milli Q water 16 hours in advance. [b] Calculated from the slope of the kinetic curves at
340 nm. Calculation is based on 10% of the reaction, while discarding the initial 15%. Errors are calculated
from standard deviations. [c] 0.1 μM of fully complementary template was used.
An ATP sensor based on DNA-directed split mDHFR reassembly
103
Figure 4. Kinetic curves for the consumption of NADPH in the reduction of dihydrofolate to tetrahydrofolate
in the presence (—) and the absence (---) of ATP.
Gratifyingly, the results obtained in the presence of ATP are comparable to the ones
obtained when a fully complementary template (as described in the previous chapter)
was used in the same buffer (Table 1, entries 1 and 2). This indicates that indeed, the
binding of the aptamer to ATP results in the total liberation of the toehold sequence
providing a full DNA template necessary for the hybridization of the conjugates and
therefore, the correct reassembly of the active enzyme.
The changes observed when ATP was added while the reaction proceeded were small
(Figure 5). This result suggests that once the protein reassembly has occurred due to the
rapid dilution, the structural change of the system to a more active conformation is
difficult.
Figure 5. Kinetic curves for the consumption of NADPH in the reduction of dihydrofolate to tetrahydrofolate
in the absence (---) of ATP and with addition of ATP during the course of the reaction (—). The arrow
indicates the moment of the addition of ATP.
The liberation of the toehold sequence by addition of ATP would allow the total
hybridization of the conjugates, however, the changes undergone by the reassembled
Chapter 5
104
enzyme are apparently minimal. Possibly, the assembly of the two protein fragments in a
non-optimal conformation is not only due to the contribution of NADPH and
dihydrofolate as explained before, but also to strong hydrophobic interactions between
the protein fragments that prevent any subsequent structural change.
Despite the small difference observed in the slope of the kinetic curves, the results were
encouraging and further investigation towards the optimization of the sequence was
carried on.
In order to increase the sensitivity of the sensor, aptamer sequences that would lead to
bigger differences in activity in the presence and absence of ATP had to be designed.
Since the activity of the system when ATP was bound was already high, a reduction in the
catalytic activity had to be induced in the absence of ATP. The activity might be reduced
using more stable misfolded aptamer structures. The use of a toehold containing a higher
number of nucleobases would increase the stability of the misfolded structures due to
stronger hybridization. Furthermore, the number of available bases for the hybridization
of the enzymatic system in the absence of ATP would be smaller, thereby reducing the
driving force for enzyme reassembly. This would result in a lower enzymatic activity as
was demonstrated in the previous chapter. In order to elucidate the required stability of
the system, sequences containing a toehold with a higher number of nucleobases were
investigated. However, the stability of the misfolded aptamer has to be such that it also
allows for the liberation of the toehold sequence once bound to ATP. Thus, excessively
stable structures had to be avoided. With this purpose, different number of mismatches
was incorporated in the sequence complementary to the toehold in the misfolded
aptamer (Figure 6).
The sequences where tested in the absence of ATP under the same conditions as
described before. Similar activities to the one obtained with the first aptamer sequence
were observed when 13 (Figure 6a) and 11 nucleobases (Figure 6b) were embedded in
the misfolded aptamer (3 and 5 nucleobases available for hybridization when ATP is not
present, respectively) (Table 1, entries 3 and 4). In principle, in these cases, a lower
number of bases was initially available for hybridization of the enzymatic system and
therefore, lower activities were expected. However, the results indicate otherwise.
Apparently, the presence of mismatches resulted in the destabilization of the structures
to a larger extent than expected leading to comparable activities. The lack of stability
would result in the exposure of the toehold allowing for the enzymatic system to
hybridize with a higher number of bases in the absence of ATP and, therefore, resulting in
a higher activity. This hypothesis was supported by the results obtained when a toehold
containing all the nucleobases necessary for the hybridization of one of the protein-DNA
fragments, that is 16 nucleobases, was used (Figure 6c; Table 1, entry 5). The activity
obtained in this case without ATP was comparable to the obtained in the previous cases.
An ATP sensor based on DNA-directed split mDHFR reassembly
105
Figure 6. DNA sequences containing toehold sequences with high number of nucleobases (in pale gray) in
the absence of ATP. a) Sequence containing a toehold of 13 nucleobases and 4 mismatches, b) sequence
containing a toehold of 11 nucleobases and 3 mismatches and c) sequence containing a toehold of 16
nucleobases and 5 mismatches. Nucleobases in dark gray correspond to the blocking sequence.
Incorporated mismatches are indicated with arrows. The hairpin in the aptamer and the nucleobases
available for hybridization on the 3’ end are removed for simplification.
This suggests that indeed, the incorporation of mismatches in the aptamer results in a
large destabilization of its misfolded structure and therefore, the hybridization with the
oligo-mDHFR conjugates presents a too strong driving force. Additionally, the stability of
the folded DNA structures is known to be highly influenced by the presence of salts. Since
the presented results correspond to aptamer sequences which were prehybridized in Milli
Q water, significant changes were expected when the prehybridization of the aptamers is
done in the reaction buffer.
In order to increase the difference in the initial rates in the presence and absence of ATP
different aptamer sequences can be used. As mentioned above, the stability of the initial
and final structures is crucial for the performance of the system. Therefore, further
experiments were performed using new aptamer designs prehybridized in the reaction
buffer which contains high concentration of salts.
The DNA sequences used possessed toehold sequences of 13 nucleobases (3 nucleobases
available for hybridization with the conjugates) and contained a lower number of
mismatches in the aptamer. A deletion was not incorporated in these sequences resulting
in more stable misfolded structures. The results obtained using these sequences were
encouraging (Table 2). Although the initial rates obtained in the absence of ATP were
similar to those obtained previously, the activity of the resulting enzymatic systems by
addition of ATP turned to be slightly higher than obtained for the reassembled enzyme by
the hybridization with the fully complementary DNA template (Table 1, entry 1). This is
not only very interesting because the differences between the activities in the presence
Chapter 5
106
and absence of target molecule can be enlarged by tuning the aptamer sequence but
also, because it suggests the creation of an enzymatic system that would resemble even
closer to the wild type mDHFR.
Table 2. Initial rates for the reduction of dihydrofolate to tetrahydrofolate catalyzed by the split enzyme
mDHFR system in the detection of ATP using aptamers prehybridized in the reaction buffer.[a]
Entry Bases in
toehold
Mismatches in
aptamer
Initial rate with ATP
(· 10-9
M · s-1
) [b]
Initial rate without ATP
(· 10-9
M · s-1
) [b]
1 13 2 35.11 ± 5.90 14.27 ± 1.05
2 13 1 29.30 ± 5.21 10.73 ± 2.03
3 13 - 20.00 ± 2.62[c]
9.16 ± 0.95[c]
[a] All experiments were performed four times using equimolar amounts of protein-DNA conjugates and
aptamers to a final concentration of 0.1 μM, 100 μM dihydrofolate, 100 μM NADPH, 2 mM ATP in buffer (20
mM Tris-HCl, pH 8.3, 300 mM NaCl, 5 mM MgCl2) at 25 ⁰C, unless noted otherwise. All aptamers where
prehybridized in the reaction buffer 16 hours in advance. [b] Calculated from the slope of the kinetic curves
at 340 nm. Calculation is based on 10% of the reaction, while discarding the initial 15%. Errors are
calculated from standard deviations. [c] Corresponds to the average of two experiments.
The results depicted in Table 2 show maximum differences in initial rates of a factor of 3
when one mismatch is incorporated in the aptamer sequence. These differences,
although not large, are significant and represent an improvement over the previous
sequences tested.
It is interesting to observe what the influence is of the reduction in the number of
mismatches in the aptamer sequence on the activity of the sensor. The decrease in the
number of mismatches from two to zero gradually reduces the activity of the system
when no ATP is present indicating that since the misfolded structure becomes more
stable, the availability for hybridization of the nucleobases in the toehold sequence gets
also reduced. Adversely, this increase in stability affects the capability of the system to
liberate the toehold sequence once the ATP is recognized as can be seen from the values
of the initial rates. The higher the number of mismatches is, the higher the activity of the
system indicating that the liberation of the toehold becomes also easier. The biggest
differences in activity found between ATP containing and non-containing samples
corresponded to the presence of one mismatch for this particular sequence (Table 2,
entry 2). These results illustrate once more how important the balance in stability
between the structures formed in the presence and absence of ATP is. The construction
of a sensor with higher sensitivity will be possible by screening multiple DNA sequences
designed to present a balance between the stabilities of the DNA structures.
Combined, these results show the potential of the presented approach for the
development of an aptamer-split protein-reassembly-based molecular sensor.
An ATP sensor based on DNA-directed split mDHFR reassembly
107
5.4. Summary and conclusions
In this chapter the DNA-based split enzyme reassembly approach was used for the
development of a molecular sensor. The studied system involves small molecule-triggered
enzymatic catalysis using an ATP aptamer appended to the DNA template necessary for
the enzyme reassembly. The results demonstrated that the design of the DNA sequence is
of major importance and a careful balance between the stability of the misfolded
aptamer in the absence of ATP and the hybridized enzymatic system is required. Despite
the challenges in the design of the optimum DNA sequences, noticeable differences in
activity were observed in the presence and absence of ATP. Further optimization of the
DNA sequence can provide more significant differences resulting in enhanced sensor
efficiency. The optimization will involve the screening of many DNA sequences, taking
into account the stability of the structures adopted before and after binding of the target
molecule.
5.5. Experimental section
General remarks
Oligonucleotides were purchased from BioTez Berlin-Buch GMbH. NADPH, adenosine 5’-
triphosphate (ATP) disodium salt hydrate and dihydrofolate were purchased from Sigma-
Aldrich. Concentration determinations were done using a Nanodrop ND-1000 from
Thermo Scientific. UV-Vis measurements were recorded on a JASCO V-560/V-570 UV-Vis
Spectrometer at 25 ⁰C. mDHFR assays were conducted in quartz cuvettes with a 1 cm
path length.
Nterm-mDHFR and Cterm-mDHFR were expressed and purified as described in the previous
chapter. Protein-oligonucleotide conjugates were synthesized and purified as described in
the previous chapter.
General procedure for the aptamer prehybridization:
The freeze-dried oligonucleotides were dissolved in the corresponding volume of Milli Q
water or Tris-HCl buffer (20 mM, pH 8.3, 300 mM NaCl, 5 mM MgCl2) to a final
concentration of 20 μM. The oligonucleotide solutions were warmed to 93 ⁰C for 15 min.,
cooled slowly to room temperature and stored at 5 ⁰C overnight.
General procedure for the ATP detection experiments:
A solution of equimolar amounts of Nterm-mDHFR and Cterm-mDHFR- oligonucleotide
conjugates in 100 mM NaH2PO4, pH 4, 100 mM Tris-HCl, 8 M urea (as obtained from the
final purification step from uncoupled oligonucleotide on the Ni-NTA column) was
prepared. 5 μL of an ATP stock solution (400 mM in buffer) (when required), 5 μL of 20
μM aptamer solution and 2 μL of NADPH stock solution (50 mM in Milli Q water) were
added to a 100 μM dihydrofolate (H2F), Tris-HCl 20 mM, pH 8.3, 300 mM NaCl, 5 mM
Chapter 5
108
MgCl2 buffer solution to obtain final concentrations of 2 mM ATP, 100 nM aptamer and
0.1 mM NADPH. The amount of buffer used corresponds to the volume needed to obtain
a total reaction volume of 1 mL. The experiment was started by the addition of the
corresponding volume of the pre-mixed conjugate solution to obtain a final concentration
of 100 nM in the reaction mixture. Progress of the reaction was monitored by following
the decrease of the NADPH absorption at 340 nm with UV-Vis spectroscopy.
Oligonucleotide sequences:
Table 1, entry 2: 5’- ACC TTG ACC TGG GGG AGT ATT GCG GAG GAA GGT CAG GTC AAG
GTC AGA CAT GTC AGC GTT CTC ACC AGT C – 3’
Table 1, entry 3: 5' - A TAT CTA ACT TCG ACC TGG GGG AGT ATT GCG GAG GAA GGT CAG
GTC AAG GTC AGA CAT GTC AGC GTT CTC ACC AGT C - 3'
Table 1, entry 4: 5' - AT CTT ACA TTG ACC TGG GGG AGT ATT GCG GAG GAA GGT CAG GTC
AAG GTC AGA CAT GTC AGC GTT CTC ACC AGT C - 3'
Table 1, entry 5: 5' - T ATA TGT CCG ATC TTA ACC TGG GGG AGT ATT GCG GAG GAA GGT
CAG GTC AAG GTC AGA CAT GTC AGC GTT CTC ACC AGT C - 3'
Table 2, entry 1: 5’- ATG TCT AAC TTT GAC CTG GGG GAG TAT TGC GGA GGA AGG TCC
AGG TCA AGG TCA GAC ATG TCA GCG TTC TCA CCA GTC – 3’
Table 2, entry 2: 5’- ATG TCT GAC TTT GAC CTG GGG GAG TAT TGC GGA GGA AGG TCC
AGG TCA AGG TCA GAC ATG TCA GCG TTC TCA CCA GTC – 3’
Table 2, entry 3: 5’- ATG TCT GAC CTT GAC CTG GGG GAG TAT TGC GGA GGA AGG TCC
AGG TCA AGG TCA GAC ATG TCA GCG TTC TCA CCA GTC – 3’
5.6. References
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Recommended