Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases

Article abstract of DOI:10.1093/nar/gkz008

Six 1,5-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12 I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

Full text of DOI:10.1093/nar/gkz008

Nucleic Acids Research, 2019
1
doi: 10.1093/nar/gkz008
Kinetic analysis of N-alkylaryl carboxamide hexitol
nucleotides as substrates for evolved polymerases
Marleen Renders1,†, Shrinivas Dumbre1,†, Mikhail Abramov1, Donaat Kestemont1,
Lia Margamuljana1, Eric Largy2, Christopher Cozens3,4, Julie Vandenameele5, Vitor
1,7,*
B. Pinheiro3,4, Dominique Toye6, Jean-Marie Fre`re5 and Piet Herdewijn
1KU Leuven, Rega Institute for Medical Research, Medicinal Chemistry, Rega, Herestraat 49 box 1041, 3000 Leuven,
Belgium, 2ARNA laboratory, Universite´ de Bordeaux, INSERM U1212, CNRS UMR5320, IECB, 2 rue Robert
Escarpit, 33600 Pessac, France, 3Structural and Molecular Biology Department, University College London, Gower
Street, London WC1E B6T, UK, 4Institute of Structural and Molecular Biology, Department of Biological Sciences,
Birkbeck College, University of London, Malet Street, London, WC1E 7HX, United Kingdom, 5Laboratory of
Enzymology and Protein Folding/Robotein Platform, Centre for Protein Engineering (CIP), Department of Life
Sciences, University of Lie`ge, Quartier Agora, Alle´e du six Aouˆt 13, Baˆt. B6a, 4000 Lie`ge, Belgium, 6Chemical
engineering laboratory, University of Lie`ge, Alle´e de la chimie, 3, Baˆt B6c, 4000 Lie`ge, Belgium and 7Universite´
d’Evry, CNRS-UMR8030/Laboratoire iSSB, CEA, DRF, IG, Genoscope, Universite´ Paris-Saclay, Evry 91000, France
Received November 21, 2018; Revised December 21, 2018; Editorial Decision December 28, 2018; Accepted January 21, 2019
ABSTRACT
in oligonucleotide therapy (1,2) and later for the develop-
ment of an orthogonal episome for the generation of genet-
ically contained organisms (3–5), for applications in nan-
otechnology (6,7) and for use in aptamer and aptazyme se-
lections (8,9). The six-membered 1ꢀ,5ꢀ-anhydrohexitol sugar
ring does not contain the glycosidic linkage present in
the natural nucleic acids (RNA and DNA), rendering it
chemically and enzymatically stable and a noteworthy al-
ternative for the generation of nucleic acid binders, cata-
lysts and nanomaterials (6,8,9). The evolution of DNA-
dependent HNA polymerases and reverse transcriptases
by Pinheiro et al. allowed the sequence-specific synthesis
and reverse transcription of HNA fragments necessary for
in vitro selection experiments and enabled the isolation of
functional HNA molecules, including an HNA aptamer
against hen egg lysozyme and an endonuclease HNAzyme
(8,9). Nonetheless, the negatively charged backbone, to-
gether with its general lack of functional groups, limit the
binding interactions that HNA (and also the natural nu-
cleic acids, DNA and RNA) can entertain. Base-modified
nucleotides have been introduced in selections in order to in-
crease the possible interactions of natural nucleic acids with
their binding target (10) or to increase their catalytic po-
tential (11). Due to the synthetic challenge and because of
the required high-fidelity and efficient incorporation of the
modified nucleotides into a DNA duplex overhang, how-
ever, sugar analogues that have modified bases appended
Six 1ꢀ,5ꢀ-anhydrohexitol uridine triphosphates were
synthesized with aromatic substitutions appended
via a carboxamide linker to the 5-position of their
bases. An improved method for obtaining such 5-
substituted hexitol nucleosides and nucleotides is
described. The incorporation profile of the nucleotide
analogues into a DNA duplex overhang using re-
cently evolved XNA polymerases is compared. Long,
mixed HNA sequences featuring the base modifica-
tions are generated. The apparent binding affinity of
four of the nucleotides to the enzyme, the rate of the
chemical step and of product release, plus the speci-
ficity constant for the incorporation of these modi-
fied nucleotides into a DNA duplex overhang using
the HNA polymerase T6G12 I521L are determined via
pre-steady-state kinetics. HNA polymers displaying
aromatic functional groups could have significant
impact on the isolation of stable and high-affinity
binders and catalysts, or on the design of nanomate-
rials.
INTRODUCTION
1ꢀ,5ꢀ-Anhydrohexitol nucleic acids (HNA) have been devel-
oped in our laboratory, first for their potential applications
*To whom correspondence should be addressed. Tel: +3216322657; Fax: +3216330026; Email: piet.herdewijn@kuleuven.be
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
Present addresses:
Vitor B. Pinheiro, KU Leuven, Rega Institute for Medical Research, Medicinal Chemistry, Rega, Herestraat 49 box 1041, 3000 Leuven, Belgium.
Christopher Cozens, LabGenius Ltd, B201.3 Biscuit Factory, 100 Drummond Road, London SE16 4DG, UK.
ꢁ
C
The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work
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2 Nucleic Acids Research, 2019
onto them have rarely been used for in vitro selection strate-
gies (12).
Prism v6 was used for the analysis of the pre-steady-state
kinetics, together with Matlab for the global fitting of the
generated data.
We have synthesized six 1ꢀ,5ꢀ-anhydrohexitol nucleoside
triphosphates with 5-substituted uracil bases involving aro-
matic residues. These substitutions are linked via a carbox-
amide group to the uridine nucleotides. The 5-substituted
uridine nucleotides can provide an ambiguous hydrogen
bonding pattern via the rotation of the exocyclic carbamoyl
group, tautomerization, salt concentration (13) and the
static interaction of the aryl substituent on the carbamoyl
nitrogen can yield a better orientation of the 5-substituent
on the base (Figure 1) for increased interactions with a pro-
tein target in aptamer selections (14). Carboxamide link-
ers have been described before for the functionalization at
position-5 of the nucleobase and the subsequent isolation of
modified nucleic acid aptamers (14). However, in the litera-
ture, the synthesis of carbamoyl-modified nucleosides is de-
scribed starting from 5-iodouracil nucleosides and the con-
version to the respective modified compounds is multi-step
(15,16) or poor-yielding after Pd (II)-catalyzed carboxyami-
dation (14,17). Below, we describe an improved chemical
pathway to obtain these compounds.
Elongation reactions
All elongation reactions were carried out on a C1000 Touch
Thermal Cycler. The reactions were quenched by addition
of gel loading buffer containing 90% formamide, 50 mM
EDTA and 0.05% of orange G and heated for 10 min at
95◦C before loading onto the gel for analysis.
Annealing of primer and template
The primer was annealed 1:2 to template in 2×
ThermoPol® reaction buffer by heating to 95 ◦C for
5 min and slowly cooling down to room temperature.
Binding affinity determination
The binding affinity between the primer-template complex
P3T4 and the enzyme was determined using an octet HTX
from Pall – Forte´Bio. Samples and reagents were placed
in black, flat, polypropylene microplates (Greiner 655209),
filled with 200 ␮l/well. Eight channels were used simul-
taneously, with streptavidin biosensors (Pall – Forte´Bio
18–5019). Data were recorded using Octet Data Acquisi-
tion 8.2.0.9. After biosensors hydration in buffer (89% KB
10×, 10% ThermoPol® buffer 10×, 1% MgSO4 100 mM),
loading of biotinylated primer-template duplex P3T4 at 20
␮g/ml was realized during 600 s and remaining accessible
sites were quenched during 300 s using biocytin at 10 ␮g/ml.
A baseline in buffer was then recorded during 60 s, followed
by 600 s of enzyme association and 600 s of enzyme dissocia-
tion in buffer. Five different concentrations of enzyme were
used, ranging from 1.56 to 25 nM. Analysis was done with
Octet Data Analysis 9.2. The buffer signal was substracted
from the sample signals and kinetics were aligned according
to the baselines. A global fitting with a 1:1 model was then
performed to get Kd(DNA), kon and koff values.
The base substituents that we have synthesized are in-
spired by the successes that have been obtained using aro-
matic groups to select high affinity binding aptamers and
aptazymes (14,18–27). This is in line with the observation
that antibody binding sites often contain a multitude of aro-
matic residues in their hypervariable domains, critical for
recognition (28,29).
Here, via the generation of base-modified HNA nu-
cleotides and their polymerization into highly functional-
ized HNA sequences, we pave the way for the generation
of tight binding, chemically and metabolically stable ap-
tamers. Because the high-fidelity recognition of the modi-
fied nucleotides by polymerases is a requirement for their
use in in vitro selections, the incorporation kinetics of the
functionalized hexitol nucleotides by an engineered DNA-
dependent HNA polymerase are determined. The synthesis
and evaluation of new, hypermodified nucleotides is neces-
sary to advance the field of aptamer therapeutics and di-
agnostics as only by increasing the number of available ap-
tamer chemistries, we can gain knowledge on the attributes
that are essential for success.
Pre-steady-state burst kinetics
All pre-steady-state kinetic experiments were carried out on
an SFM-3000 Quench flow instrument from Bio-Logic with
the internal temperature maintained at 50◦C using a wa-
ter bath (5 l bath volume) circulating at 10 l/min. The first
syringe was filled with enzyme and primer-template in the
appropriate concentration in 2× Thermopol buffer. In the
second syringe the nucleotide or nucleotide analogue was
pipetted, together with MgSO4, yielding an additional 1
mM of Mg2+ in the final reaction mixture. The ageing time
of the samples varied between 5 ms and 60 s. The samples
were quenched with 0.3 M EDTA after which they were
mixed with loading dye and analysed on an 18% denatur-
ing PAGE gel. The concentrations of the various modified
compounds were as follows: hTTP: 6.25, 12.5, 25, 50 and
75 ␮M; 1c, 1d and 1f: 6.25, 12.5, 25 and 100 ␮M; 1e: 1.6,
6.25, 12.5 and 25 ␮M. The sampling times were 3, 5, 10, 20,
30 and 60 s for all compounds with the exception of 25 ␮M
1e in which case the reaction was also stopped after 0.5, 1.0
MATERIALS AND METHODS
General methods
All oligonucleotides were obtained from Integrated DNA
Technologies (Leuven, Belgium) and PAGE purified on a 15
or 20% (depending on the length of the oligo) denaturing
gel. Afterwards the oligonucleotides were lyophilized and
resuspended in Milli-Q water. ThermoPol® buffer 10× and
MgSO4 (100 mM) were purchased from NEB. Ultrapure
dTTP, PCR grade, was purchased from Qiagen. Accugel
(19:1 acrylamide/bisacrylamide) 40% from National Diag-
nostics was used to prepare all denaturing polyacrylamide
gels. A Typhoon FLA 9500 scanner (GE Healthcare, elon-
gation experiments) was used for the visualization of the
PAGE gels containing the fluorescently labelled reactions.
The ImageQuant TL™ version 8.1 image analysis software
(GE Healthcare) was used for gel band analysis. Graphpad

Nucleic Acids Research, 2019 3
Figure 1. (A) Schematic presentation of the modified nucleotides used in this study, 1a–f, (B) the aryl (R) moieties in 1a–f.
and 2.5 s. When times were larger than 2 s, samples were
withdrawn manually. All experiments were carried out in
duplicate. The complete set of data were fitted to the Equa-
tions (1–4) and (6) using the lsqcurvefit function in Matlab
9.1 and the default optimization parameters, with the two
independent variables t and [N] fixed. The initial values for
the parameters Kd(N), k2 and k3 were set to 10 ␮M, 0.8 s−1
the analytical data for the compounds and their synthetic
intermediates can be found in the Supporting Information.
The reaction scheme starts from the commercially available
1,5:2,3-dianhydro-4,6-O-benzylidene-D-allitol (2) and 1ꢀ,5ꢀ-
anhydro-4,6-O-benzylidene-3-deoxy-D-glucitol (3) building
blocks. The regioselective opening of the epoxide moiety in
(2) by the DBU salt of 5-iodouracil provides the nucleo-
side (5) (32). Alternatively, the 3-deoxy analogue of 5 (4)
was obtained via the Mitsunobu-type condensation reac-
tion between the alcohol (3) and N3-benzoyl-5-iodouracil
(33). For the generation of the base modifications, molybde-
num hexacarbonyl [Mo(CO)6] catalysed (34,35) aminocar-
bolylation was used. This catalyst itself serves as a source
of molecular carbon monoxide allowing an easy reaction
execution with 65–92% yield for the 5-substituted uracil
nucleosides of 1ꢀ,5ꢀ-anhydrohexitol (6a-e). The xanthate
ester of compounds 6a and 6b affords the correspond-
ing 3-deoxy-4,6-diols upon Barton–McCombie deoxygena-
tion and acetic acid-mediated benzylidene cleavage. These
compounds were treated with POCl3 in trimethylphos-
phate (TMP) in the presence of Proton-Sponge. Addition
of tetrabutylammonium pyrophosphate (TBAPP) directly
to the intermediate phosphorodichloridate affords the 5-
substituted hUTPs 1a–e in 30–40% yield. The nucleotide 1f
was obtained from nucleotide 1e by transfer hydrogenolysis
catalyzed by palladium on carbon in cyclohexene.
and 0.025 s−1. The values for the unknowns were solved by
minimizing the difference between the calculated and the
experimental values. The final fitted values were checked to
not depend on these initial values. The standard deviations
were computed from the corresponding diagonal elements
of the parameter covariance matrix. For the single burst ki-
netic experiment as shown in Figure 4, Graphpad Prism v6
was used for data fitting. Non-linear regression curve anal-
ysis was applied using least squares fit and initial values of
6.0 for A0, 0.001 for k1 and k2. Again, the final fitted values
were checked to not depend on these initial values.
Enzyme active site titration
The enzyme active site was titrated by incubating the en-
zyme (20 nM concentration as determined by A280) at 50◦C
with a series of DNA substrate concentrations (between
5 and 100 nM). The reaction was initiated by adding the
dTTP substrate at 200 ␮M and quenched after 50 ms us-
ing a quench-flow instrument. The concentrations of prod-
uct obtained were then fitted to the quadratic equation
[(E0 + Kd(DNA) + [DNA]) – [(E0 + Kd(DNA) + [DNA])2 –
The incorporation of 5-substituted hexitol uracil nucleotides
opposite a homopolymeric overhang in a DNA duplex using
the engineered HNA polymerases T6G12 and T6G12 I521L
(4E0[DNA])]0.5]/2 to give the effective enzyme concentra-
tion E0 and the dissociation constant of the DNA substrate
from the polymerase Kd(DNA) (30,31).
To be able to use a modified nucleotide in an in vitro selec-
tion, the faithful incorporation of this modified nucleotide
into a growing DNA duplex is required. For this reason,
we tested whether the six 5-substituted hUTPs are a sub-
strate for polymerases. We used a ten-mer polyA overhang
in a DNA primer-template duplex and two variants of the
HNA polymerase evolved by Pinheiro et al. (8); T6G12, a
RESULTS AND DISCUSSION
Synthesis of 5-substituted hexitol uridine nucleotides
Six 5-substituted hUTPs were synthesized as depicted in
Scheme 1. The detailed procedures for the synthesis and

4 Nucleic Acids Research, 2019
Table 1. The oligonucleotide sequences used
Name
Sequence
P1
P2
P3
T1
T2
T3
T4
5ꢀ-FAM-CGGATCCGTTTAAGCTAGG-3ꢀ
5ꢀ-Cy5-CAGGAAACAGCTATGAC-3ꢀ
5ꢀ-CAGGAAACAGCTATGAC-3ꢀ
5ꢀ-GGCCGCAAAAAAAAAACCTAGCTTAAACGGATCCG-InvdT-3ꢀ
5ꢀ-TGGTCCAGCATCGTGAGATCGATTACCGAACAGCACTACGTGGCTAAGTGCTTATCTCCTAGCTTAAACGGATCCG-3ꢀ
5ꢀ-mUmUmUmUAGTCATAGCTGTTTCCTG-3ꢀ
5ꢀ-CCCCAGTCATAGCTGTTTCCTG-Biotin-3ꢀ
The positions in the sequences where a thymine or uracil nucleotide is expected to be incorporated, are underlined. The template overhangs are highlighted
in bold. The template strand T1 is protected at its 3ꢀ-end with an inverted dT to prevent 3ꢀ-extension of the template strand (36). 2ꢀ-O-methyl RNA
nucleotides are indicated by ‘m’.
Scheme 1. Synthesis of the 5-substituted hUTP nucleotides 1a–f; (i) 5-iodouracil, DBU, CH3CN, 80◦C, 16 h, 63%; (ii) N3-benzoyliodouracil, PPh3,
DEAD, dioxane, 18 h, RT, 75%; (iii) NH3, MeOH, 90 min, RT, 65%; (iv) Mo(CO)6, NEt4Cl, Bu3N, RNH2, diglyme, 100–120◦C, 2 h, 65–92%; (v) 1,1ꢀ-
Thiocarbonyldiimidazole, dichloromethane, reflux, 8 h, then Bu3SnH, AIBN, toluene, 1–1.5 h; 80–82%; (vi) 80% AcOH, 40◦C, 1 h, 30–60%; (vii) POCl3,
TMP, Proton-Sponge, 0◦C, 5 h; (viii) Bu3N, (NBu4)3HP2O7, DMF, 0–25◦C, 30 min, 30–40%; (ix) Pd, cyclohexene.

Nucleic Acids Research, 2019 5
Thermococcus gorgonarius DNA polymerase variant har-
bouring 18 mutations compared to the wild type enzyme,
and T6G12 I521L, which contains an additional mutation
in the palm domain, because these enzymes are specifically
evolved to synthesize HNA. The elongation reactions were
carried out as described in detail in the Methods section.
The primer was 5ꢀ-FAM labelled (P1, Table 1) allowing visu-
alization of the elongation products after denaturing PAGE
separation. The results are shown in Figure 2.
For both T6G12 and T6G12 I521L, the compounds 1b,
1c and 1f show the best incorporation profiles. Whereas
compound 1c leads to the formation of primarily three
products: main product 1, compounds 1b and 1f lead to a
ladder of products. With compound 1c the primer is almost
entirely consumed, while with 1b and 1f still a significant
fraction of the primer remains unreacted.
A ‘no template control’ reaction was carried out for
each of the reactions, to exclude template-independent
elongation of the primer with the modified nucleotides
(Supplementary Figure S1). This shows that the template-
independent extension of the primer is higher with T6G12
than with T6G12 I521L.
Interestingly, when one compares compounds 1a and 1b,
the extra carbon in 1b seems to improve the incorporation
substantially. Likewise, a substitution in the para-position
of the phenylgroup in 1a with a hydroxyl- (as in 1f) or a
methoxy-functionality (as in 1c) greatly enhances its capac-
ity as a substrate for the enzymes. A benzyl-functionality (as
in 1e) seems to reduce this beneficial effect on the incorpora-
tion, although it still shows an improved incorporation pro-
file compared to 1a––the compound with the unsubstituted
phenylgroup. Histidine-like functionalities have been ap-
pended to nucleobases before and the resulting nucleotides
are known to be difficult substrates for polymerases (37–
40). This seems to remain the case when using evolved poly-
merases and a hexitol-backbone (1d).
continued with the optimization of the reaction conditions
using this enzyme.
The optimal Mg2+ concentration for the elongation was
determined. 5-(p-benzyloxy)benzylcarboxamide hUTP 1e
was chosen for this experiment, because the polymerase
stalls the most when incorporating this compound into a
mixed sequence (data not shown). The results are shown
in Supplementary Figure S3. The elongation seems optimal
in the presence of 2 mM extra MgSO4 in addition to the
MgSO4 (2 mM) of the Thermopol buffer 1×, i.e. at a con-
centration of 4 mM MgSO4 final.
Finally, after the optimization of the reaction tempera-
ture to cycling at 1 min at 94◦C, followed by 5 min at 50◦C
and 2 h at 65◦C, for 16 h, the compounds were incorporated
into the P1T2 duplex using T6G12 I521L (1 ␮M final con-
centration of enzyme). For all modifications, except for 1e
(truncated product) and 1f (overshooting) full-length elon-
gation can be obtained (Figure 3). Note that the sequences
containing the bulky 5-substituted hU-building blocks are
retarded on the gel compared to the HNA sequences with-
out base-substituents. The control reaction in which the
template strand is omitted, is shown in Supplementary Fig-
ure S4.
Pre-steady-state
kinetics
of
incorporation
using
T6G12 I521L and the 5-substituted hexitol uracil nu-
cleotides
For an analysis of the incorporation of the heavily modified
nucleotides into a growing DNA duplex (P2T3) by the poly-
merase T6G12 I521L, pre-steady-state kinetics were car-
ried out. The template T3 with the 2ꢀ-O-methyl RNA over-
hang immediately after the position where the modified nu-
cleotide has to be incorporated, was chosen for the kinetic
studies, to avoid misincorporation of a second modified nu-
cleotide. Given the heavily engineered polymerase that is be-
ing used for the incorporation, the likelihood of misincor-
poration is much higher than with a natural DNA or RNA
polymerase.
First, an enzyme (E) active site titration was performed
to determine the effective enzyme concentration in the poly-
merase preparation ‘E’ of 14.9 0.4 nM (74.5% active frac-
tion) and a binding affinity of the DNA substrate to the
polymerase, Kd(DNA) of 12.6 nM 0.3 nM, which is within
the range expected for wild type DNA polymerases and
very similar to the value obtained using Bio-Layer Interfer-
ometry measurements (12.14 0.1 nM) (data not shown)
(31,42).
The incorporation of 5-substituted hexitol uracil nucleotides
opposite a defined mixed sequence overhang in a DNA duplex
using in vitro evolved polymerases
Given the long-term goal of aptamer/aptazyme selec-
tions, we set out to probe the incorporation of the 5-
substituted hUTPs into mixed HNA sequences using dif-
ferent mutant polymerases. We tested a series of recently
in vitro evolved polymerases for this purpose; T6G12,
T6G12 I521L, K6G12 - the KOD DNA polymerase vari-
ant that has mutations in the same relative positions as the
6G12 TgoT derivative, and TgoT EPL and TgoT EPLH -
both variants of the TgoT enzyme that were recently de-
scribed for having improved tPhoNA polymerization activ-
ity (8,41). A template (T2, Table 1) was used that is designed
to contain all combinations of dinucleotides in such a way
that each hN has to be incorporated before and after each of
the four hNs at least once, accounting for a 57-mer overhang
in total (P1T2 duplex). 5-(p-Methoxy)benzylcarboxamide
hUTP (1c) was chosen for optimization of the reaction con-
ditions. Only with T6G12 I521L in the absence of MnCl2
a defined band of reaction product compatible with full-
length incorporation could be obtained. For this reason, we
The nucleotide incorporation by a polymerase obeys a
rather simple model (43):
In a first approximation and in order to compare the in-
corporation of the various compounds, we considered the

6 Nucleic Acids Research, 2019
Figure 2. The incorporation of hTTP (H) and the 5-substituted hUTPs 1a-f (lanes a–f) opposite a poly-dA template overhang in a DNA duplex using the
evolved HNA polymerases T6G12 I521L and T6G12. The modified nucleotides (hTTP and 1a–f) are used at a concentration of 125 ␮M. After optimization
of the reaction conditions, the enzymes are used at a final concentration of 51 nM for T6G12 I521L and 82 nM T6G12. The reactions with the enzyme
T6G12 contain 0.5 mM freshly prepared MnCl2. All reactions are carried out in 1× Thermopol buffer (NEB) supplemented with 1.5 mM MgSO4. The
reactions are incubated at 50◦C overnight. The positions where a 5-substituted hUTP has to be incorporated opposite the template oligonucleotide, are
underlined in the sequence below the gel image. The lanes indicated with ‘P’ show the primer control (no enzyme and no nucleotides added). The position
for the full-length material of HNA (signifying the incorporation of ten hT nucleotides) is indicated by ‘FL HNA’ on the side of the gel image.
chemical step (k2) to be irreversible under our experimental
conditions. Indeed, the equilibrium constant of this reac-
tion
where
k2 [N]
Kd(N) + [N]
k2ꢀ =
(6)
[EDn+1] [PPi]
For instance, Figure 4 shows the time-course of P accu-
mulation when [E]0 is 14.9 nM, [Dn]0 = 100 nM and [N] =
75 ␮M. From the [P]/[E]0 versus time curve, the following
values can be deduced using fitting of Equation (1) as de-
scribed in the Materials and Methods section:
[EDn N]
can be approximated to 20 M and, with initial nM concen-
trations of EDn, the k–2 step can be neglected. Our analyti-
cal technique measures both the enzyme-bound and the free
Dn+1 so that
λ = (0.58 0.02) s−1
[P] = [EDn+1] + [Dn+1
and, under these conditions,
]
A0 = 0.96 0.02
ꢀ
ꢁ
kss = (0.025 0.001) s−1
(P)
= A0 1 − e−λt + ksst
(1)
E0
In a first approximation, good estimations would be 0.58
s−1 and 0.026 s−1 for the values of kꢀ2 and k3, respectively.
We verified that the amplitude of the burst phase in-
creased when the enzyme concentration increased and that
all modified nucleotides in this study exhibited an initial
burst in their incorporation profiles. This indicates that a
step after the chemical one (e.g. DNA product dissociation
from the enzyme) is rate-limiting for product formation in
these modified nucleotide incorporations. Additionally, we
controlled the saturation of the enzyme with the DNA sub-
strate at the concentrations used (data not shown) (44,45).
Finally, the burst kinetics were determined for a range of
nucleotide concentrations for hTTP and the modified nu-
cleotides 1c–1f. The results are shown in Table 2, where kpol
represents the maximum rate of incorporation and Kd(N) the
dissociation constant of the complex EDnN. The large er-
rors (particularly for 1c) are due to the relatively low num-
ber of data points (due in turn to the limited amounts of
modified nucleotides available) and to the fact that, in most
In this equation, the first term represents the exponential
burst phase (accumulation of EDn+1) and the second one
(ksst) the linear steady-state phase. The values of the three
parameters (A0, λ and kss) are as follows:
k2ꢀ2
k2ꢀ + k3
A0 =
(2)
ꢀ
ꢁ
2
λ = k2ꢀ + k3
(3)
(4)
k2ꢀ k3
k2ꢀ + k3
kss
=
and
ꢀ
ꢁ
k3 k2ꢀ + k3
kss
A0
=
(5)
k2ꢀ

Nucleic Acids Research, 2019 7
Figure 4. The burst kinetic profile of the incorporation of hTTP at a
concentration of 75 ␮M into the growing duplex P2T3 (100 nM) using
T6G12 I521L at a concentration of 14.9 nM as determined via the titra-
tion of the enzyme active site. Graphpad Prism v6 was used for data fitting
as described in the Materials and Methods section (R2 value of 0.997).
least one order of magnitude lower than those of the other
compounds.
Although the Kd(N)s in Table 2 are within the expected
ranges for DNA polymerases, the incorporation rates (kpol
)
are lower than would be obtained with natural thermophilic
polymerases. This is likely the collateral damage caused by
the mutations that are needed to push the enzyme towards
HNA polymerization. Especially for the compounds 1d and
1f the incorporation rates are somewhat lower than that for
an hT building block.
Figure 3. The incorporation of the 5-substituted hUTPs together with
hATP, hCTP and hGTP into the P1T2 duplex overhang using T6G12 I
521L after cycling for 1 min at 94◦C, followed by 5 min at 50◦C and 2 h
65◦C, for 16 h in total, in Thermopol buffer 1× containing an additional
2 mM MgSO4. ‘P’ indicates the primer control. Lane H shows the hNTP
control. Lanes a + hACG to f + hACG show the incorporation of the hA,
hC and hG building blocks together with 1a–f respectively.
CONCLUSION
cases, the samples were manually pipetted, resulting in a rel-
atively large error on the sampling time. Despite the very
large errors recorded for 1c, it is interesting to note that the
We have successfully synthesized 5-substituted hexitol
uracil nucleotides, using an improved chemical synthesis
pathway compared to the previously described methods to-
wards obtaining 5-substituted nucleotides. We have then
used the modified nucleotides for polymerizing HNA se-
quences with aromatic substituents on the U-bases. Such
polymers might be important tools in the selections of ap-
tamers to difficult protein or small molecule targets, as has
been seen for DNA (18,21,24,46). The kinetic parameters
for the incorporation of four of the modified nucleotides us-
ing an in vitro evolved HNA polymerase were determined.
Despite the quite large errors on the experimental values
due to manual pipetting of the samples within a short time
frame, the data are sufficient to conclude that, with the ex-
ception of compound 1d, all compounds have a similar in-
corporation rate using the evolved enzyme and the base
substitutions do not have a large influence on the speci-
ficity of the incorporation. Although the apparent bind-
ing affinities of the enzyme for the 5-substituted hUTPs
are within the range expected for thermophilic nucleic acid
polymerases, the incorporation rates seem markedly low.
This indicates that, although the mutated polymerase has
been evolved towards HNA polymerization and does ac-
cept the 5-substituted hexitol building blocks as a substrate,
the incorporation is impeded. This information is impor-
tant for future in vitro evolution experiments. The muta-
tions that have been introduced in the evolved polymerase
k
pol/Kd(N) ratio is similar to that of hTPP. Note that, for
compounds 1a and 1b, small levels of misincorporation or
primer degradation were observed, preventing the determi-
nation of the incorporation parameters.
The deduced k3 values for compounds the 1d and 1e are
significantly lower than for the three other ones. This is sur-
prising since one would not expect such a large influence of
the added nucleotide on the dissociation rate of the EDn+1
complex. Moreover, in some cases, the kss values, when de-
termined directly from the linear portions of the curves
could be as high as 0.01 s−1 (not shown). Since kss ≤ k3
(Equation 4), the fitting procedure was repeated for 1d and
1e with fixed k3 values of 0.01 and 0.02 s−1. The value of
0.02 s−1 for the dissociation of the EDn complex has been
described before in the literature (43). The results are also
shown in Table 2 and indicate that the value of k3 has only
a minor influence on those of the other two parameters.
The ratio kpol/Kd(N) represents the specificity constant for
the incorporation during the processive synthesis, a more
accurate estimation than kcat/Km obtained under steady-
state conditions. Globally, our results show that all the mod-
ified nucleotides synthesized in this study can be incor-
porated into a DNA duplex with kpol/Kd(N) values simi-
lar to that of hTTP. The only exception is the imidazole-
substituted 1d for which the second-order rate constant is at

8 Nucleic Acids Research, 2019
Table 2. The values of the kinetic constants for the incorporation of hTTP and the nucleotide analogues 1c–1f into the growing DNA duplex P2T3 using
T6G12 I521L deduced by global fitting using Matlab 9.1 (least square method)
k2 = kpol (s−1
0.62 0.13
)
Kd(N) (␮M)
22.0
91 90
38 6.3
k
pol/Kd(N) (mM−1 s−1
)
k3 (s−1
)
hTTP
1c
1d
1d
1d
1e
1e
1e
1f
8
29
22
0.028 0.002
0.026 0.002
0.002 0.0015
0.01
0.02
0.004 0.001
0.01
2
1.8
0.09 0.05
0.07 0.04
0.05 0.005
0.29 0.04
0.25 0.05
0.15 0.04
0.12 0.01
2.4
2.2
2.1
240
104
42
32
24
5
5
1.2 0.6
2.4 1.2
3.6
2
0.02
0.015 0.001
2.4 0.2
50
For 1d and 1e, the fitting was repeated with fixed values of k3 (0.01 and 0.02 s−1, as indicated in bold).
The standard deviations were computed from the corresponding diagonal elements of the parameter covariance matrix. An example of parity plot is given
in the SI (Supplementary Figure S5).
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DNA down in the enzyme, near the active site, and plays
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of the enzyme. We previously thought the detrimental fac-
tor in the polymerase mechanism, yielding poor polymer-
ization, was the unsuccessful translocation of the enzyme
after the incorporation of a modified nucleotide. The ki-
netic studies performed here show that the polymerase is
also lacking in speed when it comes to the chemical step of
the reaction. Future evolution experiments towards improv-
ing these polymerases might, for this reason, benefit from
mutations focussed in the region surrounding (if not imme-
diately in) the active site (47). The evolution of efficient and
accurate HNA polymerases is essential to the advancement
of this field of research.
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Supplementary Data are available at NAR Online.
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Peak-Chew,S., Cozens,C., Weeks,K.M., Herdewijn,P. and Holliger,P.
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ACKNOWLEDGEMENTS
Special thanks to Chantal Biernaux for editorial help.
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FUNDING
European Research Council under the European Union’s
Seventh Framework Program (FP7/2007–2013)/ERC
[ERC-2012-ADG 20120216/ 320683 to P.H.]; FWO
Flanders Research Foundation [1247114N to M.R.]; Wal-
loon Region (SPW, DGO6, Belgium) for supporting the
ROBOTEIN project (2014–2016) within the frame of the
EQUIP2013 program [convention 1318159]. The salary
of Julie Vandenameele is currently supported by the AU-
TOBMP2 project [SPW, DGO6, convention 16100518].
Funding for open access charge: Institutional budget.
Conflict of interest statement. None declared.
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