Enzymatic polymerisation involving 2′-amino-LNA nucleotides

Article abstract of DOI:10.1016/j.bmcl.2012.03.073

The triphosphate of the thymine derivative of 2′-amino-LNA (2′-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2′-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2′-Amino-LNA-T in the template and 2′-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2′-amino-LNA-T nucleotides.

Full text of DOI:10.1016/j.bmcl.2012.03.073

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3522–3526
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enzymatic polymerisation involving 20-amino-LNA nucleotides
Marie W. Johannsen a, Rakesh N. Veedu a,b, , Andreas Stahl Madsen a, Jesper Wengel a,
⇑
⇑
a Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
b School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia
a r t i c l e i n f o
a b s t r a c t
The triphosphate of the thymine derivative of 20-amino-LNA (20-amino-LNA-TTP) was synthesised and
found to be a good substrate for PhusionÒ HF DNA polymerase, allowing enzymatic synthesis of modified
DNA encoded by an unmodified template. To complement this, 20-amino-LNA-T phosphoramidites were
incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of
unmodified DNA using either KOD, KOD XL or Phusion polymerases. 20-Amino-LNA-T in the template
and 20-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified
DNA, especially for sequences with multiple 20-amino-LNA-T nucleotides.
Article history:
Received 7 March 2012
Revised 19 March 2012
Accepted 21 March 2012
Available online 28 March 2012
Keywords:
20-Amino-LNA
Locked nucleic acid
Modified nucleosides
Primer extension
Triphosphate
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
The use of oligonucleotides (ONs) for such applications as drugs
or diagnostic probes is appealing but often requires ONs that are
structurally altered from natural ONs in order to improve biostabil-
ity, pharmacokinetics and function.1–5 One example is the aptamer
Macugen (Pegaptanib). Aptamers are single stranded ONs that are
folded to form a stable three-dimensional structure capable of spe-
cifically binding a target molecule with high affinity.6,7 Macugen is
modified with an inverted nucleotide at the 30-end, 20-OMe-RNA
purine nucleotides, and 20-F-DNA pyrimidine nucleotides and con-
jugated to a 40 kDa PEG to improve biodistribution.8,9
With respect to both stability and function, the conformational-
ly locked nucleotide modification 20-amino-LNA10–13 (20-amino
locked nucleic acid) has interesting characteristics. 20-Amino-LNA
monomers thus protect antisense gapmer ONs from degradation
by nucleases,14 they increase duplex stability which may improve
target affinity and specificity, and the 20-nitrogen may serve as a
handle for attachment of functional groups.11,15–19 As an example,
N20-pyrene-functionalised 20-amino-LNA monomers have been
used in the design of probes suitable for signalling of hybridisation
events.20,21
into an avidin aptamer, the binding affinity towards avidin is not
affected significantly.22 However, this study refers to aptamer
modification after initial identification of the unmodified DNA
aptamers by SELEX, only few amino-LNA incorporation sites were
studied, and it is possible that a more systematic study15,23 would
reveal optimal incorporation sites.
SELEX in the context of aptamers relies on PCR amplification of
target binders after each round of selection, gradually increasing
the proportion of active species in the library. To improve func-
tional oligonucleotide characteristics, one approach is working
from a library of chemically modified oligonucleotides.24,25 Direct
amplification of a chemically modified pool for the next round of
selection can be difficult, so it can be necessary to add intermediate
steps, such as transcription of modified oligonucleotides into natu-
ral DNA, which can then be amplified by PCR and subsequently
transcribed back into modified ONs through primer extension
using modified dNTPs. Various bicyclic and other sugar-modified
nucleosides have previously been triphosphorylated and used as
substrates for polymerases, including cyclohexenyl nucleic acids,26
LNA,27–29 and several other 20,40-linked nucleotide analogues.30,31
While these modifications are allowed both in the template strand
and as substrates in enzymatic polymerisation reactions, they may
decrease the efficiency of the reactions compared to reactions with
natural substrates and templates, and limits on their positioning
and number of incorporations may exist. With respect to LNA,
KOD and PhusionÒ HF DNA polymerases have proven among the
most successful enzymes.
Aptamers can be evolved by a process called SELEX (Systematic
Evolution of Ligands by EXponential enrichment). SELEX is an
elegant solution to the inherent difficulty in selection of individual
components from large libraries of oligonucleotides.4,6,7 It has
previously been found that when 20-amino-LNA is incorporated
We herein present the first results on DNA-dependent 20-ami-
no-LNA polymerisation and 20-amino-LNA-dependent DNA poly-
merisation. We investigated primer extension from templates
⇑
Corresponding authors. Tel.: +61 7 33654611; fax: +61 7 33654699 (R.N.V.);
tel.: +45 65502510; fax: +45 66158780 (J.W.).
E-mail addresses: rakesh@uq.edu.au (R.N. Veedu), jwe@sdu.dk (J. Wengel).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.073

M. W. Johannsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3522–3526
3523
modified with 20-amino-LNA-T to give natural DNA as well as pri-
mer extension with DNA templates with modified 20-amino-LNA-
TTP as a substrate.
20-Amino-LNA-thymine triphosphate 4 was synthesised from
the DMT protected nucleoside 1.11 One potential route would
involve deprotection of the 50-hydroxy group followed by phos-
phorylation with POCl3, treatment with tri-n-butylammonium
pyrophosphate, and neutral hydrolysis, corresponding to what
we earlier used for preparation of the triphosphorylation of
LNA.27 Here, however, we employed a slight modification of the
method described by Ludwig and Eckstein.32,33 As shown (Scheme
1), nucleoside 111 was first acetylated at the 30-hydroxy group by
reaction with acetic anhydride followed by deprotection of the
50-hydroxy group by reaction with dichloroacetic acid and
triethylsilane. The resultant nucleoside was then reacted with
2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by
a
double displacement reaction with pyrophosphate to yield a cyclic
intermediate which is first oxidised and then hydrolysed. Removal
of the 20- and 30-protecting groups under basic conditions eventu-
ally yielded the triphosphate 4 in an overall yield of 5% from 1 (see
Supplementary data for details).
Appropriate primers and templates were designed, and template
strands modified with 20-amino-LNA-T monomers were synthes-
ised by automated DNA synthesis using known 20-amino-LNA-T
phosphoramidite with the N20-functionality protected by trifluoro-
acetylation. Phosphoramidites and modified ONs were synthesised
as previously described.11,34
Figure 1. Enzymatic extension using Phusion polymerase of primer P1.2 annealed
to modified template T1.1 (43n) by Phusion or KOD DNA polymerase. 1: 43n FAM
labelled DNA marker M1.2; 2: extension reaction, dATP, dTTP, dCTP, dGTP (10 min);
3: extension reaction, dATP, dTTP, dCTP, dGTP (5 min); 4: negative control reaction,
dTTP, dCTP, dGTP (10 min); 5: primer P1.2.
We first tested the enzymatic synthesis of an unmodified DNA
strand by Phusion polymerase using a modified template (T1.1)
which contains LNA-A at positions 31 and 33 and 20-amino-LNA-
T at positions 36 and 39 (see Supplementary data for details). As
seen in Figure 1, full length product (43n) was dominant after
10 min reaction. Pausing bands are observed corresponding to
the first four–five nucleotides, possibly due to poor extension
and/or premature termination of the primer past the initial all-G
sequence, and at positions 37n (after 1st incorporation), 40n (after
2nd incorporation), 42n and 43n. When the reaction time was
extended much beyond 10 min, the (all-DNA) product began to
be degraded likely because of the exonuclease activity of the en-
zyme. As bands are also seen below the 19n band representing
the primer, some of the bands representing less than full length
product may be due to degradation products. In the negative con-
trol, a few bands were seen indicating that the growing ON product
strand can be extended past the first amino-LNA-T, but not the sec-
ond. This points to some promiscuity in the system, but impor-
tantly no full length product was observed.
phenol-chloroform extraction is omitted) a band appeared above
the full length product in the gel, that is, representing a species
with decreased gel mobility compared to the 43n product (Figs.
S3.1 and S3.2, Supplementary data). We hypothesise that this band
represents non-denatured double stranded reaction product. Due
to the presence of two LNA-A’s, two LNA-T’s, and a high GC content,
the stability of the resulting duplex is likely to be very high and the
duplex difficult to denature, especially in the presence of salts, buf-
fers, polymerase, and excess dNTPs. Other possibilities are that this
band represents an oligonucleotide–polymerase complex, or some
tertiary structure such as a G-quadruplex.
When extension reactions were performed using template T2.1
(Table 1), a 44n template with three incorporated 20-amino-LNA-T
nucleotides at positions 29, 35, and 41, non-denatured double
stranded product was not seen, even when the samples were loaded
directly from the reaction mixture without phenol-chloroform
extraction. This points toward the effect described above being
template-specific. Phusion polymerases gave full length product in
the case of reaction with all four dNTPs, as well as a (44+1)n product.
The negative control showed that almost all extension was halted
opposite to the first incorporation site (29n) or one nucleotide past
In the reaction depicted in Figure 1, the product was isolated
from the reaction mixture by phenol-chloroform extraction fol-
lowed by precipitation from ethanol. The isolated product was
redissolved in H2O (half the original reaction volume), and 1
lL
of this solution was added to 2 L of a formamide loading solution
l
and denatured by heating prior to loading onto a gel. Using this
template, if less denaturing conditions are used (such as a lower
proportion of loading solution to reaction sample, or if the
O
N
O
N
O
N
O
N
NH
NH
NH
NH
O
P
O
O
P
O
O
P
O
O
O
O
O
a
c
b
DMTO
DMTO
HO
O
O
O
O
O
O
O
O
N
N
N
NH
OH
O
O
OH
CF3
CF3
CF3
1
2
3
4
O
O
O
O
O
Scheme 1. Reagents and conditions: (a) (CH3CO)2O, pyridine, CH2Cl2, rt, 17 h. (b) CHCl2CO2H, (CH3CH2)3SiH, CH2Cl2, rt, 3 h (60% over two steps). (c) (i) 2-Chloro-4H-1,3,2-
benzodioxaphosphorin-4-one, dioxane, pyridine (ii) (HNBu3)2H2P2O7, DMF, Bu3N (iii) I2, pyridine, H2O (iv) Na2SO3 (aq) (v) H2O, NH3 (aq) (8%).

3524
M. W. Johannsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3522–3526
Table 1
Sequences of templates (T1.1–T4.1), primers (P1.1–P3.1), and markers (M1.1–2.1) used in primer extension reactions
Name (length)
Sequence
30-ATTATGCTGAGTGATATCCGGGGCCGACCCALCALCCTGGTCTGG-50
T1.1 (43)
T2.1 (44)
30-ATTATGCTGAGTGATATCCGAACAGCCATACAGGTGAAAGTCGG-50
T3.1 (35)
T4.1 (44)
30-TAAATCCACTGTGATATCTTCCTCGCTAGCGTCTC-50
30-ATTATGCTGAGTGATATCCGTTCTGCCTTTCTGGGGATAGACGG-50
P1.1 (19)
P1.2 (19)
P3.1 (21)
50-(32P)-TAATACGACTCACTATAGG-30
50-(FAM)-TAATACGACTCACTATAGG-30
50-(Cy5)-ATTTAGGTGACACTATAGAAG-30
M1.1 (43)
M1.2 (43)
M2.1 (44)
50-(32P)-TAATACGACTCACTATAGGCCCCGGCTGGGTGTGGACCAGACC-30
50-(FAM)-TAATACGACTCACTATAGGCCCCGGCTGGGTGTGGACCAGACC-30
50-(FAM)-TAATACGACTCACTATAGGCTTGTCGGTATGTCCACTTTCAGCC-30
T = Amino-LNA-T monomer, AL = LNA-A monomer, FAM = fluorescein.
it as only very faint bands are seen at >29n. Bands were also seen at
1nt intervals from 14nt to 25nt in both the reaction and the negative
control. As described above, it is not clear whether these bands
might be due to degradation or, at least partly, to shorter extension
products (Fig. 2).
Primer extension could also be carried out using KOD or KOD XL
DNA polymerase. In both cases, the negative control continued
past 40n for T1.1 (Fig. S3.2, Supplementary data), and when using
T2.1 with KOD XL, full length extension past the modified nucleo-
tides in the template required such error-prone conditions that full
length extension was also seen in the negative control reaction
(Fig. S3.5, Supplementary data).
tended to full length product within five minutes (Fig. 3,
Fig. S3.4, Supplementary data). The negative control stops at
27nt. Degradation products smaller than the primer are seen in
both the negative and positive controls even after <1 min (Fig. 3).
After 1 h, the degradation products were the only bands seen.
The modified product was also degraded, however, in this case
degradation largely stopped at position 29, showing that the 30-
50-exonuclease activity of the polymerase may degrade the modi-
fied ON but it cannot continue past the modified nucleotide
(Fig. S3.4, Supplementary data). This supports an earlier study
showing that 20-amino-LNA nucleotides protect against nucleolytic
degradation.14
We also investigated the enzymatic incorporation of 20-amino-
LNA-T into a DNA mixmer ON. An all DNA 35nt template with a
single incorporation site opposite to position 28 was used with
Phusion polymerase under the same conditions as were used for
reading 20-amino-LNA-modified templates. The primer was ex-
Next, we performed another extension experiment using a 44n
all-DNA template (T4.1, Table 1), which encodes for incorporation
of 20-amino-LNA-T opposite positions 37, 39, and 41. Phusion DNA
polymerase efficiently afforded the full length extension product
after 120 min reaction (Fig. 4).
No difference in gel migration was observed between the all-
DNA and amino-LNA incorporated products (Fig. 4, lanes 4 and 3,
Fig. 3, lanes 2 and 3) products. The faintness of the bands in the
control reaction may be due to the degradation of the control
DNA product.
Figure 3. Enzymatic extension using Phusion polymerase of primer P3.1 annealed
to template T3.1 (35n). 1: Primer 3.1. 2: Positive control, dTTP, dATP,dCTP, dGTP
(30s). 3: Extension reaction, 20-amino-LNA-TTP, dATP,dCTP, dGTP (10 min). 4:
Negative control reaction, dATP, dCTP, dGTP (30s). 5: Negative control reaction II, no
dNTPs (30s).
Figure 2. Enzymatic extension using Phusion polymerase of primer P1.2 annealed
to modified template T2.1 (44n). 1: 44n FAM labelled marker M2.1. 2: Extension
reaction, dATP, dTTP, dCTP, dGTP (10 min). 3: Negative control reaction, dTTP, dCTP,
dGTP (10 min). 4: Primer P1.2.

M. W. Johannsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3522–3526
3525
Table 2
Summary of results
Enzyme Synthesis of DNA from template
Synthesis of ON
containing
20-amino-LNA from
DNA template
containing
20-amino-LNA
Phusion Yes
Yes
n.d.
Yesc
KOD
Yesa
KOD XL Yesb
a
b
c
Partial extension of negative control.
Partial (T1.1) or full (T2.1) extension of negative control.
Full extension of negative control. n.d. = not determined.
however, find conditions where the primer could be extended to
full length without also seeing full length product in the negative
control.
Kuwahara et al. have previously investigated the enzymatic
incorporation of very similar cyclic nucleotide analogues. Their re-
search showed that bulkiness negatively influenced the incorpora-
tion of the modified triphosphates. They also found that
incorporation of modifications was more difficult than reading a
modified template.30 We also attempted reading of templates
modified with 20-N-glycyl-amino-LNA-T. However, with the condi-
tions and polymerases used, primers could not be extended past
the first incorporation site encoded by this modified nucleotide.
Polymerisation of N20-functionalized 20-amino-LNA nucleotides
was tested using triphosphates modified with the less bulky N-
substituents methyl and acetyl, but no full length product was
observed.
Figure 4. Enzymatic extension using Phusion polymerase of primer P1.1 annealed
to template T4.1. 1: Primer P1.1. 2: Negative control reaction, dATP, dCTP, dGTP
(120 min). 3: Extension reaction, amino-LNA-TTP, dATP, dCTP, dGTP (120 min). 4:
Positive control reaction, dTTP, dATP, dCTP, dGTP (120 min).
A time-course experiment was carried out for the P1.1/T4.1 sys-
tem shown in Figure 4. The reaction with 20-amino-LNA-TTP is
much slower than that with dTTP, especially for incorporation of
the second and third modified nucleotide, and full length (44n)
product does not appear as more than a faint trace until the
60 min time point (Fig. 5). These results show that 20-amino-LNA
can be incorporated into DNA oligonucleotides using Phusion poly-
merase, but not as well or as quickly as natural nucleotides.
To avoid problems due to degradation, we also attempted DNA-
dependent 20-amino-LNA polymerisation using T3.1 and KOD XL
polymerase, which has previously been successful in the case of
LNA triphosphates.29,30 Using this template, we could not,
In conclusion, we have successfully synthesised the 50-triphos-
phate derivative of the 20-amino-LNA-T nucleoside as the triethyl-
ammonium salt. This modified nucleotide was incorporated into
DNA strands by enzymatic synthesis with a rate depending on
the sequence and the number of incorporations. Templates chem-
ically synthesised to contain 20-amino-LNA-T nucleotides are able
to efficiently direct enzymatic synthesis of DNA oligonucleotides.
Of the polymerases assayed, Phusion is the most suitable, as it pro-
vides full length product without strong nuclease activity, and does
not extend the negative control to full length (Table 2). However,
the efficiency and ease of analysis of the reaction is template
dependent and both product degradation and partial extension of
the negative control remain challenging.
Acknowledgements
The authors would like to thank Lykke H. Hansen for excellent
technical assistance. This research was made possible by funding
from the Danish National Research Foundation, the Danish Na-
tional Advanced Technology Foundation, and the European Re-
search Council under the European Union’s Seventh Framework
Programme (FP7/2007-2013)/ERC Grant agreement No. 268776.
R.N.V. acknowledges The University of Queensland Fellowship
and Early Career Grant Schemes.
Supplementary data
Supplementary data (experimental procedure for synthesis of
20-amino-LNA-TTP, NMR spectra, additional gel images, and gen-
eral protocols for primer extension reactions) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2012.03.073.
References and notes
Figure 5. Enzymatic extension using Phusion polymerase of primer P1.1 annealed
to modified template T4.1, time-course reaction. 1–4: Extension reaction, 20-amino-
LNA-TTP, dATP, dCTP, dGTP, time points as shown at the top of the gel. 5–8: Positive
control reaction, dTTP, dATP, dCTP, dGTP, time points as shown at the top of the gel.
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