Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA

Article abstract of DOI:10.1039/b917312a

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteenmismatched targets, and high

Full text of DOI:10.1039/b917312a

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Optimized DNA-targeting using triplex forming C5-alkynyl
functionalized LNAw
Sujay P. Sau,a Pawan Kumar,ab Brooke A. Anderson,a Michael E. Østergaard,a
Lee Deobald,c Andrzej Paszczynski,c Pawan K. Sharmab and Patrick J. Hrdlicka*a
Received (in Austin, TX, USA) 21st August 2009, Accepted 17th September 2009
First published as an Advance Article on the web 12th October 2009
DOI: 10.1039/b917312a
Triplex forming oligonucleotides (TFOs) modified with
C5-alkynyl functionalized LNA (locked nucleic acid) monomers
display extraordinary thermal affinity toward double stranded
DNA targets, excellent discrimination of Hoogsteen-
mismatched targets, and high stability against 30-exonucleases.
Substantial efforts have been invested to develop probes for use
in the antigene technology, i.e., sequence-specific targeting of
double stranded DNA (dsDNA).1 The efforts are motivated by
the prospect of developing fundamental research tools and new
types of therapeutic and diagnostic agents.2,3 (TC)-motif triplex
forming oligonucleotides (TFOs), which bind to polypurine
regions in dsDNA targets via Hoogsteen base pairs in the major
groove, are the most widely studied DNA-targeting agents,1
although promising alternatives are emerging.4 A drawback of
the TFO-approach is the instability of triplexes at physiological
pH due to electrostatic repulsion between the three strands and
insufficient N3-protonation of cytosines resulting in weak
Hoogsteen base pairing. Chemical modification of TFOs is
therefore essential to increase triplex stability as well as to
protect TFOs from enzymatic degradation.1 Three classes
of nucleotide building blocks have found particular use as
TFO-modifications: (a) ribonucleotides and O20-alkylated
analogs hereof as they favorably preorganize the TFO strand
for hybridization to dsDNA targets,5 (b) conformationally
restricted nucleotides with RNA-like furanose puckers such as
locked nucleic acid (LNA, monomer V, Fig. 1)6 or analogs
thereof7 in what is an extension of the TFO preorganization
concept, and (c) C5-alkynyl functionalized pyrimidine DNA
monomers, as they promote enhanced base stacking inter-
actions in triplexes.8 Interestingly, building blocks that combine
C5-alkynyl functionalization and RNA-character induce greater
triplex stabilization than the respective C5-alkynyl DNA and
RNA TFO monomers by themselves.9 In light of this, we
hypothesized that C5-alkynyl functionalized LNA monomers
Fig. 1 Structures of LNA and novel C5-alkynyl functionalized LNA
monomers.
X and Y (Fig. 1) would synergistically integrate beneficial
properties from two compound classes, i.e., (1) high thermal
affinity toward dsDNA and partial resistance toward nucleases
(LNA-component), and (2) resistance toward nucleases due to
the presence of steric shields along with favorable contributions
to binding affinity (C5-substituent).
The corresponding phosphoramidites10 of the C5-ethynyl and
C5-propargylamine functionalized LNA monomers X and Y and
the commercially available LNA thymine phosphoramidite were
incorporated one, three or six times into a 15-mer (TC)-motif
DNA TFO sequence (Table 1) that has previously been used to
evaluate hybridization properties of LNA,7d ENA7d and 20,40-
BNANC7e TFOs. TFOs were synthesized on automated DNA
synthesizers (0.2 mmol scale) using standard conditions except for
extended coupling (15 min, using 4,5-dicyanoimidazole as
activator) and oxidation times (45 s).w This resulted in stepwise
coupling yields of 498% for the LNA monomers. 5-Methyl-
deoxycytidine rather than deoxycytidine monomers were used to
ameliorate the normal pH dependence of (TC)-motif TFOs.11z
Following workup and purification, the composition and purity
(490%) of all synthesized TFOs were verified by MALDI-TOF
MS (Table S2)w and HPLC, respectively.w
Binding to a central target region in a 21-mer dsDNA was
first characterized by non-denaturing PAGE using equimolar
quantities of TFOs and dsDNA target in a pH 7.2 HEPES
buffer containing Mg2+. Conventional LNA and C5-ethynyl
functionalized LNA TFOs form stable triplexes with dsDNA
targets under these conditions (V- and X-series, Fig. S1)w as a
single band of lower mobility than the corresponding dsDNA
target was observed in all instances. Triplex formation between
C5-propargylamine functionalized LNA TFOs and dsDNA
target is incomplete as a band (B20–40% intensity) of identical
mobility as the corresponding dsDNA target is observed
in addition to the triplex band (Y-series, Fig. S2).w
Triplex formation was, however, driven toward completion
a Dept. of Chemistry, Univ. of Idaho, ID-83844, USA.
E-mail: hrdlicka@uidaho.edu; Fax: +1 208 885 6173;
Tel: +1 208 885 0108
b Dept. of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
c Environmental Biotechnology Institute, Univ. of Idaho,
ID-83844, USA
w Electronic supplementary information (ESI) available: Synthetic
outline of phosphoramidites, protocols for synthesis/purification of
TFOs, determination of Tm- and kon-values, and exonuclease studies;
MALDI-TOF-MS data of TFOs; thermal denaturation profiles;
PAGE experiments; absorption decay profiles; discussion regarding
parallel duplex formation. See DOI: 10.1039/b917312a
ꢀc
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Table 1 Tm-values and association rate constants (kon) for triplexes formed by C5-alkynyl functionalized LNA, conventional LNA or DNA
TFOs with dsDNA targeta
Tm [DTm/mod]/1C
kon/Mꢁ1 sꢁ1
Seq. 50 - 30
B =
V
X
Y
V
X
Y
ON
B1
B2
B3
B4
TTT TTmC TBT mCTmC TmCT
40.0 [+11.0]
48.5 [+6.5]
53.5 [+8.2]
61.0 [+5.3]
42.5 [+13.5]
54.0 [+8.3]
59.5 [+10.2]
67.5 [+6.4]
42.5 [+13.5]
57.0 [+9.3]
60.0 [+10.3]
74.5 [+7.6]
3800
3600
3900
4300
4400
4000
4100
4400
ND
ꢁ
TTT TBmC TTBmCTmC BmCT
3700
3900
5500
ꢁ
ꢁ
ꢁ
TTT TBmC TBT mCBmC TmCT
ꢁ
ꢁ
ꢁ
TTB TBmC BTBmCBmC BmCT
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
a
Tm’s were determined as the first derivative of differential thermal denaturation curves (A260vs. T)6cw recorded in a pH 7.0 phosphate buffer
solution containing 140 mM KCl using 1.0 mM of each strand. Tm-values are averages of at least two independent measurements within 1 1C.
kon’s were measured in pH 7.2 HEPES buffer.w kon’s are averages of at least three independent measurements. Data for DNA reference TFO T1
(B = T): Tm = 29.0 1C, kon = 4400 Mꢁ1 sꢁ1. Structures of monomers are shown in Fig. 1. mC denotes 5-methyldeoxycytidine monomers. dsDNA
target sequence (TFO binding region underlined): 50-GCT
ND = not determined.
TCG-30, 30-CGA
ACG-50.
between the triplex strands due to partial protonation of the
propargylamino substituent, or from improved p-stacking with
neighboring nucleobases.8b Consequentially, C5-propargylamine
functionalized LNA TFO Y4 containing six Y monomers as next-
nearest neighbours forms exceedingly stable triplexes with dsDNA
targets (Tm = 74.5 1C, Table 1).
Importantly, the observed stabilization of triplexes by C5-
alkynyl LNA TFOs compares favourably with the corresponding
parent LNA TFOs (V-series) as additional increases in Tm-values
of between +1.1 1C and +2.8 1C per modification are observed
(compare Tm-data for X-, Y- and V-series, Table 1). In fact,
direct comparison with previously reported hybridization data
for ENA7d and 20,40-BNANC7e TFOs in the same sequence,
strongly suggests that C5-alkynyl LNA TFOs stabilize triplexes
more efficiently than these ‘gold standard’ TFO building
blocks. Thus, incorporation of C5-alkynyl functionalized LNA
monomers into DNA TFOs is a simple,w yet powerful approach
to improve thermal affinity of TFOs toward dsDNA targets.
To evaluate the influence of C5-alkynyl functionalized LNA
monomers on association kinetics of triplex formation, the
association rate constant kon was determined for TFOs by fitting
a second order rate equation to the A260 decay curve13 obtained
by mixing pre-annealed dsDNA target with equimolar quantities
of TFOs (Fig. S6).w Generally little variation in the kon-values
between DNA, LNA or C5-functionalized LNA TFOs was
Fig. 2 Representative thermal denaturation profiles of triplexes
between reference DNA T1 or TFOs from the B4-series, and com-
plementary dsDNA target. For sequences and conditions see Table 1.
using a two-fold excess of C5-propargylamine functionalized
LNA TFOs (Fig. S2).w
After qualitatively establishing triplex formation, the thermal
affinity of C5-functionalized LNA TFOs toward the dsDNA
target was determined by UV thermal denaturation experiments
at pH 7.0 in the absence of Mg2+-ions and compared relative to
reference DNA and LNA TFOs (Table 1). The thermal denatura-
tion curves of the studied triplexes either exhibit (a) biphasic
profiles when thermal denaturation temperatures (Tm’s) of
triplexes differed substantially from the Tm of the dsDNA target
(Tm = 56.0 1C), or (b) monophasic profiles when denaturation
curves for triplexes and dsDNA targets overlapped
substantially (Fig. 2, Fig. S3 and S4).w Similar observations
in identical sequence contexts have been made with other
LNA analogs.7b,d To facilitate determination of triplex Tm’s,
differential thermal denaturation curves6c were obtained by
‘subtracting’ the denaturation curve of the dsDNA target from
the ‘raw’ triplex denaturation curve (Fig. S3).w
Singly modified C5-alkynyl functionalized LNA TFOs exhibit
dramatically increased thermal affinity toward dsDNA targets
relative to reference DNA TFO T1 (DTm E +13.5 1C, B1-series,
Table 1). Additional modification of TFOs with X/Y monomers
results in progressively more stabilized triplexes although less
pronounced DTm/mod-values are observed (e.g., compare data
of B1-, B2- and B4-series, Table 1). C5-Propargylamine
functionalized LNAs (Y-series) stabilize triplexes slightly more
efficiently than the corresponding C5-ethynyl functionalized LNAs
(X-series), which likely results from reduced electrostatic repulsion
observed (B4000 Mꢁ1
s
ꢁ1, Table 1), which verifies that
LNA-type TFOs primarily influence triplex dissociation kinetics.6a
As an exception hereto, densely modified C5-propargylamine
functionalized LNA Y4 exhibits faster triplex association kinetics
which most likely is linked to the presence of partially positively
charged ‘patches’ of the C5-propargylamine substituent.9b
Increased affinity of TFOs toward dsDNA targets should
preferably be accompanied by concomitant improvements in
mismatch discrimination to avoid off-targets effects.5b
Accordingly, the ability of singly modified C5-alkynyl LNA
TFOs (B1-series) to discriminate dsDNA targets with a single
Hoogsteen base pair mismatch opposite of the modification
was evaluated in a thermal denaturation buffer containing
10 mM Mg2+ (see footnote Table 2), which is known to
accelerate and stabilize triplex formation.14yw
As anticipated, less stable triplexes are generally formed with
mismatched dsDNA targets. Introduction of C5-functionalized
LNA monomers into TFOs greatly improves discrimination of
Hoogsteen TA- and CG-mismatches compared to reference
ꢀc
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Table 2 Tm-values of matched and mismatched triplexes in the
presence of Mg2+-ionsa
Idaho Research Office and Research Council Seed Grant, and
National Institutes of Health [grant number P20 RR016448
from the COBRE Program of the National Center for
Research Resources]. MEØ acknowledges a scholarship from
the College of Graduate Studies. We thank the EBI Murdock
Mass Spectrometry center for mass spectrometric analyses.
Tm[DTm]/1C
A : T (match)
T : A
C : G
G : C
ON
T1
V1
X1
Y1
37.0
49.0
51.5
50.0
10.0 [ꢁ27.0]
10.0 [ꢁ39.0]
10.0 [ꢁ41.5]
10.0 [ꢁ40.0]
19.5 [ꢁ17.5]
28.5 [ꢁ20.5]
24.5 [ꢁ27.0]
26.5 [ꢁ23.5]
13.5 [ꢁ23.5]
24.0 [ꢁ25.0]
27.5 [ꢁ24.0]
27.5 [ꢁ22.5]
Notes and references
z O4-Triazolyl-dT-CE phosphoramidites12 were incorporated into
TFOs, and converted to 5-methyldeoxycytidine monomers during
deprotection (32% aq. NH3).
y Triplex formation was complete within B1 h in the presence of
10 mM Mg2+, while requiring overnight incubation in absence of
Mg2+. Triplex stabilities increased by 8–10 1C (Tm-data Tables 1 and 2).
a
Tm-values (DTm = change in Tm-value relative to matched triplex).
Conditions and TFO-sequences (B1-series) as described in Table 1
except for addition of 10 mM MgCl2. dsDNA targets = 50-GCT AAA
AAG AMA GAG AGA TCG-30 : 30-CGA TTT TTC TM0T CTC
ꢁ
ꢁ
TCT ACG-50, where M : M0 = A : T, T : A, C : G and G : C,
ꢁ
ꢁ
respectively.
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Fig. 3 Exonuclease (SVPDE) degradation of singly modified C5-
functionalized LNA and reference TFOs. Nuclease degradation studies
performed in Tris-buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 9.0) at
37 1C using 2 mM of TFO strands and 0.43 mg of SVPDE (snake venom
phosphordiesterase). For sequences of T1–V1–X1–Y1, see Table 1.
TFOs T1 and V1, while discrimination of GC-mismatches is
largely unaffected (Table 2).
Consequentially, the most demanding mismatched dsDNA
targets are more efficiently discriminated by C5-functionalized
LNA TFOs (DTm = ꢁ24.0 1C and ꢁ22.5 1C for X1 and Y1,
respectively) than by reference TFOs (DTm = ꢁ17.5 1C and
ꢁ20.5 1C, for T1 and V1, respectively). This data demonstrates
that C5-alkynyl functionalized LNAs exhibit increased
thermal affinity and specificity relative to parent LNAs.
Finally, the resistance of singly modified C5-alkynyl
functionalized LNA TFOs (B1-series) against degradation by
the 30-exonuclease snake venom phosphodiesterase (SVPDE)
was evaluated, by following the increase in absorbance
(hyperchromicity) at 260 nm15 of TFOs in the presence of
SVPDE (Fig. 3). As expected, DNA TFO T1 is rapidly
degraded (485% cleavage after 1 h) whereas singly modified
LNA V1 confers some protection against SVPDE-mediated
degradation (B70% cleavage after 1 h). C5-Ethynyl and, in
particular, C5-propargylamine functionalized LNA TFOs are
considerably more resistant against nucleolytic degradation
(X1 B60%, Y1 B35% after 1 h). This suggests that the C5-
substituents act as a steric shield, to ensure greater biostability.
To conclude, C5-alkynyl functionalized LNAs exhibit
unsurpassed TFO-hybridization properties, which are likely
to enable efficient DNA-targeting under physiological
conditions. Studies along these lines are ongoing.
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We appreciate financial support from Idaho NSF EPSCoR,
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ꢀc
This journal is The Royal Society of Chemistry 2009
6758 | Chem. Commun., 2009, 6756–6758