[Manuscript in preparation]
Synthesis and isolation of singlestranded DNA with multiple LNA-A
modifications
Holger Doessing 1, Rakesh N. Veedu 2, Jesper Wengel 3, and Birte Vester 1,*
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1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense
M, Denmark; holgerd@bmb.sdu.dk
2
School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane,
Queensland, Australia-4072; rakesh@uq.edu.au
3
Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark;
jwe@ifk.sdu.dk
*
Author to whom correspondence should be addressed; b.vester@bmb.sdu.dk; Tel.: +45-6550-2406;
Fax: +45-6550-2467.
Abstract
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*ca. 200 ord
Keywords
Locked nucleic acid, KOD DNA polymerase, single-stranded, aptamer
Introduction
***fooo
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In vitro selection involves repeated partitioning, amplification, and regeneration of a sequence
library. In order for in vitro selection with LNA to work it is therefore essential to establish techniques
that are compatible with LNA chemistry. We present for the first time results demonstrating how KOD
DNA polymerase can incorporate LNA monomers into a DNA library from both double- and singlestranded templates. We also show how these full-length LNA-modified strands may be isolated by way
of an oligonucleotide capture probe.
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Results
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We addressed the issue of regenerating a pool of LNA-containing DNA strands by preparing a DNA
library that acts as a template in primer extension with LNA triphosphate. We and others have
previously shown that template-directed incorporation of LNA using KOD DNA polymerase is possible [1,
2]. Although the kinetics of LNA incorporation has not been studied ***
The library contains a randomized core flanked by primer binding sites that allow for amplification
by PCR. We chose to distribute 7 adenosines evenly within the 30-nucleotide core. The other 23
positions were randomized with deoxy-guanosine, -cytidine, and thymidine in a 1:1:1 ratio. This design
emulates the proportion of the four nucleobases in a fully randomized library. Importantly, it also
ensures that we will not merely observe primer extension of library members with little or no adenine
content in the core.
Figure 1. DNA library encoding incorporation of 7 adenosines.
Adenosine incorporation sites are indicated in lowercase.
*SKA VI OGSÅ VISE LNA-A MONOMEREN HER?
GGACAGGACCACACCCAGaBBBBaBBBaBBBaBBBaBBBaBBBaBBBBGGCCTTTTGTGTGTCGTTT
*** skal lige shines op
*HOV-HOV! 2080 VAR EN LNA OLIGO!! VI STARTEDE SQ MED DNA PCR PÅ DEN!
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We amplified the chemically synthesized DNA library by PCR. We used fluorophore-labeled primers
for the reaction (Figure 2A). Different labels allowed us to subsequently discern the forward and reverse
DNA strands on polyacrylamide gels. We then set up primer extension reactions with KOD DNA
polymerase supplemented with dGTP, dCTP, dTTP, and either LNA ATP or dATP, as well as a fluorophorelabeled forward primer. After extension for 5 minutes the reaction was denatured and re-extended. This
process was repeated for 30 cycles, effectively rendering our setup an asymmetric PCR *REF*.
Using LNA ATP we clearly saw the displacement of the forward strand of the double-stranded
template during the first cycle, followed by the appearance of non-hybridized full-length extension
products (Figure 2B and C, lanes 1-5). Yield increased until 15 cycles. When dATP was used, however, we
saw initial primer extension, but both the product and template strands were completely degraded in
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subsequent cycles (Figure 2B, lanes 6-8), indicating that the polymerase’s strong 3′-5′ exonuclease
activity *REF* readily digests DNA strands but not LNA A-modified strands. Eventually, various run-away
products appeared (Figure 2B, lanes 9-10); this was also the case when no ATP source was present at all
(Figure 2B, lanes 11-15; and C, lanes 6-10).
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Figure 2. Generation of an LNA A-modified DNA library by asymmetric PCR using KOD DNA
polymerase.
(A) Color legend for panel B and C indicating the three different fluorophores used to 5′-label the
primer and both strands in the experiment. Colors are additive, i.e. Cy3 (green) and Cy5 (red) comigration produces a yellow band.
(B,C) 0-30 rounds of asymmetric PCR using dGTP, dCTP, and dTTP supplemented with either LNA ATP
(‘LATP’), dATP, or water (‘no ATP’).
(B) Non-denaturing polyacrylamide gel electrophoresis of samples. LATP samples: The Cy3/FAMlabeled double-stranded DNA initially runs as a singlular species (lane 1, turquoise ‘dsDNA’). Cy5-labeled
primer (red) annealed onto the template strand (blue) is evident (lane 2, purple), as is also the
displacement of the top strand (lane 2, green band). Single-stranded extension products appear after
multiple extension cycles (lanes 3-5, red ‘ssLNA’). dATP samples: Successful displacement of the top
strand and extension of the primer during the first cycle (lane 7, red/purple and green, respectively) is
followed by complete degradation of all strands after 5 cycles (lane 8), after which various run-away
species are being synthesized (lanes 9-10, red and green). No ATP samples: No primer extension takes
place (lane 12). Strand degradation and run-away synthesis commences (lanes 13-15).
(C) Denaturing polyacrylamide gel electrophoresis of the LATP and ‘no ATP’ samples from panel B.
LATP samples: The primer (red) is extended to full length. No ATP samples: Only diminutive primer
extension. Strand degradation and run-away synthesis commences (lanes 8-10). Note that full-length
strands do not co-migrate due to electrophoretic shifts caused by their fluorophore labels.
A: Color legend
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B: Non-denaturing PAGE
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C: Denaturing PAGE
We speculated that purification of the extension reaction could be simplified in the absence of the
iso-sequential forward DNA strands of the template duplex, which might co-purify with the LNA product
strands. We therefore eliminated the forward strand by performing PCR on the template library using a
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5′-phosphorylated forward primer. The PCR product could then be treated with lambda exonuclease,
which preferentially digests the 5′-phosphorylated strand of a DNA duplex *REF*, leaving only the
reverse (i.e. template) strand (Figure 3A).
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We proceeded to verify that our asymmetric PCR setup would work with our single-stranded
template library as well. We found no substantial differences in reaction progress or yield when
comparing 10-cycle reactions set up with either double-stranded DNA or single-stranded DNA generated
by lambda exonuclease (Figure 3B, C).
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Figure 3. Comparison of LNA A-incorporation by asymmetric PCR on single- and double-stranded
DNA using KOD DNA polymerase.
(A) Ethidium bromide-stained agarose gel demonstrating lambda exonuclease digestion of the 5′phosphorylated strand in a DNA duplex. The complementary stand is labeled with FAM (green). When
double-stranded the DNA binds ethidium bromide (red) and retains its FAM fluorescence (producing
yellow; lane 1). After digestion the sample is single-stranded and no longer binds ethidium bromide
(lane 2).
(B, C) Color coding as in Figure 2.
(B) Non-denaturing polyacrylamide gel electrophoresis of samples. Single-stranded DNA (ssDNA)
template: Primer (red) and single-stranded template (blue) readily form a primer:template complex
(purple). 10 cycles of asymmetric PCR with dGTP, dCTP, dTTP, and LNA ATP yields free LNA strands
(‘ssLNA’). Double-stranded DNA (dsDNA) template: As with ssDNA, but the reaction contains both ssLNA
and displaced top strands (green). ‘λ exo-digest remnants’ indicates by-products of over-digestion with
lambda exonuclease.
(C) Denaturing polyacrylamide gel electrophoresis of the samples in panel A. Extension with dGTP,
dCTP, dTTP, and LNA ATP yields full-length product.
A: Lambda exonuclease digestion of forward DNA strand
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B: Non-denaturing PAGE
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C: Denaturing PAGE
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Purification of full-length LNA strands
Next, we attended to the problem of isolating the fully extended products only. The final reaction
mixture contains both template strands as well as a range of partially extended products (Figure 2C,
lanes 2-5). We opted for an oligonucleotide capture approach, where an immobilized oligonucleotide
with full complementarity to the 3′ end of the fully extended product strands is used for extraction
(Figure 4A). Such an approach has been used for *REFS-REFS-REFS*.
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Finally, we verified the presence of LNA moieties in our eluate by enzymatic digestion. We utilized
that fact that in the absence of triphosphates the 3′-5′ exonuclease activity of KOD DNA polymerase
readily digests DNA strands (cf. Figure 2), whereas LNA-modified strands are resistant to degradation.
The Cy5-labeled eluate was combined with equimolar amounts of the LNA A-modified crude library and
the all-DNA FAM-labeled template library that was generated by lambda exonuclease, and KOD DNA
polymerase was added. While the DNA strand was completely degraded over the course of 80 minutes
the LNA-modified crude library could only be degraded down until the 3′-most LNA moiety (Figure 4C,
blue and green bands, respectively). The eluate showed a degradation pattern identical to that of the
crude library, thereby demonstrating the incorporation of LNA A in the purified extension products.
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Figure 4. Extraction and quality control of full-length extension products.
(A) Strategy for purification of single-stranded extension products: Extension products can anneal
onto a biotinylated capture oligonucleotide, which is bound to streptavidin-coated magnetic
nanoparticles. Unbound reaction components can be washed away. The capture oligomer binds the 3′
end of the library, ensuring that only full-length extension products are bound. Only DNA:DNA
hybridization takes place, and therefore the interaction is readily disrupted by heating.
(B) Denaturing PAGE of primer extension mixture before and after purification by oligonucleotide
capture. Color coding as in Figure 2.
(C) Digestion assay. Equimolar amounts of purified primer extension product (5′-Cy5; red), crude
LNA A library (5′-32P; green), and template DNA generated by lambda exonuclease (5′-FAM; blue) were
combined and subjected to the 3′-5′ exonucleolytic activity of KOD DNA polymerase. Digestion of the
DNA template was complete, whereas the presence of LNA caused a complete stop, thus demonstrating
the presence of LNA in the purified primer extension products. Differences in electrophoretic mobility of
the three species involved are mainly due to the nature of their different 5′ labels.
A: Oligo capture outline
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B: Oligonucleotide capture
C: Digestion assay
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Discussion
*vores bibliotekt er begrænset – men det er jo også kun en ”tester”
*det er mærkeligt, at KOD ikke æder DNA-enderne i LNAet eller DNAet i LNA-reaktionerne. Hvorfor
mon?
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*vores bead-approach er smart, fordi den tillader os at ”smelte” oligoerne af igen – der er nemlig
ikke LNA i primersitet – og så fanger den principielt kun fuld-længde oligoer−
Conclusion
Acknowledgements
Experimental
Oligonucleotides
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LNA A library: GGACAGGACCACACCCAGaBBBBaBBBaBBBaBBBaBBBaBBBaBBBBGGCCTTTTGTGTGTCGTTT (lowercase indicates LNA adenosine); forward primer: GGACAGGACCACACCCAG; reverse primer:
AAACGACACACAAAAGGCC. Whenever appropriate primers were chemically synthesized with 5′modifications: phosphate, Cy3, Cy5, or 6-carboxyfluorescein (FAM). Fluorescence scanning was on a
Typhoon Trio system (GE Healthcare). Radiolabeling was with T4 polynucleotide kinase and 32P-γ-ATP.
DNA synthesis was by Sigma; LNA synthesis was in-house at the Nucleic Acid Center.
Polymerase chain reaction
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0.5 nM template (all-DNA or LNA-containing single-stranded DNA) and 0.5 µM of each primer were
combined in 1× Phusion HF buffer with 200 µM of each deoxyribonucleotide triphosphate and 0.04
units/µl Phusion DNA polymerase (Finnzymes). PCR conditions were typically: 98 °C/5 min., 20 cycles (98
°C/5 s, 53 °C/10 s, 72 °C/5 min.), 4 °C/hold. Products were resolved by agarose gel electrophoresis.
Asymmetric PCR
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Reaction conditions were: 1×KOD buffer #2, 3 mM MgSO4, 0.2 mg/ml BSA, 0.2 u/µl KOD DNA
polymerase (Novagen), 2.5 µM primer, 250 ng double-stranded DNA template (67 bp), and 0.25 mM of
each nucleotide triphosphate as required. Thermocycling was typically: 95°/1 min., 30 cycles (95°/15 s,
60°/35 s, 72°/5 min), 4°/hold. Reactions were quenched with 8.3 mM (final) EDTA and stored at −20 °C.
Products were resolved on 10% non-denaturing polyacrylamide gels at 4 °C or on 13% denaturing
polyacrylamide gels.
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Lambda exonuclease digestion
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Double-stranded DNA was prepared with a 5′-phosphorylated primer and a 5′ fluorophore-labeled
primer. Digestion with lambda exonuclease (New England Biolabs) was with 6.7 units/µg doublestranded DNA (50 ng/µl final DNA concentration) at 37 °C for 25 min. The reaction was stopped by
quenching with addition of 1 vol. 50 mM EDTA or by heating to 75 °C for 15 min. Full digestion was
verified by agarose gel electrophoresis and ethidium bromide staining. Fluorescence scanning was on a
Typhoon Trio system (GE Healthcare).
Oligo capture on magnetic beads
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30 µl asymmetric PCR reaction was quenched with EDTA and combined with at least 4-fold molar
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excess of biotinylated oligomer complementary to the library’s 3′ end. The mix was heated to 65 °C for 5
min. and then slow-annealed over 1 hour. The solutions were adjusted to 1×B&W (according to
manufacturer’s instructions) and combined with 1.5 mg Dynabeads M-280 Streptavidin (Invitrogen)
washed twice in 1×B&W buffer. The number of beads corresponded to an excess of biotin binding sites.
After 20 min. with gentle agitation the beads were washed twice in 1×B&W before heat-elution of the
non-biotinylated species into 50 µl phosphate-buffered saline with 0.2% Tween-20 (75 °C, 5 min.).
Digestion assay
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Reaction conditions for digestion by KOD DNA polymerase were similar to those of asymmetric PCR,
except that no primer and triphosphates were used, and KOD DNA polymerase was added to 0.02 u/µl.
Incubation was at 72 °C. Samples were quenched with EDTA and resolved by 13% denaturing PAGE.
Fluorescence scans of Cy5 and FAM and autoradiography of 32P was combined in Photoshop (Adobe)
using gel wells as reference markers.
References
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1.
2.
Veedu, R.N., B. Vester, and J. Wengel, Efficient enzymatic synthesis of LNA-modified DNA duplexes using KOD DNA
polymerase. Org Biomol Chem, 2009. 7(7): p. 1404-9.
Kuwahara, M., et al., Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and
triphosphate analogs involving 2',4'-bridged nucleosides. Nucleic Acids Res, 2008. 36(13): p. 4257-65.
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