Results
LNA is retained over multiple rounds
LNA is the natural substrate for neither Phusion nor KOD XL DNA polymerase and it is conceivable that
library members with little or no LNA content would be preferentially amplified in our selection scheme.
We therefore sought to determine whether our scheme left our sequence pool devoid of LNA strands
after a number of rounds of amplification. Importantly, we performed our rounds without selection
against a target ligand. The only selection pressure on our library is therefore imposed by the
amplification procedure itself.
Starting out from an all-DNA library with a random region of 40 nt (*FIG*) we used this setup:
1. PCR with Phusion DNA polymerase and dNTPs to obtain the corresponding double-stranded
2.
3.
4.
5.
6.
DNA. The primer for the non-template strand has a 5′ phosphate.
Purification of the dsDNA on a spin column.
Digestion of the phosphorylated strand by Lambda exonuclease. This leaves the single-stranded
template DNA.
15 rounds of asymmetric PCR with KOD XL DNA polymerase and an LNA triphosphate and the
additional three DNA triphosphates.
Purification of the full-length LNA strands by annealing to a capture oligo bound to magnetic
beads.
After careful washing the LNA strands were heat-eluted into 1×SSC. A fraction of the eluted LNA
strands were then used as template in step 1 for another round of amplification.
We performed 3 such amplification rounds using either LNA ATP or LNA TTP (in which case the template
was the library complementary strand).
The pools contain LNA after 3 rounds
The LNA content of the LNA strands obtained from rounds 1-3 were subjected to nucleolytic digestion
by KOD DNA polymerase. Unlike the XL variant this polymerase has a very aggressive 3′-5′ exonuclease
activity against single-stranded DNA for proof-reading,(*REF*) however, it is unable to digest LNA
moieties.(*REF*) Incubation of the LNA strands with KOD DNA polymerase yielded only partially
digested products (Figure 1A/B, lanes 1-6), whereas the crude DNA library was fully digested (Figure
1A/B, lanes 7-8). The digestion patterns exhibit the expected characteristics of LNA-containing pools:
First, digestion stops are confined to a region corresponding to the LNA-containing cores. Second, band
densitometry analyses of the stops demonstrate exponential decay behavior (Figure 1C/D, red curves),
which is in accordance with an even distribution of LNA moieties within the random region.
The slopes of these exponential decays decrease slightly from round 1 to round 3. This indicates that
fewer LNA moieties are present near the 3′ end of the LNA strands rounds 2 and 3 and so the
exonuclease can digest further into the random region. This effect is most pronounced with LNA Acontaining strands (compare red curves in Figure 1C). We also find that the LNA A-containing strands are
less stable against nucleolytic attack than LNA T-containing strands, as reflected in the lower decay
constants; this could be due to a higher LNA content and/or higher nuclease stability of the LNA Tcontaining strands (compare red curves in Figure 1C and Figure 1D).
Nucleotide compositions are skewed towards ***
Next, we wanted a quantitative measure of the LNA content in our pool. We approached this by
analyzing the nucleoside composition of our LNA strand pools. This can be done by digesting the DNA of
interest with nuclease P1, phosphodiesterase I, and alkaline phosphatase (*REF*) and subjecting the
resulting nucleoside mix to LC-MS. However, due to the nuclease resistance of (successive) LNA moieties
we opted to use Phusion DNA polymerase on our LNA strands to obtain the corresponding doublestranded DNAs. By using a doubly-biotinylated primer for the template-encoding strand we were able to
immobilize the DNA on streptavidin-coated magnetic beads and subsequently heat-elute the
complementary strand (*FIG*). The eluted single-stranded DNA oligomers are therefore iso-sequential
to the original LNA strands and can easily be digested and analyzed by LC-MS.
******
** NOGET OM AT LNA-INDHOLDET STABILISERES HEREFTER ***
Sequence analysis indicates fewer ‘LNA islands’
Finally, we looked at the sequence composition at the basal level by sequencing. Clones were obtained
by amplifying either the crude DNA library or LNA A- or LNA T-containing strands from round 3 with
Phusion DNA polymerase and cloning the resulting DNA into a plasmid vector. Sequencing yielded
between *XXX-XXX* clones. While the crude library encodes many adenosine or thymidine duplets and
triplets (*FIG*) these were remarkably absent from the LNA-containing pools obtained after 3 rounds of
our selection scheme (*FIG*). The average distance between LNA moieties within the random region
was *XXX.X* nt for the LNA A-containing pool and *XXX.X* nt for the LNA T-containing pool (*FIG:
søjlediagram over afstande mellem Aer eller Ter i de forskellige pools. Husk at angive n [antal kloner] og
gennemsnitsværdi.*).
Materials & Methods
Polymerase chain reaction (PCR)
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. Thermocycling was typically: 98 °C/5 min., 20 cycles (98 °C/5 s, 53
°C/10 s, 72 °C/5 min.), 4 °C/hold.
Lambda exonuclease digestion
Primer extension
Oligo capture on magnetic beads
Digestion assay
Ca. 1.5 pmol of LNA-containing strands isolated after 1, 2 or 3 rounds of amplification and regeneration
of library AT50 (*TABEL?*) were 5′-radiolabelled. Full-length oligomers were isolated on a 6%
denaturing polyacrylamide gel and eluted into 100 µl water each. Sample volumes were then adjusted
to achieve equivalent specific activity. Similarly, the crude DNA library and an LNA A oligomer (5′-GGTCTGGTCCACACCCAGCCGCCaCCCaGGGaCGCaGCCaGGCaCGGCGGGCCTATAGTGAGTCGTATTA; lower case is
LNA A) were 5′-radiolabelled to 7.5 nM. 5 µl of each oligomer was then incubated at 72 °C in digestion
buffer (1× KOD buffer #2, 3 mM MgSO4, 0.2 mg/ml BSA) with or without 0.2 units KOD DNA polymerase
(Novagen) (10 µl final volume). 1.5 µl samples were drawn at intervals, quenched in 1 vol. ice-cold 95%
formamide/50 mM EDTA and resolved on 13% denaturing polyacrylamide gels. Autoradiography was
with the PhosphoImager system, and line densitometry was with spline curves (width: 30) in ImageJ
1.44j (NIH). Data pairs (i.e. with/without polymerase) were matched to achieve identical overall
intensity and then normalized (Excel 2010, Microsoft).
LC-MS analysis
Sequencing
Figures
Første del af figuren burde måske være noget med
at vise amplifikations-setuppet. Anden del af
figuren kunne så være en illustration af den måde
biblioteket aflæses den ene hhv. den anden vej.
B
Tredje del bliver så figurerne her.
A
C
D
Figure 1: Both LNA A and LNA T are retained in libraries over 3 rounds of amplification, but LNA T offers superior
incorporation and/or stability.
A library (40 nt fully randomized) was used as template for making LNA-containing strands, which were
used in subsequent rounds of amplification in order to test whether our amplification scheme entails
intrinsic selection pressure against LNA-containing pool members. LNA content of strands from round 1-
3 was assayed by digesting with a 3′-5′ deoxyribonuclease (KOD DNA polymerase) that cannot digest
past LNA moieties. (A, B) Digestion analysis of strands amplified using LNA ATP (A) or LNA TTP (B). Lanes
1-6 show 5′-radiolabeled LNA strands incubated at 37 °C/20 min. without or with nuclease. The position
of the LNA-induced digestion stops is consistent with the expected position of the LNA moieties in the
oligomer pools. Lanes 7, 8: The all-DNA crude library is fully degraded. Lanes 9, 10: Digestion of an LNA
A-containing oligomer yields stops at the expected intervals. All the digestive patterns shown here
remained virtually identical after 80 min. (not shown). (C, D) Densitometry scans of lanes 1-6 in A and B.
Total pixel intensities of lane pairs (i.e. with and without nuclease) were matched, and all pairs were
then normalized for direct comparison. Blue curves indicate ‘without nuclease’, red curves indicate ‘with
nuclease’. Curve brightness reflects round number. (C) As expected for a fully randomized library the
intensity of the nuclease stops fits an exponential decay function. The decay slope decreases from round
1 to round 3, indicating that the LNA A content in the pool decreases. (D) Similarly, the LNA content also
decreases in a pool propagated with LNA TTP. However, the densitometry curves are higher and steeper
than for the LNA ATP-propagated pool, suggesting that LNA TTP offers higher yield and better
incorporation and/or stability.