Molecules 2011, 16, 1-x manuscripts; doi:10.3390/molecules160x000x
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Amplification and Regeneration of LNA-Containing Libraries
Holger Doessing 1, Rakesh N. Veedu 2, Anders Giessing 1, Lasse Holm Lauridsen 1, Jesper
Wengel 3, and Birte Vester 1,*
1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230
Odense M, Denmark; E-Mails: holgerd@bmb.sdu.dk (H.D.); giessing@bmb.sdu.dk (A.G.);
lhla@bmb.sdu.dk (L.H.L.)
2
School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane,
Queensland, Australia-4072; E-Mail: rakesh@uq.edu.au
2
Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark;
E-Mail: jwe@ifk.sdu.dk
* Author to whom correspondence should be addressed; E-Mail: b.vester@bmb.sdu.dk; Tel.: +456550-2406; Fax: +45-6550-2467.
Received: / Accepted: / Published:
Abstract: This is Abstract section. One paragraph only (Apply M_abstract format).
Keywords: aptamers; LNA; in vitro selection
1. Introduction
*LNA er dejligt*
The first LNA triphospate to be synthesized was LNA adenosine triphosphate (LNA ATP), soon
followed by LNA TTP, GTP, and mCTP. *REF*
This opened up the field of enzymatic applications, and we previously established how *ALLE DE
FORSKELLIGE
KOMBOS
RAKESH
LAVEDE
MED
POLYMERASER
OG
TEMPLATES/PRODUKTER*. The aforementioned properties of LNA meant that
*Vi har tidligere vist, at vi kan indbygge LNA vha. polymerase*
*SELEX er løsningen på alt*
*Vi tror, vores setup kan bruges til SELEX*
Molecules 2011, 16
2
Here we introduce a scheme for amplification and re-generation of a randomized pool of LNAcontaining oligomers. We successfully incorporated either LNA T or LNA A in two DNA libraries and
found that after three rounds of amplification and re-generation the LNA content in our pools remained
at least *PERCENT* percent, suggesting that the scheme is feasible for in vitro selection of LNAcontaining oligonucleotides.
2. Results and Discussion
2.1. Phusion DNA polymerase reads LNA-containing strands
2.1.1. Library Design
We wanted to find out whether we could employ the previously established LNA-compatible
polymerases, KOD DNA polymerase and Phusion DNA polymerase, to successfully amplify and regenerate a library of LNA-containing single-stranded sequences. We based our library design on the
following guidelines: (1) No LNA moieties in the primer binding regions; (2) *****
2.1.2. Phusion DNA polymerase Can Employ LNA Templates
Phusion DNA polymerase had previously been shown to be able to amplify an LNA-containing
template in a PCR setup, *REFS* however, the templates employed were fairly short (*XXX*
nucleotides) and their LNA content and distribution did not correspond well with a typical in vitro
selection library. We therefore asked whether Phusion DNA polymerase could indeed amplify a ‘long’
(>60 nucleotides) LNA-containing template.
We prepared the LNA A-containing template shown in Figure 1A and found that Phusion DNA
polymerase was able to successfully generate a double-stranded all-DNA product of the expected size
under standard PCR conditions (Figure 1B, lane 1). In contrast, when we tried to substitute LNA ATP
for deoxy-ATP we were unable to obtain the desired product (lane 2). We were unable to find any
experimental conditions that would generate a suitable double-stranded LNA-containing product (data
not shown), indicating that Phusion DNA polymerase is not fit for synthesis of double-stranded LNAcontaining products of this size and composition.
Figure 1. (a) Sequence of the template employed. Lowercase ‘a’ is LNA A. Primerbinding sites are indicated ****. (b) Agarose gel electrophoresis of PCR products. Lane 1:
PCR with all four deoxynucleotide triphosphates. Lane 2: PCR with deoxy-GTP, -CTP and
–TTP (dBTP), plus LNA ATP. Lane 3: Control PCR without ATP. Lane 4: Control PCR
without template.
A
GGTCTGGTCCACACCCAG-CCGCCaCCCaGGGaCGCaGCCaGGCaCGGCGGGCCTATAGTGAGTCGTATTA
*
figur
med
primere:
GGCCTATAGTGAGTCGTATTA *
B
18n
GGTCTGGTCCACACCCAG
og
21n
Molecules 2011, 16
3
*SKAL VÆRE EN STOR JPG!
2.1.3. Phusion DNA polymerase correctly reads LNA
The fidelity of Phusion DNA polymerase reading an LNA A-containing template was tested using
an LNA -containing library with a core of 25 randomized positions (C, G or T) and 7 LNA A-moieties
at fixed positions (Figure 2A). We chose this design, as: (a) its LNA content emulated that of a typical
in vitro selection library, and (b) it allowed us to quickly identify LNA A-related sequence errors in the
product. The library was amplified by PCR with deoxyribonucleotide triphosphates, and the
corresponding double-stranded DNA was then cloned into a bacterial plasmid vector and sequenced.
Figure 2B shows a sequence logo obtained from alignment of the 32 positively identified member
sequences. All LNA A positions were clearly identified in all members. Three members contained
single-nucleotide insertions and one member had a single-nucleotide deletion, but neither of these
involved adenosines. Whether these minor errors stem from our library preparation, the PCR or even
the vector’s maintenance in the bacterial host is unclear. We conclude that Phusion DNA polymerase
correctly reads LNA A moieties that are spaced three or more nucleotides apart in a DNA library.
Figure 1. (a) Sequence of the DNA library used. Lowercase ‘a’ denotes LNA A moieties;
‘B’ denotes C, G or T. (b) Sequence logo of the randomized core of the sequenced library
members. Letter height indicates relative abundance. All LNA A-positions were clearly
identifiable in all 32 members. Positions 1, 20 and 30 are alignment artifacts caused by
single-nucleotide insertions in three members. Another member had a single-nucleotide
deletion (aligned to pos. 25).
A
5′-GGACAGGACCACACCCAG-aBBBBaBBBaBBBaBBBaBBBaBBBaBBBB-GGCCTTTTGTGTGTCGTTT-3′
B
Molecules 2011, 16
4
2.1.4. ***foo my bar
*MÅSKE VI SKA INDSÆTTE PCR EFFICACY TESTEN HER?*
2.2. KOD DNA polymerase can incorporate LNA triphosphates
2.2.1. KOD DNA polymerase accepts all four LNA triphosphates
KOD DNA polymerase was previously shown to be able to incorporate LNA *XXX* triphosphates
under primer extension conditions. *REF* We extended this analysis by attempting primer extension
with three deoxy-triphosphates plus either of the four LNA triphosphates on *XXX* (*FIG*).
* primer ext med alle fire LNA-TPer *
Figure 1. (a) *** (b) ***
A
LNA ATP: GGACAGGACCACACCCAGVVVVVVVTVVVVVVTVVVVVVTVVVVVVTVVVVVVGGCCAAAAGAGAGACGAAA
* 2080-T *
LNA TTP:
GGACAGGACCACACCCAGDDDDDDDCDDDDDDCDDDDDDCDDDDDDCDDDDDDGGAAGGTTGTGTGTAGTTG
LNA GTP: GGACAGGACCACACCCAGDDDDDDDCDDDDDDCDDDDDDCDDDDDDCDDDDDDGGAAGGTTGTGTGTAGTTG
LNA mCTP: GGACAGGACCACACCCAGHHHHHHHGHHHHHHGHHHHHHGHHHHHHGHHHHHHCACCTTCCATACATCATCC
* husk at angive primer-binding sites! *
* bedre at vise produkt OG template (farvekodet, naturligvis) *
* hør, hvordan lavede jeg lige prxt med LTTP? Jeg har ingen A-template?! *
B
Molecules 2011, 16
*
5
http://www.boergedoessing.net/holger/wiki/dokuwiki-2009-02-14/doku.php?id=day-to-
day_notes:september_2009 (skal gentages – også med LNA mCTP – for LNA-T-prøverne virker stort set ikke?! *
3. Experimental Section (M_Heading1)
Main text paragraph (M_Text).
Main text paragraph (M_Text).
4. Conclusions (M_Heading1)
Main text paragraph (M_Text).
Main text paragraph (M_Text).
Acknowledgements
Main text paragraph (M_Text).
Main text paragraph (M_Text).
References and Notes
1.
2.
3.
4.
Name, A.B.; Name, C.D. Title of the cited article. Journal Title 2007, 6, 100-110. (M Refer)
Author, A.; Author, B. Title of the chapter. In Book Title, 2nd ed.; Editor, A., Editor, B., Eds.;
Publisher: Publisher Location, Country, 2007; Volume 3, pp. 154-196.
Author, A.; Author, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, 2008;
pp. 154-196.
Reference list. We recommend the use of reference management software to prepare the
references list (e.g. Endnote, http://www.endnote.com/).
Sample Availability: Samples of the compounds ...... are available from the authors (or from MDPI).
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).