Development of aptamers with locked nucleic acids
Holger Doessing
Department of Biochemistry and Molecular Biology ,
University of Southern Denmark,
Odense, February 2011
Preface
I would like to thank the following people:
The many faces of the Microbiology group that came and went over the years.
You know how it is; I would have lost my marbles a long time ago without you
to help out when things got (too) tight, go partying, go-carting, playing games,
enjoying the occasional beer, and so on. I already miss you guys.
Birte, for being my supervisor and also, to a large extent, my secretary. You
kept this wreck on track.
The ‘Aptamer group’: Rakesh, Meghan, Torben, Lasse, Marie, Niels, Frank,
Mikaela, Per, Jesper, and Birte. I hope that we soon will have accomplished
what we set out for some 3+ years ago.
Anders, for being so patient with me and for spending sooo many hours on
getting that LC/MS stuff just right.
Gregers, for letting me stop by and do some protein purification, and Laure, for
lending me a big hand with said purification.
The guys in Jørgen Kjems’ lab in Aarhus—Reza, Morten, Kasper, Thomas, Rita,
Ebbe, Rasmus, Jesper, and of course Jørgen—for letting me share their lab
facilities and lunch. And in particular for showing me that they too were
struggling to achieve in vitro aptamer selection!
Julie, for being very patient with me and my weird working schedule. That time
when I panicked, you stepped in and instructed me what to do. When things
looked grim I always thought of you and reminded myself that there are indeed
way more important things in life than this science carrier stuff.
Dad, for telling me that it is okay not to excel. I got a grade 11 in that exam. Go
figure.
2
Contents
Preface ...............................................................................................2
Contents ............................................................................................3
Summary / Resumé ..........................................................................4
Aptamers ........................................................................................... 5
In vitro selection ...............................................................................6
The advent of a new technique ........................................................................ 6
Library design.................................................................................................. 6
Development of alternative approaches .......................................................... 6
Utility of modified nucleotides.......................................................... 7
Why use alternative chemistries?..................................................................... 7
In vitro selection with non-natural nucleotides ............................................... 7
Locked nucleic acids ........................................................................ 8
Physicochemical properties of LNA ................................................................ 8
Common uses of LNA ..................................................................................... 11
LNA and enzymes ........................................................................................... 16
LNA in aptamers ............................................................................................ 21
Preamble to Manuscripts ................................................................22
Challenges associated with LNA ................................................................... 22
In vitro selection against chitin ......................................................................25
In vitro selection against IgG ........................................................................ 28
Concluding remarks ....................................................................... 30
References ....................................................................................... 31
Manuscript I .................................................................................... 37
Manuscript II ................................................................................. 38
Manuscript III .................................................................................39
3
Summary / Resumé
*Summary in English*
*Resumé på dansk*
4
Aptamers
** SKAL APTAMER-DELEN FLYTTES LÆNGERE NED? MIT PROJEKT ER
JO IKKE SPECIELT APTAMER-AGTIGT (MERE SELEKTIONS-AGTIGT)?
*definition
The field of aptamers has received a great deal of attention over the last decade
or so. As of mid-January 2011 a query on ‘aptamer’ on the MEDLINE database
returns more than 1,900 entries, excluding reviews. Bear also in mind that
researchers in related fields use different terminologies to refer to aptamers
(e.g. ‘biosensors’).
*eksempler på targets og nukleotid-typer og praktiske anvendelser af
aptamerer
*overordnet – en blød indgangsvinkel
* gerne lidt ’kant’ på prospekterne.
5
In vitro selection
The advent of a new technique
*Matematikken bag er undersøgt nøje!
*counter-selection
Library design
Libraries design for in vitro selection are typically based around a fully
randomized core consisting of any of the four standard (deoxy-)ribonucleotides
flanked by shorter regions of fixed sequences. These allow for easy
amplification of the selected library members by PCR.
*hvor lang skal den randomiserede kerne være?
Each addition of a single ‘N’ to the randomized region increases the complexity
of the pool by a factor of four (4N), meaning that experimenters rarely are able
to sample the entire pool of a library.
*nogle har udeladt de flankerende regioner
*nogle har reduceret variationen i det randomiserede ved at bruge færre
nukleotider (bl.a. os)
*”A ribozyme composed of only two different nucleotides”
*restriktionssites?
Development of alternative approaches
6
Utility of modified nucleotides
Why use alternative chemistries?
In vitro selection with non-natural nucleotides
Non-enzymatic incorporation of modifications
Incorporation using modified triphosphates
7
Locked nucleic acids
Physicochemical properties of LNA
Topography of DNA, RNA, and LNA
In aqueous the ribofuranose moiety of DNA or RNA nucleotides can adopt two
different conformations, the C2′- and C3′-endo (Figure 1a, b). This is termed
the sugar pucker. Under normal physiological conditions the nucleotides in a
DNA double helix adopts the C2′-endo conformation. The entire arrangement
of the helical structure is then dubbed ‘B-form or ‘B-DNA’. Contrast this with
double-stranded RNA or DNA:RNA heteroduplexes, where the C3′-endo is the
predominant pucker; this configuration is dubbed ‘A-form’. These two kinds of
nucleic acid structure are discerned by several properties: For instance, the
inclination of the base pairs in relation to the helical axis are different (B-form
DNA: −1°, A-form: +19°), and the wide major groove in B-form DNA contrasts
the much narrower major groove in A-form RNA. Similarly, the minor groove
in B-type DNA is narrow and deep, whereas A-type RNA helices have a wide
and shallow minor groove (Stryer 1999). From afar, A-form helices appear
more compact, while B-form helices are more open and elongated.
Figure 1: The ribose puckering of DNA (and RNA) is able to adopt both
the C2′-endo and C3′-endo conformation (a and b). Locked nucleic acid
(LNA) is restrained due to the 2′-4’ bridge that locks the ribose in the C3′endo conformation (c). Reproduced from (Petersen and Wengel 2003).
The nucleobase attaches to the ribose via an N-glycosidic bond at C1′. The bases
are planar, and are therefore almost perpendicular to the plane of the ribose.
Per se the bond allows for some rotational freedom. This is, however, sterically
hampered by the presence of e.g. hydrogen on the C2′. In effect, only two
principal conformations are therefore allowed: anti and syn. The ‘anti’
conformer sees the nucleobase pointing ‘away’ from the ribose and this is the
8
conformation normally seen in double helices, where the bases in the
individual strands are orientated towards their complementary counterparts in
the opposite strand. The ‘syn’ conformer is rotated 180° and puts the O2 of
pyrimidines or N3 of purines above the ribose moiety. The ‘syn’ conformer is
not normally found in DNA, save in a few particular cases, where stretches of
DNA may adopt the so-called Z-form (Stryer 1999).
Locked nucleic acid (LNA) is a ribonucleoside homologue that features a 2′O,4′-C-methylene linker or bridge (Figure 1c) (Koshkin, Singh et al. 1998;
Singh, Nielsen et al. 1998). This makes this class of nucleotide analogues
similar to 2′-O-methyl RNA, a modified ribonucleotide that is commonly found
in ribosomes (Yu, Shu et al. 1997). However, in LNA the 2′-O-methylene is
bridged to the C4′. This locks the ribose moiety in the C3′-endo conformation,
effectively making the nucleotide an RNA mimic. LNA was first synthesized by
Imanishi’s group (Obika, Nanbu et al. 1997), who dubbed it ‘bridged nucleic
acid’ (BNA). Concurrently, Wengel’s group developed the same structure and
dubbed it ‘locked nucleic acid’ (Koshkin, Singh et al. 1998). The molecule and
its uses have been extensively patented by Exiqon A/S; LNA is currently
available from other, licensed vendors as well.
LNA conformational steering
The C3′ endo conformation of the ribose moiety of LNA results in a dramatic
change in the structure of neighboring nucleotides in a duplex structure.
Petersen et al. prepared two LNA thymidine-modified DNA strands, one
carrying a single modification and the other modified with three LNA residues.
Upon annealing these strands to their complementary RNA sequence and
assessing the duplex structures by nuclear magnetic resonance (NMR)
spectroscopy they found that while the LNA nucleotides engaged in normal
Watson-Crick base pairing with bases in their ‘anti’ conformation the LNA
would also steer the overall shape of the heteroduplex towards the A-form. In
fact, replacing every third residue with its corresponding LNA moiety yielded a
near-canonical A-form heteroduplex. They speculated that the presence of the
2′-4′ bridge affects the degree of pseudo-rotational freedom of the nucleotide
to the 3′ of the LNA and causing its ribose to shift toward the C3′-endo
conformer, i.e. assume an ‘LNA-like’ conformation (Petersen, Bondensgaard et
al. 2002). This effect appears to be substantial enough to extend until two
nucleotides in the 3′ direction, meaning that the full benefits of LNA-induced
steering of the nucleotide structure can be attained by modifying a structure to
include LNA moieties at every third position. Complete modification of one
strand to obtain an LNA:RNA heteroduplex does not lead to further
9
perturbations, and the structure adopts the canonical A-form (Nielsen,
Rasmussen et al. 2004).
An fully modified LNA:DNA heteroduplex mimics RNA:DNA hybrid duplexes,
which do not form perfect A-form structures, although the C3′-endo
deoxyribose pucker does dominate (Stryer 1999; Nielsen, Rasmussen et al.
2004). In particular, the ribose moieties of the DNA strand all predominantly
remain in the C2′-endo conformation, while the deoxyribonucleotide 3′ to a
single LNA residue showed partial C3′-endo puckering. Again, having every
third residue modified with LNA caused all nucleotides in the modified strand
to adopt the C3′-conformation (Petersen, Nielsen et al. 2000).
Based on these observations Petersen et al. hypothesized that the introduction
of LNA into one strand of a duplex results in a local organization of the
phosphate backbone. This change includes the pucker steering in the 3′
direction, a process that effectively reduces the conformational freedom of the
affected nucleotides and reduces the loss of entropy upon base pairing.
Moreover, the nucleobases become arranged in a way that leads to more
efficient base stacking, offering increased loss in enthalpy upon duplex
formation. Together, they speculate, the LNA-modified strand can be described
as being pre-organized to duplex formation with highly favorable
thermodynamics over that of the unmodified duplex (Petersen, Nielsen et al.
2000).
Modification with LNA leads to increased affinity
One of the effects of the aforementioned LNA-induced changes is that the
presence of one or more LNA nucleotides in a duplex greatly increases the
affinity of the LNA strand towards its complementary sequence. When dealing
with oligonucleotide affinities the melting temperature (Tm), i.e. the
temperature at which half of a population of complementary DNA molecules
exists on the duplex form, is the most convenient comparison. Introduction of
LNA into the DNA strand of a DNA:RNA duplex has been shown to increase
the Tm up to 7.3 °C per each LNA moiety incorporated (compared to the
unmodified duplex) (Wengel 1999). Modification of the RNA strand in a
similar heteroduplex has led to increases in Tm of no less than 9.3 °C per LNA
moiety (Wengel 1999). There are currently no commercially available
alternative to LNA that offers such significant improvements in thermal
stability, and this feature is by far LNA’s ‘claim to fame’. A range of various
nucleotide analogues based on LNA have been prepared, a few of which are
shown in Figure 2. The original LNA moiety has by far been the most
successful, though.
10
Figure 2: The LNA monomer (e) and its derivatives (a-d, f-g). LNA is also denoted
β-D-LNA to discriminate it from α-L-LNA (a). Illustration from (Petersen and
Wengel 2003).
Common uses of LNA
The remarkable ability of LNA to increase the Tm of a modified strand towards
its complementary sequence is often put to use in applications where very high
affinity is desirable, e.g. gene silencing (Jepsen and Wengel 2004), modulation
of RNA splicing (Childs, Disney et al. 2002; Aartsma-Rus, Janson et al. 2003;
Ittig, Liu et al. 2004), RNA interference (Braasch, Jensen et al. 2003),
molecular beacon probes (Wang, Yang et al. 2005), and DNAzymes (Vester,
Lundberg et al. 2002; Vester, Lundberg et al. 2004).
11
Another application of LNA modifications is fine-tuning and matching of the
Tm of primer pairs to achieve optimal hybridization and fewer off-target effects
under the given experimental conditions. In all these cases special software
tools based on experimental thermodynamic parameters can be used to
determine the optimal placement of LNA within a sequence (Tolstrup, Nielsen
et al. 2003; McTigue, Peterson et al. 2004; Owczarzy, Tataurov et al. 2008).
Accurate predictions on the efficiency of the LNA-modifications have proven
difficult, though (Latorra, Arar et al. 2003; Kaur, Arora et al. 2006).
*stabiliserer sekundære/tertiære strukturer
LNA can improve specificity
Placement of LNA within an antisense sequence can also improve mismatch
discrimination. Generally, LNA triplets around the mismatch site show the best
distinction between the specific target and mismatch sequence (You, Moreira
et al. 2006).
*genotyping
*arrays
** UDDRAG FRA MIT SEPCIALE:::: LNA confers substantially increased
hybridization stability and improved target discrimination (Wengel et al.,
2001). These features make LNA-modifications particularly suitable for
antisense oligos, including primers, components of combinatorial arrays
(“DNA chips”) (Mouritzen et al., 2003), and in situ hybridization (Thomsen et
all., 2005)., as well as reduced susceptibility towards nucleases (Wahlestedt et
al., 2000; Grunweller et al., 2003) suggests that LNA is a well-suited candidate
for in vivo application.**
Toxicity
Nucleoside- and backbone analogues can be grouped into generations
according to the concept behind their chemistries. The key first-generation
nucleotide analogue is phosphorothioate DNA (Figure 3). This variant of
normal DNA has one of the non-bridging oxygen atoms replaced by sulfur,
thereby making the backbone much less susceptible to nucleases. However,
non-specific protein binding often led to sequence-independent side-effects
12
(Brown, Kang et al. 1994) and severe toxicity rendered the use of this
modification problematic (Levin 1999).
The second generation of nucleotide analogues focused on modification of the
2′-O group. 2′-O-alkylated RNAs (Figure 3) are not prone to solvent-induced
hydrolysis (like RNA) and they also offer nuclease resistance. 2′-O-methyl RNA
in particular, is interesting, as this modification is found naturally in ribosomes
(Yu, Shu et al. 1997) and so poses much reduced cellular toxicity. However,
antisense oligomers fully modified with these second generation nucleotide
analogues fail to activate of RNase H. This can probably be explained by the
near B-form adopted by 2′-O-methyl RNA:RNA duplexes. (Contrast this with
the near A-form duplex structure of the DNA:RNA hybrids that are the natural
substrate for RNase H.) This is seen as a major obstacle in their use in
antisense technologies (Zamaratski, Pradeepkumar et al. 2001).
LNA is grouped along with a broad range of third-gen nucleotide analogues
that undertake many different concepts (Figure 3). However, one of the widely
explored strategies is that of conformational restriction. The diversity of this
group cannot be covered here, but select analogues have been reviewed
elsewhere (Kurreck 2003).
13
Figure 3: LNA belongs to the class of so-called third-gen nucleosides.
Phosphorothioate DNA is the major representative of First-gen nucleosides.
14
Second-gen nucleosides introduced 2′-O modifications. Third-gen nucleosides span
across a much wider range of structures and chemistries. Illustration from
(Kurreck 2003).
The toxicity of LNA is generally believed to be low. **HERFRA!
*noget og giftighed?
The lack of toxicity in rats *Wahlestedt et al., 2000 *Zhang et al., 2004*
*miRNA
*antisense i det hele taget
*PCR
*SNP
*arrays
*molecular beacons
*LNA-modified ribozymes, DNAzymes (LNAzymes)*
*struktur af alle fire LNAer (er C ikke stadig mC?)
LNA
*Specificitet
*Kortere prober
*Nukleasestabilitet
*Kan fx ikke aktivere RNase H (kræver gap-mer)
*Stickyness
15
*RNA-agtigt, men ikke helt alligevel: Veedu 2009: Efficient enzymatic
synthesis of... p1407 tv o.m., refs 36-38
*Post-modifikation
*Ikke giftigt (modsat fx PS) – men jeg har et paper, der måske modsiger dette?
LNA and enzymes
Biostability in the body
As mentioned previously, one of the motivations for employing LNA is its
stability towards nucleases. Once inside the body oligomers are bombarded by
a barrage of nucleases in both the extra- and intracellular environment. The
unfavorable pharmacokinetics of unmodified DNA or RNA oligomers—be it
antisense oligomers, siRNAs, (deoxy-)ribozymes, or aptamers—are hampering
their successful application medicine.
Ribonuclease levels and composition within the human body vary widely, but
was reported to fall in the range 200-10,100 units per ml, with the former
activity levels found in serum and the highest activity in kidney tissue
(Weickmann and Glitz 1982). Incubation of siRNA in human serum led to
substantial degradation of the unmodified RNA duplex after 1½ hours,
whereas introducing two LNA moieties at both 3′ termini offered protection
beyond 6 hour, a ~4-fold improvement. When one of the strands was replaced
by a mix-mer with a 12 DNA/9 LNA nucleotide configuration the duplex
remained surprisingly stable, even after 48 hours. Similar results were
obtained in 10% foetal bovine serum or 100% mouse serum (Elmen, Thonberg
et al. 2005) and with similar oligo designs (Mook, Baas et al. 2007). Finally,
Gao et al. injected mice with radiolabeled siRNA, extracted total RNA from
blood drawn at intervals and demonstrated that while the unmodified siRNA
was degraded in less than 5 minutes the LNA mix-mer siRNA could still be
detected after 30 minutes (Gao, Dagnaes-Hansen et al. 2009).
Deoxyribonuclease activity in blood is generally believed to be shared among
deoxyribonuclease I (DNase I), DNase II, and phosphodiesterase I with the
former being the main component. The concentration of DNase I in plasma
differs, depending on the methodology employed: 3.2 ng per ml (Prince, Baker
et al. 1998), 18.4 ng per ml (Miyauchi, Ogawa et al. 1986), or ~0.79 Kunitz
units per liter (Nadano, Yasuda et al. 1993). However the concentration of the
active catalysts, the effects of endogenous nucleases contra LNA are most
clearly demonstrated by the influence on oligomer half-lives in human serum:
A DNA 18-mer exhibited a half-life of 1.5 hours, whereas an isosequential
16
oligomer with single LNA moieties at both termini displayed a half-life of 4
hours, a >2.5-fold improvement. Increasing the number of LNA nucleotides at
both termini to three improved the half-life further to 17 hours (>11-fold)
(Kurreck, Wyszko et al. 2002). Another study reported that while a DNA
oligomer showed a half-life of less than 10 minutes in rat serum the half-life of
an equivalent DNA sequence flanked by four and five LNA moieties on each
side was ~150 minutes (>15-fold), and the corresponding LNA-DNA mix-mer
(6 DNA, 9 LNA nucleotides) achieved a half-life in excess of 5 hours (>30-fold)
(Wahlestedt, Salmi et al. 2000).
Exonucleases
Stability towards exonucleolytic attack is often assayed with snake venom
phosphodiesterase I (SVPD) from Crotalus adamanteus. This enzyme is a 3′-5′
exonuclease that digests single-stranded DNA and RNA in a non-processive
manner. Differences in SVPD’s rate of degradation of RNA versus DNA led
Razzell and Khorana to suggest that SVPD discriminates between these two
nucleic acids on basis of the sugar moiety alone (Razzell and Khorana 1959).
This could help explain why SVPD is unable to digest successive LNA moieties
in DNA or RNA; indeed, two or more LNA moieties towards the 3′ end can
offer efficient protection against SVPD (Frieden, Christensen et al. 2003).
SVPD is, however, able to completely digest substrates of a scattered
arrangement with singular LNAs (Morita, Takagi et al. 2003; Nagahama,
Veedu et al. 2009)
BAL-31 is a do-it-all nuclease in that it acts exonucleolytically on singlestranded DNA, it cleaves across nicks in double-stranded DNA and digests
both 3′ and 5′ termini (presumably due to their transiently frayed ends), and,
lastly, it also displays ribonuclease activity (Gray, Ostrander et al. 1975). BAL31 attack on duplex DNA does not seem hindered by the presence of singular 3′
and 5′ terminal LNA moieties together on both strands. Modification with two
LNA moieties at either ends improved the duplex’ half-life two-fold, with
further gains conferred by adding 2-4 LNAs to the duplex’ center region (halflives in excess of 6 hours) (Crinelli, Bianchi et al. 2002).
Archaean KOD DNA polymerase from Thermococcus kodakarensis KOD1 is
similar to that of Pyrococcus furiosus (Pfu DNA polymerase; 79% amino acid
identity) and the two enzymes also show similar fidelity (Takagi, Nishioka et al.
1997). Its proof-reading is articulated as a comparatively aggressive 3′-5′
deoxyriboexonuclease activity (Nishioka, Mizuguchi et al. 2001; Veedu, Vester
et al. 2009). KOD DNA polymerase is remarkable in that it is one of the few
17
polymerases known to be able to read and incorporate LNA (Kuwahara, Obika
et al. 2008; Veedu, Vester et al. 2009), as well as other modifications
(Kuwahara, Takano et al. 2010). We observed that prolonged incubation (> 3
min.) with KOD DNA polymerase could cause degradation of all-DNA products
in primer extension reactions (Veedu, Vester et al. 2009). Indeed, we now
utilize KOD DNA polymerase’s inability to efficiently digest past a single LNA
position to assay for the presence of LNA in extension products generated by
the ‘KOD XL’ formulation (Doessing, Veedu et al.). (KOD XL is a commercially
available mixture of exonuclease-deficient and –positive KOD DNA polymerase
(Nishioka, Mizuguchi et al. 2001).)
*NB! Kuwahara 2008 har mås(Burmeister, Lewis et al. 2005)ke et argument
for pausing i deres kinetik-paper med KOD og Phusion
Endonucleases
Protection against endonucleases poses a special challenge as a single natural
nucleotide within an oligomer is often sufficient to make the strand susceptible
to attack.
For instance, S1 nuclease—an endonuclease that acts on single-stranded DNA
and, to a lesser degree, RNA—can degrade a chimeric oligomer with a fourdeoxynucleotide gap flanked by fully LNA-modified chains, albeit at a
considerably slower rate than that of the unmodified DNA oligomer.
Decreasing the DNA nucleotide gap yields increasingly stable oligomers, and a
1-DNA gapmer or even oligomers composed entirely of LNA nucleotides are
very stable against this enzyme (Frieden, Christensen et al. 2003).
The main blood DNase, DNase I, is an endonuclease that preferentially cleaves
double-stranded over single-stranded DNA. Cleavage is technically nonspecific
but often occurs adjacent to pyrimidines. Remarkably, by using 5′ and 3′
terminal LNA nucleosides together on both strands in a DNA duplex Crinelli et
al. saw a marked improvement in stability over that observed with the
unmodified DNA duplex (Crinelli, Bianchi et al. 2002). Internal modifications
did not improve the nuclease resistance further, though.
Polymerases
The first studies on the enzymatic compatibility of LNA triphosphate was with
LNA TTP and LNA ATP pitted against 11 different enzymes: Taq DNA
polymerase, Klenow fragment (DNA polymerase I), T4 DNA polymerase, Pfu
DNA polymerase, Pfx DNA polymerase, Speed STAR HS DNA polymerase,
mutant T7 R&DNA polymerase, AMV reverse transcriptase, T7 RNA
18
polymerase, E. coli RNA polymerase, and Phusion High-Fidelity DNA
polymerase, of which only the latter was found to offer efficient incorporation
(Veedu, Vester et al. 2007; Veedu, Vester et al. 2007). Later, 9°Nm (Veedu,
Vester et al. 2007; Veedu, Vester et al. 2008) and KOD DNA polymerase
(Veedu, Vester et al. 2009) were added to the list of compatible polymerases.
Phusion High-Fidelity DNA polymerase is a proprietary enzyme described by
its manufacturer as: ‘a unique dsDNA-binding domain [that] is fused to a
Pyrococcus-like proofreading polymerase.’ (Finnzymes 2010) The first
attempts at using this enzyme with LNA TTP and –ATP were unsuccessful in
obtaining incorporation of 7 consecutive LNA moieties, but instead
demonstrated the DNA-directed ability to incorporate up to 5 singular LNA
moieties by extending a primer 23-25 nucleotides. (Veedu, Vester et al. 2007).
This was subsequently expanded upon to also include incorporation of 2 LNA
adenosines and 2 LNA thymidines across from their complementary LNAs in a
DNA/LNA mix-mer template (Veedu, Vester et al. 2007). However, the
reaction conditions employed 10-fold higher concentrations of LNA than that
of the natural triphosphates and also required the addition of both MnCl2 and
betaine; Mn2+ increases polymerase tolerance towards non-natural nucleotides,
and betaine is a known PCR enhancer additive for GC-rich templates (Rees,
Yager et al. 1993; Veedu, Vester et al. 2007). Kuwahara et al. challenged the
utility of Phusion High-Fidelity DNA polymerase: The enzyme derives its
proofreading from a strong, intrinsic 3′-5′ exonuclease activity, and Kuwahara
et al. found substantial primer degradation in their primer extension
experiments, which were set up according to manufacturer’s instructions for
PCR. Also, they were unable to attain full-length extension with either (a)
deoxy-adenosine triphosphate on poly-T templates containing 2 or more LNA
T moieties, or (b) LNA TTP on a poly-A template. Consequently they deemed
this enzyme inferior to e.g. Vent(exo−) and KOD DNA polymerase for primer
extension (Kuwahara, Obika et al. 2008). Not deterred, Veedu et al. soon
presented PCR-based incorporation of LNA ATP at singular, distal sites in a 43
base pair product (3 and 5 site in either end, respectively), which yielded the
desired product albeit at a low level (Veedu, Vester et al. 2008). The fact that
the control reaction with all four natural deoxyribonucleotides also performed
below par indicates that the enzymatic conditions were perhaps less
appropriate for efficient PCR. We later found, however, that the standard
conditions recommended by the manufacturer allowed us to reliably amplify
double-stranded DNA from an LNA-modified template without any apparent
effects on fidelity (Doessing, Veedu et al.).
19
*Noget med at vi aldrig fik Phusion til at inkorporere under PCR på “lange”
templates!
** Der er måske mulighed for at lave et “Rakesh-længde” paper om PCR med
Phusion på DNA templates?! I så fald kan jeg jo vedhæfte det og referere til det
herfra.
*KOD DNA polymerase :: er der mulighed for at lave et kommuniké her?
KOD DNA polymerase from the hyperthermophilic archeaeon Thermococcus
kodakarensis—aptly named after the Japanese island Kodakara on which it
was first isolated—was immediately recognized for its superior elongation rate,
processivity, and fidelity (Takagi, Nishioka et al. 1997), and it is one of the
fastest high-fidelity PCR-capable polymerases commercially available today.
Two variants are currently on the market: Recombinant KOD DNA polymerase
from E. coli with a mutation frequency of 0.1%; and KOD XL DNA polymerase,
which is a proprietary mixture of regular and exonuclease-deficient KOD DNA
polymerase that trades a slight increase in mutation rate (2.2%) for the ability
to amplify targets of 5-30 kb without causing significant degradation of
primers and/or template (Nishioka, Mizuguchi et al. 2001). Despite its high
fidelity KOD DNA polymerase is able to accommodate a range of derivatized
nucleotide triphosphates, including LNA (Kuwahara, Ohbayashi et al. 2002;
Obayashi, Masud et al. 2002; Ohbayashi, Kuwahara et al. 2005; Sawai,
Nagashima et al. 2007; Kuwahara, Obika et al. 2008; Veedu, Vester et al.
2009; Kuwahara, Takano et al. 2010; Vaught, Bock et al. 2010).
*noget om KODs proofreading-domæne?
*9°N DNA polymerase
*T7 RNA polymerase
*Opsummering: Type B DNApols er bedre til at akkommodere basemodificerede nukleotider (se fx refs 45,58,59 i Kuwahara 2008 + Rakesh’ refs)
Compatibility with other enzymes
**RNase H og andre
*T4 PNK [2]
*terminal deoxynucleotidyl transferase [2].
*T4 DNA ligase
20
LNA in aptamers
*LNA-modified aptamers*
21
Preamble to Manuscripts
Challenges associated with LNA
In vitro selection of e.g. aptamers can broadly be divided into four steps: (1)
Library creation, (2) selection, (3) amplification, and (4) cloning (Figure 4).
Figure 4: Outline of the steps involved in an in vitro selection
experiment. ‘PBS’, primer binding sites. Reproduced from (Marton,
Reyes-Darias et al. 2010).
As far as in vitro selection with LNA-containing pools is concerned, step 1 is
fairly easily overcome. LNA thymine, guanine, adenine, and 5-methylcytosine
phosphoramidites are available (Koshkin, Fensholdt et al. 2001; Madsen,
Kumar et al. 2010). Our preliminary results have indicated that the coupling
efficiency of the LNA amidites is roughly 2-fold lower than that of the
corresponding DNA amidites (unpublished data), and this has been
corroborated by others (Meldgaard, Lomholt et al. 2005). This is solved by
increasing the coupling time correspondingly. In theory the de novo synthesis
of a DNA or RNA pool of the desired composition and LNA content is therefore
trivial, but the length of the library employed is another concern. The typical
library design with a 30-60 nucleotide randomized core flanked by primer
binding regions often yields libraries of 70-100 nucleotides in length. Since the
yield of the final product can be calculated by the repeated multiplication of the
coupling efficacies for each position in the library (e.g. a 80-mer with a
coupling efficacy of 99.5% yields 0.99580 ≈ 67% full-length product) it becomes
obvious that longer oligomers require a substantial amount of starting
material. During our efforts in obtaining an LNA aptamer we synthesized an
83-mer LNA A-containing pool, a new record for our in-house synthesis
22
facility. An alternative approach is to use an all-DNA library and initiate
selection by generating LNA strands off this (discussed below).
We chose to exclude LNA from the primer binding sites of our library. This
decision was based on the notion that we wanted our aptamers to be as
compact as possible, and the presence of LNA in both flanks would greatly
increase the risk of the randomized core base pairing with this fixed-sequence
region.
Step 2, selection, probably does not require any LNA-specific considerations.
In preparation for the selection step, various groups have employed different
techniques for conditioning their pools. Some opt for denaturation by brief
heating followed by slow cooling in order to allow the oligomers to adopt to
their optimal tertiary structure (Mannironi, Di Nardo et al. 1997; Fukusaki,
Kato et al. 2000; Hasegawa, Sode et al. 2008; Pan, Xin et al. 2008; Qian, Lou
et al. 2009), whereas others quickly transfer the hot pool to an ice bath to
prevent intermolecular interactions from disrupting the intramolecular folding
process (Pestourie, Cerchia et al. 2006; Wang, Liu et al. 2007; Tang, Parekh et
al. 2009; Tran, Janssen et al. 2010). It is possible that—given the inherent
‘stickiness’ of LNA strands—LNA-containing pools are more prone to engage in
intermolecular interactions that result in inactive conformers. To my
knowledge this has not been investigated, neither for LNA, DNA or RNA pools.
Step 3 is the amplification and regeneration of the pool based on the members
that were selected in step 2. Since LNA is not a natural substrate for
polymerases this is undoubtedly the key problem in sustaining a pool of LNA
molecules. LNA incorporation by amplification had previously been
demonstrated (Veedu, Vester et al. 2008; Veedu, Vester et al. 2009) but not for
strands of the length and composition required for in vitro selection. Pools of
single-stranded DNA molecules are typically prepared by asymmetric PCR
(Ellington and Szostak 1992; Huizenga and Szostak 1995; Wu and Curran
1999; Fukusaki, Kato et al. 2000; Boese and Breaker 2007) or PCR followed by
removal of the template strand by way of gel shift *REF* or affinity-based
methods *REFS*. We were intrigued by the prospect of incorporating LNA
moieties under PCR conditions and sought to investigate this further. Using the
‘43n’ PCR template employed in (Veedu, Vester et al. 2008; Veedu, Vester et al.
2009) as our starting point we introduced inserts of various sizes and
compositions in the center and attempted to incorporate LNA by substituting
LNA ATP for deoxy-ATP in PCR reactions with either KOD DNA polymerase or
Phusion. We soon realized that our PCR conditions would not allow for
incorporation of LNA A moieties in both strands when these moieties were
23
placed in a zipper-like arrangement (data not shown). We speculate that
template denaturation becomes exceedingly difficult once the LNA content
reaches a given level. Alternatively, one could opt for a library design, where
LNA is incorporated into one strand only. However, such a design would imply
that the random region of the pool would only consist of 3 different nucleotides
(in order not to encode for LNA-incorporation into the template strand), of
which one (i.e. the LNA species) might be selected against. We deemed the risk
of such a radical decrease in variation unacceptable and pursued other means
of achieving step 3.
In the course of testing LNA incorporation by PCR I found that asymmetric
PCR was promising (data not shown). We therefore went for the two-step
strategy: (3a) Amplification, and (3b) re-generation. First, we focused on
amplification of the LNA library (step 3a). This turned out to be readily
achieved with Phusion High Fidelity DNA polymerase (Doessing, Veedu et al.).
Second, we optimized the reaction conditions for KOD DNA polymerase for
primer extension of longer templates with incorporation of LNA A moieties.
*REF TIL ET MINI-MANUS?* KOD DNA polymerase has a very strong 3′-5′
exonuclease activity (see previous chapter) which could potentially attack and
degrade the library’s DNA-only primer binding sites. Alternatively, it was
postulated that KOD DNA polymerase might be able to digest the template
DNA once one or more of the nucleotide triphosphates became unavailable.
Our attempts to resolve whether this was the case yielded conflicting results,
indicating that the exact nature of KOD DNA polymerase in the context of LNA
and LNA triphosphates is not yet very well understood. I bypassed the
exonuclease issue by switching to KOD XL DNA polymerase, which has a much
lower exonucleolytic activity (Nishioka, Mizuguchi et al. 2001). This meant that
I could assume no chance detrimental effects on our enzymatic products. My
final strategy for amplifying and regenerating an LNA-containing library is
presented in (Doessing, Veedu et al.).
Step 4 is the cloning and evaluation of the pool once a sufficient number of
selection rounds have been performed and the pool’s affinity towards the target
molecule has increased satisfactorily. The challenge here is very similar to step
3(a) in that the pool of selected molecules must be amplified. In this case the
goal is to obtain double-stranded DNA, which may subsequently be ligated into
a vector for sequence analysis. Phusion High Fidelity DNA polymerase can take
an LNA-modified DNA and produce blunt-ended DNA with no apparent loss of
fidelity (Doessing, Veedu et al.).
24
In vitro selection against chitin
Strategy rationale
*hvorfor valgte viat gøre som vi gjorde
*lambda frem for noed andet
In vitro selection roundup
Fukusaki et al. used a all-DNA library to perform selections towards ‘chitin’,
poly-β-1,4-N-acetylglucosamine (Figure 5a). Their library encoded a 59nucleotide randomized region. A single selection cycle consisted of:
(1) Amplification by PCR followed by gel purification.
(2) Regeneration by asymmetric PCR followed by gel purification and
ethanol precipitation.
(3) Conditioning of the single-stranded DNA pool by heat denaturation and
slow-cooling to room temperature in binding buffer ((100 mM NaCl,
100 mM KCl, 5 mM MgCl2, 50 mM Tris-acetate (pH 8.0)).
(4) Selection by leaving the pool on a prewashed chitin bead column for 30
minutes followed by rinsing and subsequent elution with distilled
water. The eluate was then ethanol precipitated and subjected to
another round of selection (Fukusaki, Kato et al. 2000).
After 8 rounds they sequenced a fraction of their pool and were able to extract
seven binding motifs, of which five showed notably high guanosine content
(Figure 5b).
I decided to test our amplification/regeneration scheme using this target and
selection protocol. Apart from the experiment already having been done by
others my decision to choose this particular setup as my point of reference was
based on several parameters:
(a) Number of selection rounds. Fukusaki et al. managed to obtain
convergence in a very complex pool (N59) after eight selection rounds*. I
would be using a much smaller pool and so should see convergence
sooner (albeit at the cost of affinity).
(b) Nucleochemistry of the library. Their library was an all-DNA library. I
would be maintaining my library as a DNA library with LNA adenine.
(c) Composition of the converged pool. Their resulting aptamers showed a
high guanine content; given the non-natural nature of LNA I was
Note that while the N59 library encodes 459 ≈ 3.3×1035 unique members only 95×1012
were used in the initial round.
*
25
expecting to see the adenine content in my pool reduced over a number
of selection rounds.
(d) Stability and availability of target. Unlike protein targets chitin does not
denature or require special handling. Also, chitin beads are readily
available at a low cost, meaning that rather than attempting to strip the
column of residual DNA and reusing it for the next selection round, I
could simply discard it and prepare a new column.
26
(a)
(b)
Figure 5: (a) Chitin (poly-β-1,4-N-acetylglucosamine) is a long-chain polymer found
in the exoskeletons of crustaceans, insects, and fungal cell walls. Illustration from
[Wikimedia Commons]. (b) Hypothesized secondary structures of five of seven
motifs extracted after 8 rounds of in vitro selection against chitin. Numbers in
parenthesis indicate guanine content. Reproduced from (Fukusaki, Kato et al.
2000).
*foo
3 µg ssDNA / 36000 g/mol = 83.3 pmol = 50×1012 members
27
Sequence coverage is nada in this case. I can get complete sequence coverage
of NAC2080 156 fmol; I can achieve 100× coverage with 15.6 pmol!
*foo
In vitro selection against IgG
Strategy rationale
*hvorfor valgte vi at gøre som vi gjorde
Due to their negatively charged backbone nucleic acids will tend to bind to
positively charged pockets on proteins. In case of in vitro selection this may
lead to isolation of binding motifs that have low specificity for the objective
target and rather binds proteins in a more generic fashion. We therefore
supplied our selection buffer with sheared salmon sperm DNA as a competitive
inhibitor of DNA binding. This approach is typically used in applications where
specific DNA binding is paramount (e.g. Southern blotting), and it has also
been employed in the field of aptamer selections (Ogasawara, Hasegawa et al.
2007)
Adsorption of nucleic acids to plastic may occur to some extent, but this
depends on the types of plastic involved and its coating, if any. When the
partitioning of the selection pool into binding and non-binding species is
followed by amplification by PCR even minute amounts of carry-over of nonspecific contaminants can become a problem. We have previously used sodium
dodecyl sulfate (SDS) to reduce the adsorption of RNA to the walls of plastic
vials with great success *REF*. Here, we decided to include another detergent,
*Tween-20*, at a final concentration of *NNNN*%.
Finally, isolation of motifs that exhibit generic protein-binding capabilities and
so are not very specific for the target molecule is also a potential risk. We
attempted to counter this by adding an unrelated competitor protein (bovine
serum albumin, BSA) to our selection buffer. This technique has also been
employed by others in order to increase the specificity of their selected
aptamers (Ogasawara, Hasegawa et al. 2007; Hasegawa, Sode et al. 2008;
Doyle and Murphy 2009).
28
*forhold ml Lna og protein
*beads frem for søjlematrix
In vitro selection roundup
29
Concluding remarks
*manuskripterne beskriver en fungerende protokol, der bare venter på at blive
taget i brug. (copy-paste fra konklusionen deri.)
*har aptamer-hypen levet op til forventningerne? er de virkelelig så gode som
man sir, eller de bare et ny omgang antisense eller siRNA, der aldrig realiseres
helt?
30
References
Boese, B. J. and R. R. Breaker (2007).
"In vitro selection and
characterization of cellulosebinding
DNA
aptamers."
Nucleic Acids Res 35(19):
6378-6388.
Brown, D. A., S. H. Kang, et al. (1994).
"Effect of phosphorothioate
modification
of
oligodeoxynucleotides
on
specific protein binding." J Biol
Chem 269(43): 26801-26805.
Braasch, D. A., S. Jensen, et al. (2003).
"RNA
interference
in
mammalian
cells
by
chemically-modified
RNA."
Biochemistry 42(26): 79677975.
Burmeister, P. E., S. D. Lewis, et al.
(2005). "Direct in vitro
selection of a 2'-O-methyl
aptamer to VEGF." Chem Biol
12(1): 25-33.
Childs, J. L., M. D. Disney, et al. (2002).
"Oligonucleotide
directed
misfolding of RNA inhibits
Candida albicans group I intron
splicing." Proc Natl Acad Sci U
S A 99(17): 11091-11096.
Crinelli, R., M. Bianchi, et al. (2002).
"Design and characterization
of decoy oligonucleotides
containing locked nucleic
acids." Nucleic Acids Res
30(11): 2435-2443.
Doessing, H., R. N. Veedu, et al.
Amplification
and
regeneration of LNA-containing
libraries.
Doyle, S. A. and M. B. Murphy (2009).
Aptamers and methods for
their in vitro selection and
uses
thereof.
http://www.faqs.org/patents/
app/20090075834. U. S. P. T.
O. S. h. U. S. P. a. T. Office.
U.S., THE REGENTS OF THE
UNIVERSITY OF CALIFORNIA
Ellington, A. D. and J. W. Szostak
(1992). "Selection in vitro of
single-stranded
DNA
molecules that fold into
specific
ligand-binding
structures." Nature 355(6363):
850-852.
Elmen, J., H. Thonberg, et al. (2005).
"Locked nucleic acid (LNA)
mediated improvements in
siRNA
stability
and
functionality." Nucleic Acids
Res 33(1): 439-447.
Finnzymes. (2010). "Finnzymes web
site."
Retrieved January,
2011,
from
http://www.finnzymes.fi/pcr/
phusion_products.html.
Frieden, M., S. M. Christensen, et al.
(2003). "Expanding the design
horizon
of
antisense
oligonucleotides with alpha-LLNA." Nucleic Acids Res
31(21): 6365-6372.
Fukusaki, E., T. Kato, et al. (2000).
"DNA aptamers that bind to
chitin." Bioorg Med Chem Lett
10(5): 423-425.
Gao, S., F. Dagnaes-Hansen, et al.
(2009). "The effect of chemical
modification and nanoparticle
formulation on stability and
biodistribution of siRNA in
mice." Mol Ther 17(7): 12251233.
Gray, H. B., Jr., D. A. Ostrander, et al.
(1975).
"Extracellular
nucleases of Pseudomonas
BAL 31. I. Characterization of
single
strand-specific
deoxyriboendonuclease and
double-strand
31
deoxyriboexonuclease
activities." Nucleic Acids Res
2(9): 1459-1492.
Hasegawa, H., K. Sode, et al. (2008).
"Selection of DNA aptamers
against VEGF165 using a
protein competitor and the
aptamer blotting method."
Biotechnol Lett 30(5): 829-834.
Huizenga, D. E. and J. W. Szostak
(1995). "A DNA aptamer that
binds adenosine and ATP."
Biochemistry 34(2): 656-665.
Ittig, D., S. Liu, et al. (2004). "Nuclear
antisense effects in cyclophilin
A pre-mRNA splicing by
oligonucleotides:
a
comparison of tricyclo-DNA
with LNA." Nucleic Acids Res
32(1): 346-353.
Jepsen, J. S. and J. Wengel (2004).
"LNA-antisense rivals siRNA for
gene silencing." Curr Opin
Drug Discov Devel 7(2): 188194.
Kaur, H., A. Arora, et al. (2006).
"Thermodynamic, counterion,
and hydration effects for the
incorporation of locked nucleic
acid nucleotides into DNA
duplexes."
Biochemistry
45(23): 7347-7355.
Koshkin, A. A., J. Fensholdt, et al.
(2001). "A simplified and
efficient route to 2'-O, 4'-Cmethylene-linked
bicyclic
ribonucleosides
(locked
nucleic acid)." J Org Chem
66(25): 8504-8512.
Koshkin, A. A., S. K. Singh, et al. (1998).
"LNA (Locked Nucleic Acids):
Synthesis of the adenine,
cytosine,
guanine,
5methylcytosine, thymine and
uracil
bicyclonucleoside
monomers, oligomerisation,
and unprecedented nucleic
acid recognition." Tetrahedron
54(14): 3607–3630.
Kurreck,
J.
(2003).
"Antisense
technologies.
Improvement
through
novel
chemical
modifications." Eur J Biochem
270(8): 1628-1644.
Kurreck, J., E. Wyszko, et al. (2002).
"Design
of
antisense
oligonucleotides stabilized by
locked nucleic acids." Nucleic
Acids Res 30(9): 1911-1918.
Kuwahara, M., S. Obika, et al. (2008).
"Systematic
analysis
of
enzymatic DNA polymerization
using oligo-DNA templates and
triphosphate analogs involving
2',4'-bridged
nucleosides."
Nucleic Acids Res 36(13):
4257-4265.
Kuwahara, M., T. Ohbayashi, et al.
(2002).
"Enzymatic
incorporation of chemicallymodified nucleotides into
DNAs." Nucleic Acids Res
Suppl(2): 83-84.
Kuwahara, M., Y. Takano, et al. (2010).
"Study on suitability of KOD
DNA polymerase for enzymatic
production of artificial nucleic
acids
using
base/sugar
modified
nucleoside
triphosphates."
Molecules
15(11): 8229-8240.
Latorra, D., K. Arar, et al. (2003).
"Design considerations and
effects of LNA in PCR primers."
Mol Cell Probes 17(5): 253259.
Levin, A. A. (1999). "A review of the
issues in the pharmacokinetics
and
toxicology
of
phosphorothioate antisense
oligonucleotides."
Biochim
Biophys Acta 1489(1): 69-84.
Madsen, A. S., T. S. Kumar, et al.
(2010).
"LNA
5'phosphoramidites for 5'-->3'-
32
oligonucleotide synthesis." Org
Biomol Chem 8(21): 50125016.
Mannironi, C., A. Di Nardo, et al.
(1997). "In vitro selection of
dopamine
RNA
ligands."
Biochemistry 36(32): 97269734.
Marton, S., J. A. Reyes-Darias, et al.
(2010). "In Vitro and Ex Vivo
Selection
Procedures
for
Identifying
Potentially
Therapeutic DNA and RNA
Molecules." Molecules 15(7):
4610-4638.
McTigue, P. M., R. J. Peterson, et al.
(2004). "Sequence-dependent
thermodynamic parameters
for locked nucleic acid (LNA)DNA
duplex
formation."
Biochemistry 43(18): 53885405.
Meldgaard, M., C. Lomholt, et al.
(2005). Technical Note: Locked
Nucleic
Acid,
LNA
Oligonucleotide Synthesis. E.
A/S, Exiqon A/S: 1.
Miyauchi, K., M. Ogawa, et al. (1986).
"Development
of
a
radioimmunoassay for human
deoxyribonuclease I." Clin
Chim Acta 154(2): 115-123.
Mook, O. R., F. Baas, et al. (2007).
"Evaluation of locked nucleic
acid-modified small interfering
RNA in vitro and in vivo." Mol
Cancer Ther 6(3): 833-843.
Morita, K., M. Takagi, et al. (2003).
"Synthesis and properties of
2'-O,4'-C-ethylene-bridged
nucleic acids (ENA) as effective
antisense oligonucleotides."
Bioorg Med Chem 11(10):
2211-2226.
Nadano, D., T. Yasuda, et al. (1993).
"Measurement
of
deoxyribonuclease I activity in
human tissues and body fluids
by a single radial enzymediffusion method." Clin Chem
39(3): 448-452.
Nagahama, K., R. N. Veedu, et al.
(2009). "Nuclease resistant
methylphosphonate-DNA/LNA
chimeric
oligonucleotides."
Bioorg Med Chem Lett 19(10):
2707-2709.
Nielsen, K. E., J. Rasmussen, et al.
(2004). "NMR studies of fully
modified locked nucleic acid
(LNA)
hybrids:
solution
structure of an LNA:RNA
hybrid and characterization of
an
LNA:DNA
hybrid."
Bioconjug Chem 15(3): 449457.
Nishioka, M., H. Mizuguchi, et al.
(2001). "Long and accurate
PCR with a mixture of KOD
DNA polymerase and its
exonuclease deficient mutant
enzyme." J Biotechnol 88(2):
141-149.
Obayashi, T., M. M. Masud, et al.
(2002). "Enzymatic synthesis
of labeled DNA by PCR using
new fluorescent thymidine
nucleotide
analogue
and
superthermophilic KOD dash
DNA polymerase." Bioorg Med
Chem Lett 12(8): 1167-1170.
Obika, S., D. Nanbu, et al. (1997).
"Synthesis
of
2′-O,4′-Cmethyleneuridine
and
cytidine.
Novel
bicyclic
nucleosides having a fixed C3'endo
sugar
puckering."
Tetrahedron Letters 38(50):
8735–8738.
Ogasawara, D., H. Hasegawa, et al.
(2007). "Screening of DNA
aptamer against mouse prion
protein
by
competitive
selection." Prion 1(4): 248-254.
Ohbayashi, T., M. Kuwahara, et al.
(2005).
"Expansion
of
33
repertoire of modified DNAs
prepared by PCR using KOD
Dash DNA polymerase." Org
Biomol Chem 3(13): 24632468.
Owczarzy, R., A. V. Tataurov, et al.
(2008). "IDT SciTools: a suite
for analysis and design of
nucleic
acid
oligomers."
Nucleic Acids Res 36(Web
Server issue): W163-169.
Pan, W., P. Xin, et al. (2008). "Minimal
primer and primer-free SELEX
protocols for selection of
aptamers from random DNA
libraries." Biotechniques 44(3):
351-360.
Pestourie, C., L. Cerchia, et al. (2006).
"Comparison of different
strategies to select aptamers
against a transmembrane
protein
target."
Oligonucleotides 16(4): 323335.
Petersen, M., K. Bondensgaard, et al.
(2002). "Locked nucleic acid
(LNA) recognition of RNA:
NMR solution structures of
LNA:RNA hybrids." J Am Chem
Soc 124(21): 5974-5982.
Petersen, M., C. B. Nielsen, et al.
(2000). "The conformations of
locked nucleic acids (LNA)." J
Mol Recognit 13(1): 44-53.
Petersen, M. and J. Wengel (2003).
"LNA: a versatile tool for
therapeutics and genomics."
Trends Biotechnol 21(2): 7481.
Prince, W. S., D. L. Baker, et al. (1998).
"Pharmacodynamics
of
recombinant human DNase I in
serum." Clin Exp Immunol
113(2): 289-296.
Qian, J., X. Lou, et al. (2009).
"Generation of highly specific
aptamers via micromagnetic
selection." Anal Chem 81(13):
5490-5495.
Razzell, W. E. and H. G. Khorana
(1959).
"Studies
on
polynucleotides. III. Enzymic
degradation;
substrate
specificity and properties of
snake
venom
phosphodiesterase." J Biol
Chem 234(8): 2105-2113.
Rees, W. A., T. D. Yager, et al. (1993).
"Betaine can eliminate the
base
pair
composition
dependence of DNA melting."
Biochemistry 32(1): 137-144.
Sawai, H., J. Nagashima, et al. (2007).
"Differences
in substrate
specificity of C(5)-substituted
or
C(5)-unsubstituted
pyrimidine nucleotides by DNA
polymerases
from
thermophilic
bacteria,
archaea, and phages." Chem
Biodivers 4(9): 1979-1995.
Singh, S. K., P. Nielsen, et al. (1998).
"LNA (locked nucleic acids):
synthesis and high-affinity
nucleic acid recognition."
Chemical Communications 4:
455-456.
Stryer, L. (1999). DNA Structure,
Replication,
and
Repair.
Biochemistry, W. H. Freemand
and Company. 1: 787-818.
Takagi, M., M. Nishioka, et al. (1997).
"Characterization of DNA
polymerase from Pyrococcus
sp. strain KOD1 and its
application to PCR." Appl
Environ Microbiol 63(11):
4504-4510.
Tang, Z., P. Parekh, et al. (2009).
"Generating aptamers for
recognition of virus-infected
cells." Clin Chem 55(4): 813822.
Tolstrup, N., P. S. Nielsen, et al. (2003).
"OligoDesign: Optimal design
34
of LNA (locked nucleic acid)
oligonucleotide
capture
probes for gene expression
profiling." Nucleic Acids Res
31(13): 3758-3762.
Tran, D. T., K. P. Janssen, et al. (2010).
"Selection
and
characterization
of
DNA
aptamers for egg white
lysozyme." Molecules 15(3):
1127-1140.
Vaught, J. D., C. Bock, et al. (2010).
"Expanding the chemistry of
DNA for in vitro selection." J
Am Chem Soc 132(12): 41414151.
Veedu, R. N., B. Vester, et al. (2007).
"Enzymatic incorporation of
LNA nucleotides into DNA
strands." Chembiochem 8(5):
490-492.
Veedu, R. N., B. Vester, et al. (2007).
"In vitro incorporation of LNA
nucleotides."
Nucleosides
Nucleotides Nucleic Acids
26(8-9): 1207-1210.
Veedu, R. N., B. Vester, et al. (2007).
"Novel applications of locked
nucleic acids." Nucleic Acids
Symp Ser (Oxf)(51): 29-30.
Veedu, R. N., B. Vester, et al. (2008).
"Polymerase chain reaction
and transcription using locked
nucleic
acid
nucleotide
triphosphates." J Am Chem
Soc 130(26): 8124-8125.
Veedu, R. N., B. Vester, et al. (2009).
"Efficient enzymatic synthesis
of LNA-modified DNA duplexes
using KOD DNA polymerase."
Org Biomol Chem 7(7): 14041409.
Vester, B., L. B. Lundberg, et al. (2002).
"LNAzymes: incorporation of
LNA-type monomers into
DNAzymes markedly increases
RNA cleavage." J Am Chem Soc
124(46): 13682-13683.
Vester, B., L. B. Lundberg, et al. (2004).
"Improved RNA cleavage by
LNAzyme
derivatives
of
DNAzymes." Biochem Soc
Trans 32(Pt 1): 37-40.
Wahlestedt, C., P. Salmi, et al. (2000).
"Potent
and
nontoxic
antisense
oligonucleotides
containing locked nucleic
acids." Proc Natl Acad Sci U S A
97(10): 5633-5638.
Wang, L., B. Liu, et al. (2007).
"Selection of DNA aptamer
that specific binding human
carcinoembryonic antigen in
vitro." Journal of Nanjing
Medical University 21(5): 277281.
Wang, L., C. J. Yang, et al. (2005).
"Locked nucleic acid molecular
beacons." J Am Chem Soc
127(45): 15664-15665.
Weickmann, J. L. and D. G. Glitz (1982).
"Human
ribonucleases.
Quantitation of pancreatic-like
enzymes in serum, urine, and
organ preparations." J Biol
Chem 257(15): 8705-8710.
Wengel, J. (1999). "Synthesis of 3‘-Cand
4‘-C-Branched
Oligodeoxynucleotides and the
Development
of
Locked
Nucleic Acid (LNA)." Acc Chem
Res 32: 301-310.
Wu, L. and J. F. Curran (1999). "An
allosteric synthetic DNA."
Nucleic Acids Res 27(6): 15121516.
You, Y., B. G. Moreira, et al. (2006).
"Design of LNA probes that
improve
mismatch
discrimination." Nucleic Acids
Res 34(8): e60.
Yu, Y. T., M. D. Shu, et al. (1997). "A
new method for detecting
sites of 2'-O-methylation in
RNA molecules." RNA 3(3):
324-331.
35
Zamaratski, E., P. I. Pradeepkumar, et
al. (2001). "A critical survey of
the structure-function of the
antisense
oligo/RNA
heteroduplex as substrate for
RNase H." J Biochem Biophys
Methods 48(3): 189-208.
Aartsma-Rus, A., A. A. Janson, et al.
(2003).
"Therapeutic
antisense-induced
exon
skipping in cultured muscle
cells from six different DMD
patients." Hum Mol Genet
12(8): 907-914.
36
Manuscript I
Manuscript in preparation.
“Synthesis and isolation of a single-stranded DNA library with
multiple LNA-A modifications”
Holger Doessing, Rakesh N. Veedu, Jesper Wengel, and Birte Vester.
37
Manuscript II
Manuscript in preparation.
“Amplification and re-generation of LNA-containing libraries”
Holger Doessing, Anders Giessing, Rakesh N. Veedu, Lasse Holm Lauridsen,
Jesper Wengel, and Birte Vester.
38
Manuscript III
Manuscript in preparation.
*Paper sammen med Poul Nielsen og co.*
39