L N A ®: L O C K E D N U C L E I C A C I D S
U N L O C K Y O U R I M A G I N AT I O N
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
OLIGONUCLEOTIDES
LNA®
WHAT IS LNA®
Features & Benefits
>> Increased thermal stability of duplexes
because of its RNA-like structure
>> Reduction of oligonucleotide length
while maintaining desired Tm
>> Shorter oligonucleotide length improves
mismatch discrimination
>> Increased resistance to certain exo- and
endonucleases
>> DNA-LNA® chimeras readily activate RNase H
>> Highly soluble in water
Locked Nucleic Acid (LNA®) is a bicyclic RNA analogue
where the ribose sugar is structurally constrained by
a methylene bridge between the 2’-oxygen and the
4’-carbon atoms (Obika et al. 1997, Koskhin et al. 1998,
Singh et al. 1998). This bridge “ locks ” the ribose in a
3’-endo structural conformation that is characteristic
of A-type DNA.
When it anneals with its DNA complement, an
oligonucleotide containing LNA® bases changes
the conformation of this duplex in comparison to a
standard DNA:DNA duplex. Specifically, LNA® bases
inserted in every third position of an oligonucleotide
changes the structure of the double helix from the B
to A type. The bases are more optimally stacked in the
A type conformation, thereby increasing the stability
of the duplex, and its Tm too.
Studies have shown that each LNA® addition can
increase the Tm by 3-6 °C. This feature allows for
oligonucleotide length reduction, increasing the
specificity of the DNA-LNA® sequence.
In fact, LNA® is recommended for use in any
hybridisation assay that requires high specificity and/or
reproducibility, e.g. Double-Dye oligonucleotide probes,
in situ hybridisation probes, Molecular Beacons and
PCR primers. Furthermore, LNA® offers the possibility
to adjust Tm values of primers and probes in multiplex
assays. As a result of these significant characteristics,
the use of LNA® modified oligonucleotides can be used
in antisense drug development.
Experimental parameter or application
LNA®
Tm increase/monomer against DNA (°C)
Tm increase/monomer against RNA (°C)
∆Tm at single mismatch against DNA
Compatible with standard oligonucleotide synthesis
Chimera with DNA
Compatible with standard molecular biology
Works in PCR primers
Enhances allele specific priming
Water solubility
Hybridisation performance is predictable
Homogenous assay performance
Eurogentec Headquarters +32 4 372 76 65
2-6
3-8
LNA® >> DNA
Yes
Yes
Yes
Yes
Yes
High
Yes
LNA®>>DNA
+32 4 372 75 00
[email protected] www.eurogentec.com
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
OLIGONUCLEOTIDES
LNA®
ENHANCE YOUR APPLICATIONS WITH LNA®
Strategically replacing DNA with LNA® can be used to help enhance the sensitivity and specificity of the
following applications.
Real-Time qPCR
In situ hybridisation
Fluorescence (dR)
40000
30000
20000
10000
2 4 6 8 10 12 14 16 18 20 22 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
The increasing use of quantitative PCR for the
detection of pathogens and in other applications has
provided the possibility of faster and more accurate
diagnostics. A validated PCR-based Salmonella
method targeting a 94-bp sequence of the ttr
gene was used as a model to compare six different
combinations of reporter and quencher dyes of a
TaqMan® probe, on three different instruments, to
improve the detection limit in a Real-Time PCR assay
with the aim of a same-day analysis.
When comparing different probe technologies, the
LNA® probe (FAM-BHQ-1®) was the most sensitive
with the strongest fluorescence signal (Reynisson
et al., 2006).
The combination FAM-BHQ-1® or Cy5®-BHQ-3®,
both dark quenchers, gave the best results (Cycle
threshold (Ct) of 25.42 ± 0.65 and 24.47 ± 0.18
at 103 DNA copies).
One of the most frequently used techniques to study
gene expression is in situ hybridisation.
One drawback of this method is that shortening
the probes greatly reduces their stringency and thus
limits their use for in situ hybridisations to detect
short transcripts.
This limitation has hampered cellular studies of
differentially spliced exons and small RNAs such as
miRNAs. LNA® allows a significant increase in the
hybridisation temperature and thereby an enhanced
stringency for short probes as required for miRNA
detection. Recent experiments have shown that the
expression of mammalian miRNAs can be regulated
at the post-transcriptional level. In particular, miR138 is spatially restricted to distinct cell types,
while its precursor, pre-miR-138-2, is ubiquitously
expressed throughout all tissues analyzed. Thus,
differential processing of pre-miRNAs might be an
alternative mechanism to control miRNA function
(Obernosterer et al., 2006).
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
OLIGONUCLEOTIDES
LNA®
RNAi and antisense studies
SNP analysis
The half-life of unmodified siRNA in vivo is very
short (seconds to minutes) (Soutschek et al., 2004),
essentially due to their rapid elimination by kidney
filtration or degradation by endogenous serum
RNases. Several studies showed that nucleotide
analogue Locked Nucleic Acids (LNA®) is substantially
compatible with the siRNA machinery, preserving
molecule integrity.
With an increased emphasis on genotyping of
single nucleotide polymorphisms (SNPs) in disease
association studies, the genotyping platform of
choice is constantly in evolution. In addition, the
development of more specific SNP assays and
appropriate genotype validation applications is
becoming increasingly critical to elucidate ambiguous
genotypes. Detection of single base substitutions
using a high affinity DNA analogue known as Locked
Nucleic Acid (LNA®) for use in allelic discrimination
assays has been achieved by several methods as
ELISA or Real-Time qPCR.
siLNA, modified at the 3’ overhangs and at the 5’ sense
end, inhibit the sense target (pS3Xs) and to the same
extent indicating that both siRNA and siLNA are
effective in loading the antisense strand into RISC.
Indeed, siLNA exhibit greatly improved bio-stability and
show enhanced inhibition at certain RNA targets.
In addition, LNA® can be used to reduce sequencerelated off-target effects by either lowering
incorporation of the siRNA sense strand and/or by
reducing the ability of inappropriately loaded sense
strands to cleave the target RNA.
Brugé et al. have shown that the implementation
of LNA® probes in Real-Time qPCR genotyping of
SOD3 760 G >C is able to unambiguously detect
single nucleotide polymorphism using annealing
temperature as low as 66.4 °C. They also
demonstrated that LNA® dual-labelled fluorogenic
probes discriminate better than the Taqman® probes
in SNP analysis (Brugè et al., 2009).
Other modifications than LNA® have been shown to
provide benefits to siRNA and could be conceivable
when successfully combined with LNA® (Elmèn
et al., 2005).
Eurogentec Headquarters +32 4 372 76 65
+32 4 372 75 00
[email protected] www.eurogentec.com
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
OLIGONUCLEOTIDES
LNA®
CUSTOM LNA® OLIGONUCLEOTIDES
Design
Synthesis
Because of the high affinity properties introduced
by LNA®, three basic design rules should be
followed for all assays that include LNA® modified
oligonucleotides:
OCH3
O
O
4. Oxidation
OCH3
N
H3CO
O
O
O
HN
N
N
N
N
O
Synthesis
Cycle
N
N
O
O
N
O
N
N
N
O
OCH3
CPG
O
HN
N
O
O
N
HO
H3CO
O
O
O
N
HN
HN
N
1. Deblocking
N
OCH3
O
CPG
HN
CPG
O
Eurogentec is able to provide you with the
optimal design for your primers, probes and
siRNA. These services include primers, Double-Dye
oligonucleotides, Molecular Beacons, siRNA design
suitable for gene expression and SNP analysis. We
continuously update our software and design rules
to reflect the latest scientific developments as well
as integrate customer requirements.
O
O
N
O
O
N
CPG
N
HN
CN
N
O
N
N
O P O
As LNA® will bind very tightly to other LNA®
residues, avoid self-complementarity and
complementarity to other LNA® containing
oligonucleotides in the assay.
CN
O
N
N
O
N
N
O
O
O P O
HN
2
3
O
HN
N
H3CO
®
As LNA® hybridizes very tightly when several
consecutive residues are substituted with LNA®,
avoid stretches of more than 4 LNA®.
H3CO
O
O
1 Introduce LNA at the positions where specificity
and discrimination are needed.
O
HN
N
OCH3
N
H3CO
N
O
O
N
3. Capping
CPG
N
O
HN
O
N
O P O
O
CN
O
N
N
N
O P N
2. Coupling
CN
N
N
O
CPG
LNA® can be mixed with DNA, RNA, and other nucleic
acid analogues – using standard phosphoramidite
DNA synthesis chemistry. Therefore, oligonucleotides
containing LNA® can be tagged with modifications
such as amino linkers, biotin, fluorophores and
are water soluble, and can be separated by gel
electrophoresis and precipitated by ethanol.
Oligonucleotides containing LNA® are deprotected,
desalted, and are routinely controlled by MALDITOF Mass Spectrometry. RP-Cartridge•Gold™
purification is highly recommended for those
applications that require higher purity.
Delivery schedules
LNA®
Unmodified LNA®
DNA Oligonucleotides
5-7 working days
Modified or purified LNA®
DNA Oligonucleotides
7 – 8 working days
Double-Dye Oligonucleotides
with LNA®
7 –10 working days
N
N
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
OLIGONUCLEOTIDES
LNA®
ORDERING INFORMATION
Web Ordering
Oli&GO™
Enhanced ordering interface (which can be found
behind the login button on the right page of the
Eurogentec’s website)[A]:
The Oli&GO™ is an exclusive and revolutionary
ordering concept developed by Eurogentec. It
simplifies and streamlines the ordering process for
your oligonucleotides, whatever your requirements.
Login
[A]
The web-ordering interface has been developed
and optimised to simplify your ordering process.
To have access to this amazing interface, register
on www.eurogentec.com [B]. And let’s go to your
first order!
With only one order and one invoice, the Oli&GO™
allows you to order your oligonucleotides, whenever
you want, even if your needs are spread over several
months.
Upon request of your order, you will receive a client
ID number and a password by e-mail, to login on
Eurogentec’s homepage to the corresponding
ordering interface.
An integrated counter gives you the status of your
account throughout its use.
If your counter is close to zero, indicated by a
coloured light, your account can easily be reloaded
directly on the Oli&GO™ ordering interface, by
e-mail or fax.
[B]
You have access to the complete Eurogentec
oligonucleotide range: Bases, Modifications,
Purifications and Additional Services.
Once the ordering form is complete and sent to
Eurogentec, you receive an e-mail confirmation. The
online tracking allows you to check the status of
your orders at any time.
Eurogentec Headquarters +32 4 372 76 65
By e-mail
To get the template form and ordering instructions, please contact us at
[email protected] or visit our website.
+32 4 372 75 00
[email protected] www.eurogentec.com
OLIGONUCLEOTIDES
LNA®
LIFE SCIENCE CUSTOM SERVICES , KITS & REAGENTS
REFERENCES
Description
Synthesis Scale
Recommended
Purification*
Included
Purification
AD-DGDDP
AD-DGDDP-SNP
N/A
N/A
N/A
N/A
N/A
N/A
BA-LN001-004
BA-LN001-020
BA-LN001-100
40 nmol
200 nmol
1000 nmol
RP-Cartridge
RP-Cartridge
RP-Cartridge
Se POP
Se POP
Se POP
PB-DD601-004
PB-DD601-020
PB-DD601-100
PB-DD610-004
PB-DD610-020
PB-DD610-100
PB-DD612-004
PB-DD612-020
PB-DD612-100
PB-DD640-004
PB-DD640-020
PB-DD640-100
40 nmol
200 nmol
1000 nmol
40 nmol
200 nmol
1000 nmol
40 nmol
200 nmol
1000 nmol
40 nmol
200 nmol
1000 nmol
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
SR-NP001-001
SR-NP001-004
SR-NP001-020
SR-NP001-100
SR-HP001-001
SR-HP001-004
SR-HP001-020
SR-HP001-100
10 nmol
40 nmol
200 nmol
1000 nmol
10 nmol
40 nmol
200 nmol
1000 nmol
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Se POP
Se POP
Se POP
Se POP
RP-HPLC
RP-HPLC
RP-HPLC
RP-HPLC
CD-OG002-XS
CD-OG002-M
-
-
-
CD-OGR02-S
CD-OGR02-M
CD-OGR02-L
CD-OGR02-XL
-
-
-
Reference
CUSTOM OLIGONUCLEOTIDES - ADDITIONAL SERVICES
Additional Services for Custom Oligonucleotides + Design
Double Dye Probe design (including LNA®)
Double Dye Probe design (including LNA®) for SNP Analysis
CUSTOM OLIGONUCLEOTIDES - BASES
LNA® Bases
LNA® Base
LNA® Base
LNA® Base
REAL - TIME qPCR OLIGONUCLEOTIDES
Double-Dye probes 3’ BHQ
DD Probe 5’ FAM + 3’ BHQ-1®
DD Probe 5’ FAM + 3’ BHQ-1®
DD Probe 5’ FAM + 3’ BHQ-1®
DD Probe 5’ HEX + 3’ BHQ-1®
DD Probe 5’ HEX + 3’ BHQ-1®
DD Probe 5’ HEX + 3’ BHQ-1®
DD Probe 5’ TET + 3’ BHQ-1®
DD Probe 5’ TET + 3’ BHQ-1®
DD Probe 5’ TET + 3’ BHQ-1®
DD Probe 5’ Yakima Yellow® + 3’ BHQ-1®
DD Probe 5’ Yakima Yellow® + 3’ BHQ-1®
DD Probe 5’ Yakima Yellow® + 3’ BHQ-1®
RNAi OLIGONUCLEOTIDES
Custom siRNA Duplexes
siRNA Duplex - Desalted
siRNA Duplex - Desalted
siRNA Duplex - Desalted
siRNA Duplex - Desalted
siRNA Duplex HPLC
siRNA Duplex HPLC
siRNA Duplex HPLC
siRNA Duplex HPLC
OLI&GO™
Oli&GO™ activation
Oli&GOTM Activation XS
Oli&GOTM Activation M
Oli&GO™ Reloads
Oli&GOTM Reload S
Oli&GOTM Reload M
Oli&GOTM Reload L
Oli&GOTM Reload XL
*Recommended Purification: With more than 20 years expertise, Eurogentec recommends you the best Purification according to your modification(s).
References
• Brugè F., et al. A novel Real Time PCR strategy to detect SOD3 SNP using LNA probes. Mutation Research (2009)
• Elmén J, et al. Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res. (2005 Jan) 14;33(1):439-47
• Koshkin AA, et al. LNA (Locked Nucelic Acid): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron (1998) 54: 3607-3630.
• Obernosterer G,et al. Post-transcriptional regulation of microRNA expression. RNA (2006), 12:1161-1167
•Obika S, et al. Stability and structural features of the duplexes containing nulcoeside analogs with a fixed N-type conformation. 2’-O, 4’-C methylene ribonucleosides. Tetrahedron (1998)
Lett 39: 5401-5404.
• Singh SK, et al. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Commun (1998) 4: 455-456.
• Soutschek J, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. (2004 Nov) 11;432(7014):173-8
• Reynisson E, et al. Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR.Journal Microbiol Methods (2006), 66 (2), 206 – 216.
LNA® is a registered trademark of Exiqon A/S. TaqMan® is a registered trademark of Applied Biosystems.
Yakima yellow® is a registered trademark of Epoch Biosciences, Inc. BHQ-1® is a registered trademark of Biosearch Technologies, Inc.
G O L - L N A L E A F L E T- 0 5 1 4 - V 5
L N A ®: L O C K E D N U C L E I C A C I D S
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