Custom LNA Oligonucleotide Design Guidelines

Design your own LNA oligo 
Incorporation of LNA into a sequence strongly affects the properties of the oligonucleotide, and great care must be taken to find the right design for your experimental purpose. While you will want to take advantage of the properties of LNA to achieve high target specificity, it is important not to use too much LNA, because this can result in a very "sticky" oligonucleotide that is difficult to handle experimentally. The design must be optimized by varying the length and LNA content of the oligonucleotide, such that you achieve good mismatch discrimination and high binding specificity, while avoiding unacceptable secondary structure and self-complementarity.

General design guidelines:

• LNA will bind very tightly to other LNA residues. Avoid self-complementarity and cross-hybridization to other LNA-containing oligonucleotides.
• Keep the GC-content between 30–60%.
• Avoid stretches of more than 4 LNA bases, except when designing very short (9–10 nucleotides) oligos.
• Avoid stretches of 3 or more Gs or Cs.
• For novel applications, design guidelines may have to be established empirically.

The following tools and calculators are also available to support you:
T_m Prediction Calculator
LNA Oligo Optimizer
Oligo Dilution Calculator
Oligo Resuspension Calculator
Oligo Concentration Converter
LNA Nomenclature Converter

To design and order Custom LNA Oligonucleotides, visit the product catalog page.

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Let us design your LNA oligo 
QIAGEN’s in-house LNA experts can help you design the best LNA oligonucleotide for your application and target of interest. Your oligonucleotide will be designed for optimal LNA content and positioning to achieve optimal specificity and minimal secondary structure and self-complementarity. To use this service, please provide us with the details ofyour request (target sequence and application) in the contact form.

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Two models for T_m prediction
QIAGEN uses two different models for predicting the melting temperature (T_m) of LNA oligonucleotides:

• DNA T_m: This model predicts the T_m of a complex formed between the LNA oligo and a complementary DNA strand.
• RNA T_m: This is a new model that predicts the T_m of a complex formed between an LNA oligo and a complementary RNA strand.

Both models are based on experimental data from thousands of oligonucleotide hybridizations. For oligonucleotides of 15–27 nucleotides in length, the resulting predictions have a precision of 1.70 and 2.07°C for RNA and DNA targets, respectively.

Recommended use of the models 
We recommend using the RNA T_m for applications in which LNA oligos target RNA sequences. RNA T_m information will be introduced to relevant products in the future.

We generally recommend starting with a hybridization temperature 30°C below the RNA T_m when detecting RNA targets. This temperature translates to approximately 20°C below the DNA T_m. For example, the oligo C+TG+AC+CGT+ATG+GTC+TA+TA will have RNA T_m and DNA T_m of 86 and 70°C, respectively.

Please note that the actual hybridization temperatures may have to be optimized for each experiment.

Assumptions and limitations of the models

• The T_m calculation models are based on a modified nearest-neighbor thermodynamic model combination and data from measurements of oligonucleotides ranging from 15–27 nucleotides in length.
• All predictions are based on the following:
• Oligos range from 6–80 bases in length
  
  • Salt concentration (Na+) is 115 mM
  • Solution is neutral (pH 7–8)
  • Oligo concentration is 1 and 2 µM for RNA and DNA T_m calculations, respectively.
  • The calculations do not account for effects of divalent cations, triphosphates or chemical modifications except LNA
• 2’ O-Me modifications can be expected to increase the T_m by 1.3°C per inserted modification. The influence of such modifications has not been taken into account in the models.

 
A phosphorothioate (PS) backbone can be expected to reduce the T_m by 0.4–1.2°C per inserted modification depending on oligo length and GC content. The influence of such modifications has not been taken into account in the models.

Dyes

Freedom dyes	5'-end	Int	3'-end	Excit	Emit	Alternative
6-FAM (Fluorescein)	+		+	495	520
MAX 550	+		+	531	560	JOE 555 VIC 554
TYE 563	+		+	549	563	Cy3
TEX 615	+		+	596	613	Texas Red-X 617
TYE 665	+			645	665	Cy5
TYE 705	+			686	705
TET	+			522	539
HEX	+			538	555
Fluorescein dT	+	+		495	520
Rhodamine dyes	5'-end	Int	3'-end	Excit	Emit	Alternative
TAMRA			+	559	583
TAMRA NHS Ester	+	+	+	559	583
ROX NHS Ester	+		+	588	608
Other	5'-end	Int	3'-end	Excit	Emit	Alternative
Dy 750 NHS Ester	+			747	776

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Attachment chemistry/linkers

Amino modifiers	5'-end	Int	3'-end
Amino Modifier C6	+		+
Amino Modifier C12	+
Amino Modifier C6 dT	+	+
Amino Modifier			+
Uni-Link Amino Modifier	+	+
Labels/Antibodies	5'-end	Int	3'-end
Biotin	+		+
Biotin dT	+	+
Biotin TEG	+		+
Biotin Dual	+
PC Biotin	+
Thiol modifications	5'-end	Int	3'-end
Thiol Modifier C3 S-S			+
Thiol modifier C6 S-S	+
Spacers and Linkers	5'-end	Int	3'-end
C3	+	+	+
PC Spacer	+	+
Hexanediol	+	+
Spacer 9	+	+
Spacer 18	+	+
1’,2’-Dideoxyribose (dSc)	+	+
I-Linker	+
Other	5'-end	Int	3'-end
Digoxigenin NHS Ester (DIG)	+		+
Cholesteryl-TEG			+

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Quenchers

	5'-end	Int	3'-end
Dabcyl			+

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Modified bases

	5'-end	Int	3'-end
LNA (Locked Nucleic Acid)	+	+	+
2-Aminopurine	+	+
Trimer-20	+	+
Fluoro Bases	+	+
2,6-Diaminopurine (2-Amino-dA)	+	+
5-Bromo dU	+	+
Deoxyuridine	+	+
Inverted dT			+
Dideoxy-C			+
5-Methyl dC	+	+	+
Deoxyinosine	+	+	+
5-Nitroindole	+	+
Ribo A			+
Ribo C			+
Ribo G			+
Ribo U			+
2'-O-Methyl RNA Bases	+	+	+

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Phosphorylation

	5'-end	Int	3'-end
Phosphorylation	+		+

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Backbone modification

	5'-end	Int	3'-end
Phosphorothioates	+	+	+

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Purification options
Our standard practice is to deprotect and desalt all oligonucleotides to remove small molecule impurities, quantify the oligo by UV spectrophotometry to provide an accurate measure of yield, and perform quality control (QC) by mass spectrometry.

Additional purification is recommended for many modified oligonucleotides including heavily LNA-substituted, unmodified oligonucleotides. For demanding applications, such as single nucleotide discrimination and antisense inhibition, purification may improve the performance of the oligonucleotide. For these situations, we typically recommend a standard RP-HPLC purification, which routinely results in >85% purity.

For long oligonucleotides (>60 bases) and for applications requiring oligonucleotides with a very high purity, we recommend PAGE, IE-HPLC (ion-exchange HPLC) or dual HPLC. RNase-free HPLC purification can be applied for oligonucleotides that will be used in applications sensitive to ribonucleases. Please contact us for further information regarding our purification offerings.

Na⁺ salt exchange is available for oligonucleotides that will be used in applications in which the presence of minute amounts of toxic salts can cause unwanted side reactions.

Modifications and secondary structure of the oligonucleotide might affect yield and purity guarantees. Please contact us to obtain information on guaranteed yield and purity.

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Delivery
By default, oligonucleotides are delivered lyophilized in standard tubes. A number of tools for post-delivery concentration adjustment can be accessed here.

• Orders of 24 oligonucleotides or more can be delivered in 96-well plates.
• Orders of 96 oligonucleotides or more can be delivered in 384-well plates.

We also offer options for delivery in plates, such as normalization to the same concentration across the plate, delivery of the full yield or mixing of normalized oligonucleotides in the same well.

Please contact us with any request for custom delivery of your oligonucleotides.

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