Product Sheet

LNA Oligonucleotide Melting Temperature
LNA is a unique base modification due to its higher Tm. This extreme duplex stability restricts the use of to many LNA bases especially in short oligos. Introduction of more than 4 LNA bases consecutively induces irreversible intra oligo LNA base binding and should be avoided. A maximum of 8 LNA bases is recommended in a 18mer oligo.

Prediction of Melting Temperature (Tm) for DNA and LNA Modified Oligonucleotides
The Tm prediction given below was calculated by the LNA Tm predictor available at http://lnatools.com/

Melting temperature
In order to design optimal probes and primers it is important to predict the melting temperature (Tm) of the LNA modified oligonucleotides. Tm is a necessary, but not sufficient, factor for determining the efficiency of capture probes and primers. Other important factors are the secondary structure, the discrimination ability and the steepness of the melting curve. LNA gives a higher degree of freedom with regard to optimising the melting temperature range of the oligonucleotides.

Parameter estimation in the Tm model
LNA modified oligonucleotides have eight possible monomers as opposed to the four DNA nucleotides, therefore the number of possible nearest neighbour pairs is 64 instead of 16. Since both entropy (?S) and enthalpy (?H) are estimated using statistical learning, up to 128 parameters needs to be estimated from the experimental data. Because of experimental error, more than one sample per parameter is necessary, thus in the order of 300 measurements of hybridisations are used, in order to get good parameter estimates. To get even better parameter estimates several parameter reduction techniques have been used. The model parameters were trained by optimising a sum-of-squared-residual error function using gradient descent (16).

Tm Comparison of Standard DNA bases and LNA substituted bases
Oligo Type	18 mer Oligo Sequence Lower case DNA and Upper case are LNA bases	# of LNA bases	Tm at 115 mM salt and 2µM Traget+Probe	Tm at 200 mM salt and 2µM Traget+Probe
All DNA	5’-caagttcctcctgtttag-3’	Zero	55oC	58oC
All LNA	5’-CAAGTTCCTCCTGTTTAG-3’	18	98oC	102oC
Mixed	5’-caagTtcctcctgtttag-3’	1 T LNA base	56oC	59oC
Mixed	5’-caAgttcctcctgtttag-3’	1 A LNA base	57oC	60oC
Mixed	5’-caaGttcctcctgtttag-3’	1 G LNA base	57oC	60oC
Mixed	5’-caagttCctcctgtttag-3’	1 C LNA base	57oC	60oC
Mixed	5’-caagTtcctcctgtTtag-3’	2 T LNA base	57oC	59oC
Mixed	5’-caagTtcctcctGtttag-3’	1 T & 1G LNA	58oC	61oC
Mixed	5’-caAgttCctcctgtTtag-3’	3 LNA (T,C & A)	60oC	62oC
Mixed	5’-caAGttCctcctgtTtag-3’	4 LNA (G,A,T& C)	61oC	64oC
Mixed	5’-caAGttCctCctgtTtag-3’	5 LNA	64oC	66oC
Mixed	5’-cAAgttCctCctGtTtag-3’	6 LNA	66oC	69oC
Mixed	5’-cAAgttCcTCctGtTtag-3’	7 LNA	69oC	72oC
Mixed	5’-cAAgttCcTCctGtTtAg-3’	8 LNA	72oC	75oC

*License Agreement: Locked-nucleic Acid (LNA) phosphoramidites are protected by EP Pat No. 1013661, US Pat No. 6,268,490 and foreign applications and patents owned by Exiqon A/S. Products are made and sold under a license from Exiqon A/S. Products are for research purposes only. Products may not be used for diagnostic, clinical, commercial or other use, including use in humans. There is no implied license for commercial use, including contract research, with respect to the products and a license must be obtained directly from Exiqon A/S for such use.