Laura Auchterlonie & Rachael Amador Locked Nucleic Acid Laura Auchterlonie & Rachael Amador

Structure The ribose ring is connected by a methylene bridge (orange) between the 2’-O and 4’-C atoms thus “locking” the ribose ring in the ideal conformation for Watson-Crick binding.

Conformational Types Two major conformational types. A-type and the B-type, dictated by the puckering of the single nucleotides. C3´-endo (N-type) conformation in the A-type. C2´- endo (S-type) conformation in the B-type. The A- type is adopted by RNA The B- types is adopted by DNA

Overall Structure and Dynamics The general structure of LNA–DNA and LNA–RNA duplexes resemble the RNA–DNA and RNA–RNA duplexes. LNA–LNA duplex exhibits a slight unwinding of the helix (see twist angles in section on base stacking), resulting in relatively shallower grooves, compared to other duplexes. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Intrastrand phosphate distances in LNA strands are shorter than DNA and slightly shorter than RNA.

Sugar Puckering LNA tunes the sugar puckering in partner DNA strand towards C3′-endo pucker or North conformations more efficiently than RNA.

Backbone Flexibility The DNA backbone is slightly more flexible than LNA or RNA backbones and LNA does not show any conformational coupling of its reduced backbone flexibility onto partner strands. Also, the LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex.

Water Structure and Dynamics LNA is less hydrated compared to DNA or RNA but has a well-organized water structure, in context of the backbone. DNA and RNA only form very infrequent multiple hydrogen-bonding bridges via water molecules. LNA strands have the most frequent hydrogen-bonding bridges, resulting in a net higher occupancy of water bridged backbone.

Base Stacking Compared to RNA helices, LNA has a decrease in helical twist, roll and propeller twists angles. Facilitates widening of major groove. Subsequent addition of consecutive LNAs stabilizes duplexes by favorable enthalpic changes that are associated with enhanced stacking interactions.

Function/Properties of LNA High melting temperature/thermal stability. High solubility High binding affinity. Resistant to exo- and endonucleases.

Melting Temperatures

LNA-LNA duplex water-mediated hydrogen bonding

Binding Affinity/ Nuclease resistance

Unlinked Nucleic Acids (UNA) UNA is an analogue of RNA in which the C2′-C3' bond has been cleaved. UNA is very flexible, as a result of the lack of the C2′-C3' bond. UNA has a destabilizing effect.

Why Use LNAs? Tm normalization – robust detection regardless of GC content Single nucleotide discrimination Broad applicability For AT-rich nucleotides, which give low melting temperatures, more LNA™ is incorporated into the LNA™ oligonucleotide to raise the Tm of the duplex. This enables the design of LNA™ oligonucleotides with a narrow Tm range which is beneficial in many research applications such as microarray, PCR and other applications where sensitive and specific binding to many different targets must occur under the same conditions simultaneously.

The power of Tm normalization. The power of Tm-normalization is demonstrated by the comparison of DNA and LNA™ probes for detection of microRNA targets with a range of CG content Signal intensity from microarray experiments using LNA™ enhanced (blue) or DNA based (gray) capture probes. MicroRNA targets with varying GC-content (pink) were added at 100amol each. The signal from DNA-based capture probes varies with GC content and results in poor detection of many microRNAs, whereas LNA™ probes offer robust detection of all microRNAs The small sizes and widely varying GC-content (5-95 %) of microRNAs make them challenging to analyze using traditional methods. DNA or RNA based technologies for microRNA analysis can introduce high uncertainty and low robustness because the melting temperature (Tm) of the oligonucleotide/microRNA duplex will vary greatly depending on the GC content of the sequences. This is especially problematic in applications such as microarray profiling and high throughput experiments where many microRNA targets are analyzed under the same experimental conditions. These challenges in microRNA analysis can be overcome by using LNA™-enhanced oligonucleotides. By simply varying the LNA™ content, oligonucleotides with specific duplex melting temperatures can be designed, regardless of the GC-content of the microRNA. Exiqon has used the LNA™ technology to Tm-normalize primers, probes and inhibitors to ensure that they all perform well under the same experimental conditions. Another challenge of studying microRNAs is the high degree of similarity between the sequences. Some microRNA family members vary by a single nucleotide. LNA™ can be used to enhance the discriminatory power of primers and probes to allow excellent discrimination of closely related microRNA sequences. LNA™ offers significant improvement in sensitivity and specificity and ensures optimal performance for all microRNA targets.

Single Nucleotide Discrimination Intelligent placement of LNA™ monomers The difference in Tm between a perfectly matched and a mismatched target is described as the delta Tm. Incorporation of LNA raises delta Tm 1. can also ensure excellent discrimination between closely related sequences down to as little as one nucleotide difference.

Broad Applicability Strand invasion properties Physical properties (e.g. water solubility) are very similar to those of RNA and DNA

Powerful Tool for Nucleic Acids Research The unique characteristics of LNA™ make it a powerful tool for detection of low abundance, short or highly similar targets in a number of other applications The unique ability of LNA™ oligonucleotides to discriminate between highly similar sequences has further been exploited in a number of applications targeting longer RNA sequences such as mRNA.

Proven LNA™ applications                                                                               Proven LNA™ applications LNA™ is a powerful tool in many applications where standard DNA or RNA oligonucleotides do not have sufficient affinity or specificity. The figure shows an overview of some of the LNA™ applications that have been used for the study of RNA and DNA.

mRNA in situ hybridization Fast and specific mRNA in situ hybridization with specific LNA™ oligonucleotide in fixed cells Improved signal and less background using a LNA™ mRNA in situ hybridization probe (left picture) compared to a DNA probe (right picture). Images from Thomsen et al., RNA 2005, (11), 1745 - 48.

References Potent and nontoxic antisense oligonucleotides containing locked nucleic acids Claes Wahlestedt*†, Peter Salmi*, Liam Good*, Johanna Kela*, Thomas Johnsson*, Tomas Ho¨ kfelt‡, Christian Broberger‡, Frank Porreca§, Josephine Lai§, Kunkun Ren§, Michael Ossipov§, Alexei Koshkin¶, Nana Jakobsen¶, Jan Skouv¶i, Henrik Oerum¶, Mogens Havsteen Jacobsen¶, and Jesper Wengel** Locked Nucleic Acids (LNA) Gene Link. LNA (Locked Nucleic Acid):  High-Affinity Targeting of Complementary RNA and DNA†Birte Vester*‡ and Jesper Wengel§ Nucleic acid analogues.atbBIO http://www.atdbio.com/content/12/Nucleic-acid-analogues LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV‐1 1. Fabien Darfeuille1,2, Jens Bo Hansen3, Henrik Orum3 Carmelo Di Primo*,1,2 and Jean‐Jacques Toulmé1,2 Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulation Vineet Pande and Lennart Nilsson* LNA (Locked Nucleic Acid): High-Affinity Targeting of Complementary RNA and DNA†Birte Vester*,‡ and Jesper Wengel Locked and Unlocked Nucleosides in Functional Nucleic Acids. Holger Doessing and Birte Vester http://www.geneworks.com.au/library/LNA_-_Locked_Nucleic_Acid.pdf http://www.exiqon.com/lna-technology CONTAINS LNA MOVIE http://www.exiqon.com/ls/Documents/Scientific/LNA_folder.pdf MORE INFO ON LNA