Amino acids attached to 2′-amino-LNA: synthesis and excellent duplex stability

Abstract

The synthesis of 2'-amino-LNA (the 2'-amino derivative of locked nucleic acid) has opened up a number of exciting possibilities with respect to modified nucleic acids. While maintaining the excellent duplex stability inferred by LNA-type oligonucleotides, the nitrogen in the 2'-position of 2'-amino-LNA monomers provides an excellent handle for functionalisation. Herein, the synthesis of amino acid functionalised 2'-amino-LNA derivatives is described. Following ON synthesis, a glycyl unit attached to the N2'-position of 2'-amino-LNA monomers was further acylated with a variety of amino acids. On binding to DNA/RNA complements, the modified ONs induce a marked increase in thermal stability, which is particularly apparent in a buffer system with a low salt concentration. The increase in thermal stability is thought to be caused, at least in part, by decreased electrostatic repulsion between the negatively charged phosphate backbones when positively charged amino acid residues are appended. Upon incorporation of more than one 2'-amino-LNA modification, the effects are found to be nearly additive. For comparison, 2'-amino-LNA derivatives modified with uncharged groups have been synthesised and their effect on duplex thermal stability likewise investigated.

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

“…that the 2΄- O -methyl RNA interaction with an RNA target is stronger than with a DNA target based on T m measurements, which could help to explain the reduced efficiency of the tested ON (77). 2΄-Glycylamino-LNA is another recently developed modification carrying a positively charged glycyl group at the N2΄-position enhancing duplex stability (78), but did not show any improvement in the anti-gene context. It is plausible that these delivery methods are not optimal for positively charged modified ONs, and further optimization might be needed.…”

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

“…The amino-glycyl modification adds a glycyl residue to the N2′ position of 2′-amino-LNA, thereby providing a positive charge (51). UNA is known to decrease the Tm and destabilize the duplex, which potentially could increase target specificity if appropriately designed (52).…”

Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation

“…The gapmer ASOs (ASO 1-12) were prepared by automated oligonucleotide synthesis using commercial DNA and LNA phosphoramidites and palmitoyl-and myristoyl-amino-LNA phosphoramidites using standard methods for oligonucleotide synthesis, workup, purification, and isolation with minor modifications as published for oligonucleotides containing palmitoyl-amino-LNA nucleotide monomers. 34 Sequences of all ASOs are listed: ASO 1: 5 0 -TAGcctgtcactt CTC-3 0 , ASO 2: 5 0 -PAGcctgtcacttCTC-3 0 , ASO 3: 5 0 -PAGcctgtcactt CPC-3 0 , ASO 4: 5 0 -TAGcctgtcacttCPP*-3 0 , ASO 5: 5 0 -MAGcctgtcactt CTC-3 0 , ASO 6: 5 0 -MAGcctgtcacttCMC-3 0 , ASO 7: 5 0 -TAGcctgt cacttCTC-3 0 , ASO 8: 5 0 -PAGcctgtcacttCTC-3 0 , ASO 9: 5 0 -PAGcctgtca cttCPC3-3 0 , ASO 10: 5 0 -TAGcctgtcacttCPP*-3 0 , ASO 11: 5 0 -MAG cctgtcacttCTC-3 0 , ASO 12: 5 0 -MAGcctgtcacttCMC-3 0 . P and M denote palmitoyl-amino-LNA and myristoyl-amino-LNA thymine monomers, respectively; P* and M* denote palmitoyl-amino-LNA and myristoyl-amino-LNA 5-methyl-cytosine monomers, respectively; A, C, G, and T denote LNA monomers, and a, c, g, and t denote DNA monomers.…”