Alexei A. Koshkin

LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition

Abstract LNA (Locked Nucleic Acids), consisting of 2′- O ,4′- C -methylene bicyclonucleoside monomers, is efficiently synthesized and its nucleic acid recognition potential evaluated for six different nucleobases, namely adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil. Unprecedented increases (+3 to +8 °C per modification) in the thermal stability of duplexes towards both DNA and RNA were obtained when evaluating mixed sequences of partly or fully modified LNA. Studies of mis-matched sequences show that LNA obey the Watson-Crick base pairing rules with generally improved selectivities compared to the corresponding unmodified reference strands.

LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition

A novel class of nucleic acid analogues, termed LNA (locked nucleic acids), is introduced. Following the Watson–Crick base pairing rules, LNA forms duplexes with complementary DNA and RNA with remarkably increased thermal stabilities and generally improved selectivities.

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2′‐O, 4′‐C‐methylene bridge. We have investigated a modified 9‐mer single stranded oligonucleotide as well as three 9‐ and 10‐mer modified oligonucleotides hybridized to unmodified DNA. The single‐stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson–Crick base‐pairing with all the nucleotides in anti‐conformation. By use of selective DQF‐COSY spectra we determined the ratio between the N‐type (C3′‐endo) and S‐type (C2′‐endo) sugar conformations of the nucleotides. In contrast to the corresponding single‐stranded DNA (ssDNA), we found that the sugar conformations of the single‐stranded LNA oligonucleotide (ssLNA) cannot be described by a major S‐type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N‐type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N‐type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S‐type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N‐type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases. Copyright

Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing [2.2.1]bicyclo nucleoside monomers. A novel and significantly improved method for convergent synthesis of LNA [2.2.1]bicyclo nucleosides using a 4-C-tosyloxymethyl-1, 2-di-O-acetyl furanose as a key synthon is described. In addition, an alternative, robust linear approach allowing selective formation of the desired [2.2.1]bicyclo LNA nucleosides via a tricyclic nucleoside intermediate is introduced.

Lna (Locked Nucleic Acid)

Abstract LNA (Locked Nucleic Acid) forms duplexes with complementary DNA, RNA or LNA with unprecedented thermal affinities. CD spectra show that duplexes involving fully modified LNA (especially LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMR examination of an LNA:DNA duplex confirm the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA is demonstrated suggesting strand invasion by LNA. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has been accomplished.

Evaluation of Oligonucleotides Containing Two Novel 2′-O-Methyl Modified Nucleotide Monomers: A 3′-C-Allyl and a 2′-O-3′-C-Linked Bicyclic Derivative

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.