Miriam Frieden

On the in vitro and in vivo Properties of Four Locked Nucleic Acid Nucleotides Incorporated into an Anti‐H‐Ras Antisense Oligonucleotide

Locked nucleic acid (β‐D‐LNA) monomers are conformationally restricted nucleotides bearing a methylene 2′‐O, 4′‐C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, β‐D‐amino LNA (amino‐LNA), β‐D‐thio LNA (thio‐LNA), β‐D‐LNA (LNA), and its stereoisomer α‐L‐LNA in an antisense oligonucleotide (ODN). A well‐known antisense ODN design against H‐Ras was modified at the 5′‐ and 3′‐ends with the different LNA analogues (LNA‐DNA‐LNA gapmer design). The resulting gapmers were tested in cancer‐cell cultures and in a nude‐mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H‐Ras ODNs were very efficient in H‐Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti‐H‐Ras ODN containing α‐L‐LNA had clearly the highest efficacy in H‐Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino‐LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both α‐L‐LNA and LNA gapmer ODNs had a high efficacy of tumor‐growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor‐growth inhibition could be observed at dosages as low as 0.5 mgu2009kg−1 per day. These results indicate that α‐L‐LNA is a very promising member of the family of LNA analogues in antisense applications.

Nuclease Stability of LNA Oligonucleotides and LNA-DNA Chimeras

Abstract The stabilizing properties of LNA and α-l-LNA oligonucleotides against endo- and 3′-exonucleases have been evaluated.

Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFOs is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.

Locked Nucleic Acid Holds Promise in the Treatment of Cancer

Providing novel treatments to help cancer patients live longer and have better lives remains one of the biggest challenges of the pharmaceutical industry. Today much is known about the molecular and genetic causes of cancers thus facilitating the development of novel targeted cancer drugs with improved risk-benefit ratios compared to contemporary broadly-acting, cytotoxic cancer drugs. Antisense therapy, e.g. the use of single stranded oligonucleotides as therapeutic modalities, provides the means to develop such targeted drugs, and in recent years this concept has enjoyed a major renaissance. Locked Nucleic Acid (LNA) is a novel, third generation RNA analogue that displays most if not all of the characteristics required to make potent and safe antisense drugs. Here we review the key properties of LNA oligonucleotides in the context of their use as safe and effective antisense drugs and provide a status on their therapeutic development in the field of cancer.

Self-association of short DNA loops through minor groove C:G:G:C tetrads

In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson–Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d, utilizing 2D NMR spectroscopy and restrained molecular dynamics. These three molecules self-associate forming symmetric dimers stabilized by a novel kind of minor groove C:G:G:C tetrad, in which the pattern of hydrogen bonds differs from previously reported ones. We hypothesize that these quadruplex structures can be formed by many different DNA sequences, but its observation in linear oligonucleotides is usually hampered by competing Watson–Crick duplexes.

Parallel-Stranded Hairpins Containing 8-Aminopurines. Novel Efficient Probes for Triple-Helix Formation

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure.

Convenient Synthesis of 8-Amino-2′-deoxyadenosine

Abstract We studied the behaviour of 8-azido-2′-deoxyadenosine and 8-bromo-2′-deoxyadenosine in aqueous solutions of ammonia and primary and secondary amines. Unexpectedly, 8-Azido-2′-deoxyadenosine is converted to 8-amino-2′-deoxyadenosine in excellent yields. The use of this reaction for the preparation of 8-aminoadenine derivatives needed for the preparation of oligonucleotides carrying 8-aminoadenine is discussed.

Solid-Phase Synthesis of Circular Oligonucleotides

A protocol for the straightforward preparation of small circular oligodeoxyribonucleotides (2-28 nt) is reported. The assembly of the oligonucleotide chain (standard phosphoramidite chemistry) and cyclization by the phosphotriester method take place on a tailor-made nucleotide-derivatized solid support. Although cyclization yields are moderate, the procedure exploits a synthesis design that allows selective cleavage of the circular oligonucleotide from the support, which facilitates isolation of the target molecule by simple filtration.

Properties of Triple Helices Formed by Oligonucleotides Containing 8-Aminopurines

Abstract The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.

Chemistry of Locked Nucleic Acids (LNA)

Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucloetides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids.