Dale C. Guenther

Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA’s oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.

C5‐Functionalized LNA: Unparalleled Hybridization Properties and Enzymatic Stability

Antisense oligonucleotides (ONs) are widely explored as fundamental research tools and therapeutic agents against diseases of genetic origin due to their ability to modulate gene expression by interfering with target RNA. Introduction of chemically modified nucleotides into antisense ONs is crucial to increase binding affinity toward RNA targets, improve discrimination of mismatched RNA to avoid off-target effects, and enhance stability against nucleases to slow down degradation. The use of conformationally restricted nucleotides and locked nucleic acids (LNAs, Scheme 1) in particular, has to some extent addressed these challenges. Antisense LNAs are accordingly evaluated in several clinical trials. Substantial efforts have been invested to develop LNA analogues with even more desirable biophysical properties and reduced hepatotoxicity. These studies have primarily focused on modification of the oxymethylene bridge spanning the C2’and C4’-positions and/or introduction of minor-groove-oriented substituents into the bridge. Improved enzymatic stability, e, f, j] altered biodistribution, or reduced hepatotoxicity has been reported for some of the analogues, but improvements in hybridization properties relative to LNA were generally not observed. Results from comparative in vivo antisense studies must be awaited to assess if the significantly increased synthetic complexity of these conformationally restricted nucleotides is justified. C5-functionalized pyrimidine DNA building blocks have attracted considerable attention due to their ability to accommodate functional entities in the major groove of nucleic acid duplexes and straightforward synthesis. Small C5-entities are generally well tolerated in duplexes and result in small increases in thermal affinity toward DNA/RNA complements. f] In light of this, we hypothesized that C5-alkynyl-functionalized LNA monomers would synergistically integrate beneficial Scheme 1. Synthetic outline of phosphoramidites 5 W–5 Z. CAN = ceric ammonium nitrate, DMTr = 4,4’-dimethoxytrityl, TBAF = tetrabutylammonium fluoride, PCl = 2-cyanoethyl N, N’diisopropylchlorophosphoramidite.

Invaders: Recognition of Double‐Stranded DNA by Using Duplexes Modified with Interstrand Zippers of 2′‐O‐(Pyren‐1‐yl)methyl‐ribonucleotides

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.

Sandwich assay for mixed-sequence recognition of double-stranded DNA: Invader-based detection of targets specific to foodborne pathogens

A 96-well plate sandwich assay based on Invader capture/signalling probes is used to recognize 28-mer mixed-sequence dsDNA targets specific to Salmonella enterica, Campylobacter jejuni, Escherichia coli. Targets are detected down to 20-55 pM concentration with excellent binding specificity.

C5-Alkynyl-Functionalized α-L-LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.