University of Idaho

Background: Our interest in nucleic acids is linked to their pivotal role as carriers of genetic information in all Kingdoms of Life. Thus, the genetic information of a eukaryotic cell is stored in the nucleus as double stranded DNA (dsDNA). The information is processed in a highly regulated, sequence-specific and catalytic manner to give multiple copies of a corresponding RNA transcript, which is processed and transported to the cytosol where it is used as a template for protein synthesis. Revolutionary advances in genome sequencing and nucleic acid biology over the past 15 years have led to a deeper understanding of the Central Dogma (DNA→RNA→protein). As a result, the biology of nucleic acids offers many avenues for exogenous modulation of gene expression, which can be exploited for applications in fundamental research, therapeutics and biotechnology. Development of chemically functionalized oligonucleoties that enable strong, specific and stable binding to nucleic acid targets of interest is at the center of these applications.

DNA-targeting. Double-stranded DNA (dsDNA) is a considerably more complex target than single stranded RNA (see below) since the Watson-Crick hydrogen-bonding faces of the nucleobases are buried in the duplex core. This leaves few molecular ‘handles’ for sequence-specific recognition of dsDNA. Molecules targeting dsDNA in biological contexts face the additional challenge of compacted and protein-associated chromosomal DNA. Development of strategies for site-specific targeting of dsDNA has the prospect of providing uniquely empowering tools for fundamental gene function studies, and identifying novel classes of therapeutic and diagnostic agents. In fact, dsDNA-targeting agents have already been used for site-specific modulation of gene expression and induction of site-specific mutagenesis, as well as direct detection of specific dsDNA target regions in living cells. Current state-of-the-art technologies (i.e., triplex forming oligonucleotides [TFOs]; peptide nucleic acids) are subject to limitations such as the need for long homopurine dsDNA target regions and non-physiological buffer conditions to ensure binding.

Development of optimized TFO building blocks (see publication 21) or radically novel methodologies for site-specific and sequence-unrestricted targeting of double-stranded DNA (i.e., the Invader approach) is a core focus in our laboratory (see publications 23; 6; 16; 14).

RNA-targeting oligonucleotides (e.g.,. antisense; siRNA; antagomir) are widely explored as fundamental research tools and increasingly evaluated as therapeutic agents against diseases of genetic origin. Introduction of chemically modified nucleotides into oligos is essential to increase their binding affinity toward RNA targets, improve discrimination of mismatched RNA to reduce off-target effects, and enhance stability against nucleases to slow down degradation. Among the many chemistries that have been evaluated in the past two decades, phosphorothioate DNA (PS-DNA), O2’-alkylated RNA and affinity- and specifity-enhancing Locked Nucleic Acid (LNA) have emerged as the most therapeutically interesting modifications. One PS-DNA based antisense drug has already been approved by the FDA for treatment of ocular CMV infections in immuno-compromised patients, while ~40 antisense drug candidates currently are evaluated in clinical trials.

We are developing new nucleotide building blocks such as nucleobase-modified LNA monomers that display promising hybridization and diagnostic properties (see publications 22; 29; 26; 30)