Oligonucleotides
The development of solid-phase synthesis methods in the 1970s allowed the artificial generation of oligonucleotides of up to ~100 nucleotides in length. Moreover, this technology enabled the incorporation of modified bases into the growing chain composed of nucleotides. The chemical synthesis and modification of oligonucleotides opened the way for new research applications and novel therapeutic strategies.
Classification of Oligonucleotides
Various classes of oligonucleotides have been developed in the meantime: antisense oligonucleotides (ASOs), ribozymes and small interfering RNAs (siRNAs) specifically inhibit gene expression by Watson-Crick base pairing to a complementary messenger RNA (mRNA), but in contrast, oligonucleotide aptamers bind to their target by structural recognition. ASOs, being 15 to 20 nucleotides in length, can be employed to inhibit gene expression specifically. Oligonucleotide aptamers have defined three-dimensional structures that interact with their biological targets (typically proteins). Immunomodulatory oligonucleotides interact with proteins of the immune system via discrete motifs (such as CpG oligonucleotides). mRNA therapeutics aims to deliver a chemically modified RNA to cells where it can be translated to a functional protein.
Figure 1. Oligonucleotide-based therapeutics.
Mechanism of Oligonucleotides
Upon binding to mRNA, therapeutic oligonucleotides can elicit a biological response via a number of distinct antisense mechanisms. These can be broadly classified as mechanisms that promote degradation of the targeted mRNA or which are occupancy based and do not promote the degradation of RNA. Single-stranded DNA oligonucleotides activate the RNase H antisense mechanism. RNase H1 is a ubiquitously expressed enzyme that selectively cleaves the RNA strand of an RNA/DNA heteroduplex. Double- or single-stranded RNA oligonucleotides function via the RISC pathway where the enzyme Argonaute cleaves the mRNA strand of the RNA/RNA duplex. In contrast, oligonucleotides of various chemical compositions can bind mRNA at specific locations such as close to exon-intron junctions or translation initiation sites and modulate RNA splicing or arrest translation without causing RNA degradation. In addition, oligonucleotides can also bind to and antagonize endogenous microRNAs which are important regulators of gene expression. Indeed, oligonucleotides that function via all of the above antisense mechanisms have progressed in the clinic for the treatment of a wide variety of disease indications.
Figure 2. The mechanism of RNase H1-activating ASOs activity.
Despite the enormous potential of the antisense approach, it took almost three decades for the concept to be firmly established in the clinic. Key reasons that led to success included the identification of tissues which accumulate ASOs and are sensitive to antisense effects, a more sophisticated understanding of the pharmacokinetic/pharmacodynamics properties of ASOs in animal models, a steady progress in our understanding of the fundamental biology of antisense mechanisms, and advancement of oligonucleotide medicinal chemistry and production strategies that allowed for a large number of ASOs with improved drug-like properties to be synthesized and screened for biological activity.
Reference
- Crooke, S. T. (2017). Molecular mechanisms of antisense oligonucleotides. Nucleic acid therapeutics. 27(2): 70-77.
