Therapeutic tiny or short modified antisense oligonucleotides (Tiny ASOs) can block gene expression or modulate splice-switching!

Tiny antisense oligonucleotides and short, synthetic, antisense oligonucleotides, when forming base pairs with regulatory RNA, disrupt the normal RNA–RNA base-pairing or protein–RNA binding interactions. These interactions occur during transcription or post-transcription events.

For example, synthetic, modified nucleic acids, known as splice-switching oligonucleotides, can base pair with a pre-mRNA and disrupt the regular splicing repertoire of the transcript by blocking RNA–RNA base pairing or protein–RNA binding. Splice-switching oligonucleotides (SSOs) can block interactions between components of the splicing machinery and pre-mRNAs.

Tiny RNAs occur naturally in Caenorhabditis elegans, where they have regulatory functions in developmental timing. Lau et al. (2001) reported that the noncoding RNAs lin-4 and let-7 control developmental timing in C. elegans.

Tiny antisense oligonucleotides (Tiny ASOs) are short, modified synthetic strands of DNA or RNA designed to bind to and block the expression of specific genes. TAOs are typically 8 to 25 nucleotides in length and delivered to cells using various methods, including lipid nanoparticles, polymers, and viruses.

Once inside a cell, Tiny ASOs bind to their target mRNA molecule and prevent it from being translated into a protein

ASOs can be conjugated to N-acetylgalactosamine (GalNAc) units for enhanced delivery into cells and increased potency (Yamamoto et al. (2016, 2019,  2021). 

Figure 1: Structure of tandemly-conjugated monovalent GalNAc units (Yamamoto et al.)

Several approaches are possible, including: 

  • Steric blocking: Tiny ASOs can physically block the ribosome from binding to the mRNA molecule, preventing translation.

  • RNAse H recruitment: Tiny ASOs can recruit an enzyme called RNAse H, which cleaves the mRNA molecule, destroying it.

  • MicroRNA mimicry: Well-designed Tiny ASOs can mimic the structure of microRNAs, which are small RNA molecules that naturally regulate gene expression. Tiny ASO-microRNA mimics can bind to the mRNA molecule and target it for degradation. 

Tiny ASOs are promising new therapeutics for various diseases, including cancer, genetic disorders, and infectious diseases. However, therapeutic Tiny ASOs are still in the early stages of development, but clinical trials have shown encouraging results.

Medicinal scientists investigate Tiny ASOs in the treatment of several diseases:

  • Cancer: Tiny ASOs can target various cancer genes, including oncogenes, which promote cancer growth, and tumor suppressor genes, which inhibit cancer growth. For example, TAOs targeting the KRAS oncogene are promising oligonucleotide based therapeutics in the treatment of lung cancer and pancreatic cancer.

  • Genetic disorders: Tiny ASOs may allow the treatment of genetic disorders. Examples are Duchenne muscular dystrophy and Huntington's disease. For example, TAOs targeting the DMD gene have shown promise in treating Duchenne muscular dystrophy.

  • Infectious diseases: Tiny ASOs may also allow treatment of contagious diseases such as HIV/AIDS and hepatitis C virus infection. For example, Tiny ASOs targeting the HIV-1 genome.

Arzumanov et al. (2003) targeted the HIV-1 trans-activation responsive element (TAR) RNA stem-loop interaction with the HIV trans-activator protein Tat by inhibiting the trans-activation by steric blockage using 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. The research group showed that OMe/LNA mixmers are steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

Kauppinen et al. (2005) reported the use of LNA-antisense, LNA-modified siRNA (siLNA), and the detection and analysis of microRNAs by LNA-modified oligonucleotide probes. LNA-antisense oligonucleotides enable gene silencing and targeting of non-coding RNAs. Also, LNA probes allow the targeting of non-coding microRNAs.

Obad et al. (2011) utilized fully LNA-modified phosphorothioate oligonucleotides, tiny LNAs, complementary to the miRNA seed regions to inhibit single miRNAs and entire miRNA families in cultured cells and in several tissues of adult mice and in a mouse breast tumor model in vivo.

Mallory and Hastings (2016) reviewed splice-switching antisense oligonucleotides that are active in vivo. The review listed several examples of advanced SSOs and their targets that have shown promise in treating disease/pathological conditions in vivo.

To enhance delivery into cells and increase the potency of ASOs, Yamamoto et al. (2016, 2021) developed a series of N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotides. The research group conjugated the GalNAc ligand to the 5’-end of the antisense oligonucleotides and demonstrated the effect of GalNAc conjugation on anti-miRNA ASOs, specifically on tiny LNAs. The study observed an in vivo potency of ~300 to 500 fold larger than expected from previous studies of GalNAc-conjugated gamer-type ASOs. The 2021 study confirmed that GalNAc conjugation of tiny LNAs can improve poor pharmacokinetic properties of natural ASOs.

In summary, TAOs are a promising new class of therapeutics that can potentially treat many diseases. However, more research is needed to fully understand their safety and efficacy.

Clinical trials:

(1) Phase 1 Study of EZN-2968. EZN-2968, a locked nucleic acid antisense oligonucleotide against hypoxia-inducible factor 1α [NCT00466583]

(2) SPC2996 in Chronic Lymphocytic Leukemia. 
LNA Antisense Molecule Against Bcl-2, in Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia. [NCT00285103]


Arzumanov A, Stetsenko DA, Malakhov AD, Reichelt S, Sørensen MD, Babu BR, Wengel J, Gait MJ. A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues. Oligonucleotides. 2003;13(6):435-53. [PubMed]

Katoch, Y.M. "Immunomodulators in the Treatment of HIV/AIDS and Mycobacterial Diseases." Journal of Immunology and Immunopathology, 2002, 4, 1&2, 15-19. [

Kauppinen S, Vester B, Wengel J. Locked nucleic acid (LNA): High affinity targeting of RNA for diagnostics and therapeutics. Drug Discov Today Technol. 2005 Autumn;2(3):287-90. [

Hayakawa, Kazushige. "[Radiation Therapy in the Treatment of Lung Cancer]." 
Nihon Igaku Hoshasen Gakkai Zasshi. 2003 Nov;63(9):533-8. [PubMed]

Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science. 2001 Oct 26;294(5543):858-62. [

Mallory A. Havens, Michelle L. Hastings, Splice-switching antisense oligonucleotides as therapeutic drugs, Nucleic Acids Research, Volume 44, Issue 14, 19 August 2016, Pages 6549–6563. [

Obad S, dos Santos CO, Petri A, Heidenblad M, Broom O, Ruse C, Fu C, Lindow M, Stenvang J, Straarup EM, Hansen HF, Koch T, Pappin D, Hannon GJ, Kauppinen S. Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet. 2011 Mar 20;43(4):371-8. [PMC]

Yamamoto T, Sawamura M, Wada F, Harada-Shiba M, Obika S. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides. Bioorg Med Chem. 2016 Jan 1;24(1):26-32. [
Yamamoto T, Terada C, Kashiwada K, Yamayoshi A, Harada-Shiba M, Obika S. Synthesis of Monovalent N-Acetylgalactosamine Phosphoramidite for Liver-Targeting Oligonucleotides. Curr Protoc Nucleic Acid Chem. 2019 Sep;78(1):e99. [Current Protocols]

Yamamoto T, Mukai Y, Wada F, Terada C, Kayaba Y, Oh K, Yamayoshi A, Obika S, Harada-Shiba M. Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides. Pharmaceutics. 2021 May 31;13(6):817 [PMC]

Web links:

Dave Bartel’s lab: http://bartellab.wi.mit.edu/

Chris Burge’s lab: http://genes.mit.edu/burgelab

splice-switching oligonucleotides

Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cell-penetrating peptides, cholesterol, tocopherol, other peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs, miRNA, or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

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