Among many gene silencing methods, RNase H-mediated degradation stands out for its use of antisense oligonucleotides (ASOs), which confers its high specificity and long-lasting effects without permanently altering the genome. Therefore, it has broad applicability in drug discovery with its potential to target and eliminate the RNA sequences of disease-causing genes.

Here, we explore the importance of RNase H–mediated RNA cleavage and the role of ASOs in establishing targeted RNA degradation for several gene-silencing applications.

What is RNase H, and What is its Role in RNA Cleavage?

Functionality of RNase H in RNA Processing

RNase is an endonuclease that degrades the RNA strand of RNA-DNA hybrids, which could otherwise disrupt genomic stability when accumulated. For instance, DNA replication requires short RNA primers to start, but these primers must be degraded to complete the process.¹ Furthermore, RNA-DNA hybrids called R-loops may also form during transcription and potentially cause DNA damage.²

Types of RNase H and Their Specificity

There are two main types of RNase H. RNase H1 is localized to mitochondria and the nucleus and is responsible for removing R-loops and RNA primers. It requires at least four consecutive ribonucleotides to recognize a strand for cleavage.³ However, it does not recognize single ribonucleotides embedded in DNA. In contrast, RNase H2 is found dominantly in the nucleus and can identify and remove ribonucleotides incorrectly integrated into double-stranded DNA (dsDNA). ⁴

Impact of RNase H on RNA Levels

Due to its multifaceted role in degrading abnormal RNA strands, RNase H is a critical regulator of RNA levels. RNase H ensures uninterrupted transcription and mitigates DNA damage by preventing the accumulation of RNA-DNA hybrids.

The mechanism has now been leveraged using synthetic DNA molecules called Antisense oligonucleotides (ASOs) to selectively degrade disease-causing RNA strands. These oligonucleotides, usually 15-22 nucleotides long, are designed to bind complementary RNA targets to trigger RNase H activity.

How Does RNase H-Mediated Degradation Occur?

Pathway of RNase H-Mediated RNA Degradation

RNase H-mediated RNA Degradation is invoked by the annealing of an RNA strand to its complementary DNA. RNase H1 or H2 recognizes the RNA-DNA hybrid based on its length and location in the cell, catalyzing the cleavage of the RNA strand in a sequence-independent manner. During RNase H activity, the phosphodiester bonds of the RNA strand are hydrolyzed, resulting in shorter RNA fragments lacking 5'-cap and poly-A tails. Thus, exonucleases, such as FEN1, can further degrade these fragments.⁵ Furthermore, RNA degradation during replication is often followed by DNA synthesis to replace the degraded RNA.

Factors Influencing RNase H-Mediated Cleavage

Although RNase H activity does not depend on a specific target sequence, there are other factors influencing the process:

1. Hybrid structure: DNA-RNA hybrid length, stability, and mismatches within the DNA-RNA pairing may interfere with cleavage.
2. Enzyme concentration: A higher RNase H concentration means improved cleavage efficiency, but excessive amounts can cause unintended degradation.
3. Sequence content: A hybrid rich in guanine-cytosine (GC) pairings is more stable and may slow cleavage.
4. Regulatory proteins and positively charged ions (Mg²⁺ or Mn²⁺) promote RNase activity.

Comparison of RNase H-Mediated and Other RNA Degradation Pathways

There are several mechanisms of RNA degradation besides RNase-H mediation, including:

1. Exoribonuclease-mediated RNA Decay, which targets a single-stranded RNA (ssRNA) and degrades it directionally
2. Endoribonuclease-mediated RNA Decay, which involves sequence-specific cleavage of ssRNA at internal sites
3. RNA Interference (RNAi)-Mediated Degradation, where an RNA-induced silencing complex (RISC) containing a complementary small interfering RNA (siRNA) or micro-RNA (miRNA) binds and cleaves mRNA
4. Nonsense-mediated mRNA Decay (NMD), aimed at degrading mRNAs with nonsense mutations with premature stop codons

While different degradation mechanisms are tailored for various aspects of gene regulation and mRNA quality control, the requirement for a DNA pair (in a hybrid form) for progression makes RNase H-mediated degradation unique.

How Do Antisense Oligonucleotides Function in RNA Degradation?

ASOs can utilize RNase H-mediated degradation to degrade disease-causing mRNA selectively. The binding between the target mRNA and its complementary ASO DNA recruits RNase H1 to facilitate RNA cleavage. Exonucleases subsequently remove the resulting RNA fragments. Thus, ASOs assist in silencing the target gene by preventing its translation.

Typically containing 15-22 nucleotides, ASOs are designed and chemically optimized to augment their targeting and cleavage efficiency. Such ASOs, called gapmers, consist of a central region with 8-10 central deoxynucleotides, flanked on both sides by 2-5 ribonucleotides. The nucleotides in the flanked region can be modified with 2′-O- methoxyethyl (2′-OMe) or locked nucleic acids (LNAs)) to improve the stability of the RNA-DNA hybrid.⁶ Furthermore, the linkages between the ASO nucleotides can be tweaked to prevent premature degradation.⁷

What Are the Challenges in Utilizing Antisense Oligonucleotides for RNA Degradation?

Delivery Methods for Antisense Oligonucleotides

Sufficient cellular uptake is one of the key challenges of ASOs. Many drug delivery methods were employed to improve the ability to cross biological barriers to reach highly protected tissues.

Firstly, chemical modifications to the ASO backbone and ribose sugars can improve nuclease resistance and ASO half-life while promoting RNase H recognition.⁸

Bioconjugation can enhance the targeted delivery potential of ASOs by utilizing peptides, oligosaccharides, and antibodies that have binding affinities for the surface proteins on specific cells. One example is cancer immunotherapy, where conjugation to DNA aptamers that are antagonists of the immune checkpoint inhibitors (PD-L1) directs the ASOs to tumor cells enriched in PD-L1. Thus, ASOs were made more effective in reducing PD-L1 levels and blocking immune escape pathways.⁹

Nanoparticles, such as liposomes, are gaining interest as delivery vehicles due to their protection of ASO from endogenous nucleases and their biocompatibility with biological barriers, making the penetration of ASOs easier. Nanoparticles can be modified by further conjugation to improve stability and prevent immunogenicity. They can also be made stimuli-sensitive (e.g., sensitive to temperature and pH changes in cells) to release the ASO load for maximum cellular uptake at targeted cells.¹⁰

Despite the number of emerging solutions, ASO delivery may still suffer from limited tissue specificity, potential immunogenicity, and complications in formulation and manufacturing.

Off-Target Effects and Their Implications

Unwanted degradation is another setback for ASO-based RNA degradation. ASOs, including chemically modified versions with high affinities, may sometimes bind partially complementary sites. To add to this risk, RNase H may tolerate mismatches in RNA-DNA hybrids. As a result, ASO may facilitate the degradation of an unintended RNA with severe implications for adverse effects, such as liver toxicity.¹¹

Applications in Research and Medicine

Despite challenges with delivery and targeting, ASO for RNase H-mediated degradation remains one of the promising methods for gene silencing on a post-transcriptional level.

A series of ASO-based drugs have been FDA-approved for the treatment of neurodegenerative and genetic disorders. One of the recent approvals was for Tofersen, which works by binding the mutated superoxide dismutase 1 (SOD1) responsible for the degeneration of motor neurons in Amyotrophic Lateral Sclerosis (ALS).¹² More trials are underway to treat cancer and neurological disorders. By extending drug discovery to genes that are difficult to target with small-molecule inhibitors, ASOs can help overcome drug resistance while complementing and strengthening existing therapeutic strategies.

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