5. CONCEPT OF ANTISENSE MOLECULES

Concept of Antisense Molecules

Antisense technology is a modern approach used to block the production of specific proteins by interfering with the natural process of gene expression. It works by using short, synthetic DNA or RNA sequences called antisense oligonucleotides (ASOs), which bind to messenger RNA (mRNA) and prevent it from making proteins.

In simple words, antisense molecules are “gene silencers” that switch off the production of harmful or unwanted proteins.

Basic Understanding of Sense and Antisense Strands

  • During transcription, the DNA double helix separates into two strands.
  • The sense strand (coding or + strand) has the same sequence as mRNA.
  • The antisense strand (template or – strand) is used by the cell to produce mRNA.
  • Sometimes, the sense strand also produces RNA, and this RNA is called antisense RNA.

What Are Antisense Molecules?

Antisense molecules are short, single-stranded DNA or RNA sequences designed to be complementary to a particular mRNA sequence. When they bind to that mRNA, they form a DNA/RNA hybrid that blocks protein synthesis.

These molecules are scientifically prepared as oligo-deoxy-nucleotides or chemically modified nucleic acid analogs.

How Antisense Technology Works

Antisense oligonucleotides prevent protein formation by:

  • Binding to target mRNA, preventing it from reaching the ribosome.
  • Blocking translation by forming a stable duplex with mRNA.
  • Allowing the enzyme RNase H to degrade the mRNA–DNA hybrid.

As a result, the instructions for protein synthesis are interrupted, and the protein cannot be formed.

Antisense Oligonucleotides (ASOs)

These are small DNA/RNA fragments, usually 15–20 nucleotides long, complementary to specific mRNA sequences. They can be natural or chemically modified to improve stability and activity.

Discovery

The antisense effect was first shown by Zamecnik and Stephenson in the 1970s using viral RNA.

Classes of Antisense Oligonucleotides

1. RNase H–Dependent ASOs

These oligonucleotides recruit the enzyme RNase H, which cuts the RNA in a DNA/RNA hybrid. This destroys the mRNA and stops protein production.

2. Steric-Blocker ASOs

These molecules block translation physically, without degrading mRNA. They prevent ribosomes or splicing machinery from binding to mRNA.

Role of RNase H

RNase H is a cellular enzyme that recognizes DNA/RNA hybrids and cleaves the RNA part. It catalyzes the hydrolysis of the 3’-O–P bond of RNA. This is a crucial mechanism behind antisense-mediated gene silencing.

Characteristics of an Ideal Antisense Oligonucleotide

  • High specificity for the target mRNA.
  • Efficient cellular uptake.
  • Minimal binding to non-target sequences.
  • Good nuclease stability.
  • Non-toxic and safe.

Generations of Antisense Oligonucleotides

1. First-Generation ASOs

These include phosphorothioate oligonucleotides, where one non-bridging oxygen of the phosphate group is replaced with sulfur. This improves stability but reduces affinity and may cause toxicity. These can activate RNase H.

2. Second-Generation ASOs

Modified at the 2′-position of ribose, such as:

  • 2’-O-methyl
  • 2’-O-methoxyethyl

These modifications improve stability and binding with lower toxicity.

3. Third-Generation ASOs

This group includes highly stable and selective nucleic acid analogs such as:

  • PNA (Peptide Nucleic Acid)
  • LNA (Locked Nucleic Acid)
  • Morpholino oligonucleotides

They provide excellent resistance to nucleases and strong hybridization with mRNA.

Mechanisms of Blocking Protein Synthesis

1. RNase H Activation

DNA/RNA hybrid formation triggers RNase H to degrade the RNA, stopping protein translation.

2. Translation Blocking

Binding of antisense RNA to mRNA prevents ribosomes from initiating translation. The duplex may also be rapidly degraded by ribonucleases.

Applications of Antisense Technology

  • Studying gene function (functional genomics)
  • Developing targeted therapies for human diseases
  • Use in plants and animals for genetic studies
  • Treatment of viral infections and cancers

Detailed Notes:

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