Effects of Modifications on Nucleic Acid Properties

Modifications of nucleic acids can alter their physical properties, stability, interaction potential, and biological function. Naturally modified nucleic acids regulate vital cellular processes, including gene expression, DNA repair, and protein synthesis. Artificial modifications have become essential tools in biotechnology, drug development, and therapeutic strategies like gene editing and RNA-based therapies in recent decades.

Many of these modifications occur naturally. Presently there are 143 known modified ribonucleosides. Examples are methylation and acetylation. The artificial introduction of nucleic acid modifications can stabilize oligonucleotides and make them more resistant to nucleases.

Understanding the effect of modifications on nucleic acid properties is essential for insights into genetic regulation, biotechnology, and therapeutic applications.

Structural Changes

Nucleic acid modifications often alter the three-dimensional structure of DNA or RNA, affecting how these molecules fold and interact.

Examples are:

Methylation: Added methyl groups, such as 5-methylcytosine in DNA, can change the flexibility and conformation of the DNA helix. Methylation makes DNA less accessible to transcription factors, which silences gene expression.

Phosphorothioate Modification: A phosphorothioate is a common non-natural modification in which a sulfur atom replaces a non-bridging oxygen atom in the phosphate backbone. This modification enhances the resistance of nucleic acids against nucleases, making it useful in therapeutic oligonucleotides like antisense molecules.

Base Modifications: Modified RNA bases such as pseudouridine (Ψ) can improve base-pairing stability, influence RNA secondary structures, and enhance protein interactions.

Changes in Stability

Nucleic acid modifications can either enhance or reduce the stability of the modified molecules, affecting their lifespan and functionality:

Methylation of DNA (for example, in CpG islands): Methylation can protect DNA from degradation. Methylation is important in epigenetic regulation. Methylation at CpG sites can prevent recognition by specific proteins, such as transcription factors, while promoting binding by others, such as methyl-CpG-binding proteins.

RNA Modifications: Various modifications are available for RNA molecules, such as adding a 2'-O-methyl group, which increases their resistance to degradation by exonucleases and stabilizes the RNA structure. Modified mRNAs, like those used in mRNA vaccines, often have pseudouridine instead of uridine to increase stability and decrease immune recognition.

Modifications Affecting Base Pairing and Hybridization

Nucleic acid modifications may influence the hydrogen bonding patterns and base-pairing properties of oligonucleotides:

Base Methylation: Methylation of bases often alters Watson-Crick hydrogen bonding, potentially interfering with base pairing and affecting replication and transcription fidelity.

Modified Bases in tRNA: In transfer RNA (tRNA), modifications like inosine or queuosine at certain positions enhance the flexibility of base pairing, which is crucial for the accurate and efficient translation of the genetic code.

Bridged or Locked Nucleic Acids (BNAs, LNAs): Modifications like BNA and LNA constrain the ribose ring via a methylene-based bridge, significantly increasing the melting temperature of DNA/RNA duplexes and improving the affinity and stability of hybridization.

Impact on Gene Expression and Regulation

Epigenetic modifications like DNA methylation and histone modifications can regulate gene expression:

Gene Silencing: DNA methylation of promoter regions, particularly in CpG islands, often leads to the repression of gene transcription. DNA methylation is a significant mechanism in gene regulation and cellular differentiation.

Histone Modifications: While not directly modifying DNA, histone modifications, usually acetylation or methylation, alter the accessibility of DNA by loosening or tightening the DNA-histone interaction, thereby modulating the transcriptional activity of nearby genes.

Effects on Enzymatic Processes

Enzymes involved in nucleic acid metabolism, such as polymerases, endonucleases, and ligases, can be sensitive to modifications:

DNA Replication and Repair: DNA modifications such as 5-methylcytosine can interfere with recognizing and repairing DNA mismatches, impacting mutation rates.

RNA Splicing and Translation: Modifications in pre-mRNA or mRNA, for example, an added 5’-cap and poly-A tail, are needed for proper splicing, transport, and translation. Additionally, mRNA modifications like N6-methyladenosine (m6A) affect splicing efficiency and translation rates, influencing gene expression.

Therapeutic and Biotechnological Applications

Chemical modifications of nucleic acids enable drug development in biotechnology and therapeutic applications:

Antisense Oligonucleotides and siRNA: Phosphorothioate backbones or 2'-O-methyl modifications increase resistance to nuclease degradation in therapeutic oligonucleotides.

CRISPR/Cas9: Modifications added to guide RNAs (gRNAs) increase their stability and efficiency in directing Cas9 to specific genomic locations.

Immune Response Modulation

Nucleic acid modifications are also critical in immune system recognition:

Avoidance of Immune Detection: Modified RNA, such as mRNA with pseudouridine, reduces innate immune activation, essential in therapeutic applications like mRNA vaccines to prevent rapid degradation and reduce inflammatory responses.

Immunostimulatory Effects: Certain modifications, like CpG oligodeoxynucleotides, are known to stimulate the immune system and are being explored as adjuvants in cancer immunotherapy.

Modifications for siRNAs, ASOs, AMOs, and gapmers. 

• Inserting mismatches into oligonucleotides will decrease a duplex's melting temperature (Tm) and prevent hybridization or polymerization.

• A higher Tm value correlates with improved binding affinity and results in a more robust duplex. More energy is required to destabilize the connection between both molecules.

• The sugar ring and the backbone are the targets for most modifications.
• The C2′ position is the selected site for modifications. The C2′ position defines the conformation of the sugar ring. Many introduced changes at this position shift the conformation of the sugar moiety from a C2′-endo (southern conformation, typical of DNA duplexes) to a C3′-endo sugar pucker (northern conformation, typical of RNA duplexes), improving the binding affinity of ASOs and AMOs for RNA complements.

• Also, in this conformation, the 2′-modification is closer to the 3′-phosphate group, conferring higher nuclease resistance to the oligonucleotide.

• Modification at the 2′-carbon of the ribose, for example, 2′-OMe, 2′-MOE and 2′-F, increase binding affinity in the following order of increased potency: 2′-OMe ≅ 2′-MOE < 2′-F).

• These substitutions can be combined to improve potency. For example, MOE/LNA, 2′-OMe/LNA, or 2′-F/MOE are examples of oligonucleotide mixmers with enhanced binding affinity compared to oligonucleotides containing only one type of substitution.

Table 1: Effects of Modifications on Melting Temperature

Reference

BNAs

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LNAs

Lennox K a, Behlke M a. A direct comparison of anti-microRNA oligonucleotide potency. Pharm Res. 2010;27:1788–99. [PubMed]

Lennox K, R Owczarzy, Thomas DM, et al.. Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide Modifier. Mol Ther Nucleic Acids. 2013;2:e117. [PMC] [PubMed]

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