Custom Antisense Oligonucleotide Synthesis

2'-O-Methyl (2'-OMe)

2'-O-Methyl (2'-OMe) RNA bases are chemically modified nucleotides where a methyl group (-CH3) is added to the 2' hydroxyl group of the ribose sugar. This structural alteration serves two primary purposes: increasing the chemical stability of the oligonucleotide and reducing its immunogenicity when introduced into biological systems. By adding a bulky, hydrophobic methyl group, the oligonucleotide becomes more resistant to enzymatic degradation by ribonucleases, which would otherwise cleave unmodified RNA molecules. Furthermore, the 2'-OMe modification decreases the immune recognition of the oligonucleotide, which is especially important in therapeutic applications to avoid immune system activation. This modification is commonly found in antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and other RNA therapeutics, where it enhances the molecule's binding affinity to complementary RNA strands, improves pharmacokinetic properties, and prolongs the therapeutic effect. Its reduced immunogenicity also makes 2'-OMe RNA bases a preferred choice in treatments where minimizing immune response is critical for patient safety.

Phosphorothioate (PS)

Phosphorothioate is a backbone modification in which one of the non-bridging oxygen atoms in the phosphate group of an oligonucleotide is replaced with a sulfur atom. This seemingly small alteration has profound effects on the stability and function of the oligonucleotide. Phosphorothioate-modified oligonucleotides are highly resistant to degradation by nucleases, the enzymes that typically cleave unmodified oligonucleotides in biological systems. This enhanced stability makes phosphorothioate oligonucleotides ideal candidates for therapeutic applications, particularly in antisense and siRNA technologies, where sustained activity in vivo is necessary for effective gene silencing. Additionally, the sulfur substitution alters the oligonucleotide's overall charge and conformation, which can improve its interaction with proteins and other cellular components. However, the modification may also slightly reduce the binding affinity of the oligonucleotide for its target RNA or DNA sequence, so careful design is required to balance stability and efficacy in therapeutic applications. Despite this, phosphorothioate modifications remain a cornerstone in oligonucleotide therapeutics due to their excellent durability in physiological conditions.

2'-O-Methoxyethyl (MOE)

2'-O-Methoxyethyl (MOE) modifications involve the addition of a methoxyethyl group (-OCH2CH2OCH3) to the 2' position of the ribose sugar in adenosine, guanosine, or thymidine nucleotides. This bulky group significantly enhances the oligonucleotide's stability and resistance to nucleases, while also improving the affinity of the oligonucleotide for its complementary strand. MOE-modified nucleotides are often used in therapeutic oligonucleotides, such as antisense drugs and siRNAs, where durability in biological systems is essential for efficacy. MOE-modified oligonucleotides have prolonged half-lives in vivo, allowing for less frequent dosing in therapeutic settings. Additionally, the MOE modification improves the oligonucleotide's pharmacokinetics by enhancing its binding specificity and reducing the likelihood of off-target effects. This modification is particularly valuable in treating chronic diseases, where long-term stability and efficacy are required for sustained therapeutic outcomes.

N-acetyl-galactosamine (GalNAc)

N-acetyl-galactosamine (GalNAc) is a carbohydrate modification that plays a critical role in targeted drug delivery, particularly for therapeutic oligonucleotides designed to treat liver diseases. GalNAc conjugation allows oligonucleotides to bind with high affinity to the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes, the predominant cells in the liver. This receptor-mediated delivery system ensures that GalNAc-conjugated oligonucleotides are selectively taken up by liver cells, enabling precise targeting of therapeutic molecules. GalNAc modifications have revolutionized the field of RNA therapeutics by providing a highly efficient and selective method for delivering siRNAs, antisense oligonucleotides, and other RNA-based drugs to the liver. This targeted approach not only improves therapeutic efficacy but also reduces off-target effects and minimizes systemic toxicity. GalNAc conjugates are particularly valuable in the treatment of liver-associated diseases such as hypercholesterolemia, hepatitis, and liver fibrosis, where precise delivery to hepatocytes is essential for successful treatment outcomes.

2'-Fluoro (2'-F)

2'-Fluoro (2'-F) modifications involve substituting the hydroxyl group at the 2' position of the ribose sugar with a fluorine atom in adenosine, cytidine, guanosine, or uridine nucleotides. This substitution significantly enhances the stability of the oligonucleotide by increasing resistance to ribonucleases, which typically degrade unmodified RNA molecules. Fluorine is a small, highly electronegative atom that provides chemical stability without significantly altering the base pairing properties of the nucleotide, allowing the modified oligonucleotide to maintain high binding affinity to its target sequence. The increased stability of 2'-F-modified oligonucleotides makes them particularly useful in therapeutic applications, such as siRNAs, antisense oligonucleotides, and aptamers, where long-lasting activity is required. Additionally, 2'-F-modified oligonucleotides exhibit improved pharmacokinetic profiles, including enhanced serum stability and extended half-life, making them more effective in therapeutic settings. These modifications also reduce the immunogenicity of the oligonucleotide, minimizing the risk of immune activation and adverse reactions in patients.

Peptide Nucleic Acid (PNA)

Peptide Nucleic Acid (PNA) is a synthetic nucleic acid analog in which the standard sugar-phosphate backbone of DNA or RNA is replaced by a peptide-like backbone composed of N-(2-aminoethyl) glycine units. This unique structure makes PNAs neutral in charge, allowing for stronger hybridization with complementary DNA or RNA sequences due to the lack of electrostatic repulsion. PNAs exhibit high thermal stability and specificity in binding to complementary strands, making them excellent tools for molecular diagnostics, antisense therapies, and gene editing applications. PNAs are also highly resistant to enzymatic degradation, increasing their durability in biological systems. Due to their synthetic backbone, PNAs are less likely to trigger immune responses, making them suitable for therapeutic uses. PNA oligomers can bind to single-stranded DNA or RNA with high affinity, forming stable duplexes that can interfere with gene expression or inhibit protein synthesis, offering promising applications in gene regulation and targeted therapeutics.

Bridged Nucleic Acid (BNA)

Bridged Nucleic Acid (BNA) is a type of synthetic nucleotide analog in which the ribose sugar is chemically "bridged" between the 2' oxygen and the 4' carbon, locking the sugar in a constrained conformation. This structural modification enhances the thermal stability of oligonucleotides by increasing their binding affinity to complementary nucleic acid strands. BNA modifications, which are also referred to as Locked Nucleic Acids (LNA), improve the hybridization properties of oligonucleotides, making them more effective at targeting specific DNA or RNA sequences. BNAs are commonly used in antisense oligonucleotides, siRNAs, and molecular probes, where precise binding and resistance to nuclease degradation are critical. The bridged structure reduces the conformational flexibility of the nucleotides, resulting in tighter binding to complementary strands and greater resistance to degradation by enzymes. These properties make BNAs particularly useful in therapeutic applications where stability and specificity are paramount, such as in the development of gene silencing therapies and molecular diagnostics.

5-Methylcytidine

5-Methylcytidine is a modified cytosine nucleotide in which a methyl group is attached to the 5-carbon position of the cytosine ring. This modification plays a crucial role in epigenetic regulation, as it mimics the natural methylation of cytosine in DNA, which is commonly involved in gene silencing. The methylation of cytidine can influence gene expression by affecting how DNA interacts with transcriptional machinery or by altering chromatin structure. In synthetic oligonucleotides, 5-Methylcytidine enhances hybridization specificity and stability when forming duplexes with complementary strands. This modification increases the thermal stability of the nucleic acid duplex, making it more resistant to denaturation and degradation by nucleases. 5-Methylcytidine is often incorporated into antisense oligonucleotides, siRNAs, and other nucleic acid-based therapeutics to improve binding affinity to target sequences, increase biological stability, and better mimic the natural methylation patterns found in gene regulation studies.

Abasic RNA

Abasic RNA refers to RNA molecules in which a base (adenine, cytosine, guanine, or uracil) has been removed, leaving an "abasic site." These sites, also known as apurinic or apyrimidinic (AP) sites, are devoid of a nucleobase but still retain the ribose-phosphate backbone. Abasic RNA modifications are often introduced in experimental settings to study the effects of missing bases on RNA stability, structure, and function. In therapeutic and molecular biology contexts, abasic sites are used to disrupt RNA duplex formation or ribonucleoprotein interactions, affecting the biological activity of the RNA. RNA molecules with abasic sites are generally less stable because the absence of a base weakens the hydrogen bonding required for proper base pairing with complementary sequences. However, abasic modifications can also be used to intentionally destabilize RNA structures for certain applications, such as regulating gene expression or studying RNA degradation pathways.

Other Modifications