Frequently Asked Questions

Amino modifiers introduce a reactive amino group (−NH2) to oligonucleotides, enabling covalent conjugation with various molecules such as fluorophores, biotin, or surfaces.

Key points:

(1) The 5'Aminolinker (C6) modifier is attached to the 5′ sugar ring via phosphoramidite chemistry during synthesis (not to the terminal base).

(2) The amino group absorbs at 210 nm (not 260 nm) and cannot be detected by standard agarose or acrylamide gel electrophoresis.

(3) The 3'Aminolinker(C7) is compatible only with certain 5′ modifications (e.g., FAM, HEX, TET, Fluorescein, Biotin, phosphate). It is incompatible with dyes like Alexa Fluor® that also require an amine for conjugation.

(4) Amino modification remains a simple and cost-effective strategy. For most applications, the 5'C6-NH2 Modification is highly recommended.

Biotin-modified oligonucleotides leverage the strong biotin-streptavidin interaction, enabling diverse applications in research and diagnostics. Key uses include:

Non-radioactive immunoassays for protein detection

Intracellular chemical staining and cell separation

Nucleic acid isolation and hybridization to detect specific DNA/RNA sequences

Probes for studying conformational changes (e.g., in ion channels)

Immobilization on solid-phase surfaces (e.g., beads, chips)

For conjugation to a gold electrode, mix 1 µL of 1 mM TCEP with 99 µL of thiol-modified DNA (at a concentration of 0.2 µM) and incubate at room temperature for 30–60 minutes. The reduced DNA may be used directly in subsequent experiments without removing TCEP. If a higher DNA concentration is required (e.g., 100 µM), 10 mM TCEP should be used for reduction. In this case, the amount of TCEP must be at least 100 times the molar amount of DNA. For conjugation to gold nanoparticles, mix 200 µL of 15 µM thiol-modified DNA with 5 µL of 1 M TCEP and reduce at room temperature for 30 minutes. The mixture can then be directly added to the gold nanoparticle solution.

Common stabilizing modifications include:

(1) 2′-O-Methyl (2′-OMe): Enhances nuclease resistance

(2) 2′-Fluoro (2′-F): Increases binding affinity and stability

(3) Phosphorothioate backbone: Improves resistance to enzymatic degradation

(4) Locked Nucleic Acid (LNA):“Locks” the structure for extreme stability and high affinity

They enable the development of advanced therapeutics by:

(1) Enhancing target engagement and specificity (e.g., LNA, cEt)

(2) Improving pharmacokinetics (e.g., GalNAc for liver targeting)

(3) Reducing immunogenicity (e.g., 2′-OMe, pseudouridine)

(4) Facilitating efficient delivery (e.g., cholesterol conjugation)

The choice depends on your goal:

For stability: Choose 2′-OMe, 2′-F, or LNA

For delivery: Consider GalNAc or cholesterol

For epigenetics: Use 5-methyl-dC or 5hmdC

For high-affinity binding: LNA or cEt are ideal

For functional conjugation: Biotin, amines, or thiols may be used