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Affinity Tagging Made Simple

Biotinylation of DNA and RNA
The binding strength between biotin and streptavidin is close to that of a covalent bond[1] and serves for most enrichment and immobilization approaches in molecular biology. Being a small and stable epitope, biotin conjugated to nucleoside triphosphates is routinely incorporated into DNA and RNA by corresponding polymerases.[2] Tolerance towards biotinylated substrates is common to virtually all classes of enzymes participating in nucleic acid metabolism.[2-8] In result, tagged nucleic acids can be obtained simply by adding the corresponding biotinylated nucleotide to common in vitro reactions of DNA (Scheme 1) and RNA (Scheme 2) synthesis.

Available biotin-nucleotide-conjugates for enzymatic incorporation into DNA and RNA:

Available biotin-nucleotide-conjugates for enzymatic incorporation into DNA and RNA.

For product information and ordering please click here: Additionally, a highly diverse set of biotin linkers is available from our catalog. Please don’t hesitate to inquire for custom-made solutions!

Scheme 1: Single step biotinylation of DNA
Scheme 1:
Single step biotinylation of DNA. Biotinylated dNTPs are randomly incorporated into DNA by DNA-polymerases[2] (A) or reverse transcriptases[3] (B).

Scheme 2: Biotinylated nucleotides can be randomly or site-specifically incorporated into RNA
Scheme 2:
Biotinylated nucleotides can be randomly or site-specifically incorporated into RNA on several ways. (A) Similar to dNTPs in DNA synthesis, NTPs are statistically installed by RNA polymerases.[2] (B) Site-specific 5′-labeling is achieved by transcriptional priming using biotinylated initiator nucleotides.[4-5]
3′-labels are obtained by subjecting a substrate RNA to polyadenylation with suitable NTPs (C)[6] or to ligation with biotinylated pCp (D)[7-8].

Selected references:
[1] Green (1975) Avidin. Adv. Protein Chem. 29:85.
[2] Langer et al.. (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA. 78:6633.
[3] Lee et al.. (2016) Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays. Anal. Chem. 88:12272.
[4] Huang et al.. (2008) Synthesis of biotin–AMP conjugate for 5′ biotin labeling of RNA through one-step in vitro transcription. Nat. Protoc. 3:1848.
[5] Pitulle et al.. (1992) Initiator oligonucleotides for the combination of chemical and enzymatic RNA synthesis. Gene. 112:101.
[6] Moritz et al. (2014) Simple methods for the 3' biotinylation of RNA. RNA. 20:421.
[7] Yamato et al. (2017) RNA Aptamer Binds Heparin-Binding Epidermal Growth Factor-Like Growth Factor with High Affinity and Specificity and Neutralizes Its Activity. Int. J. Gerontol. 11:191.
[8] Booy et al. (2016) RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1). J. Biol. Chem. 291:5355.

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