Glen Report 33-24: New Product — 5’-Biotin II Phosphoramidite

Biotin-based assays have been widely applied in nucleic acids research since the strong affinity between biotin and streptavidin was discovered in the 1970s.1 The protein-ligand contact is one of the strongest noncovalent interactions, with a Kd of 10-15 M. A biotin label is versatile and is often used to enrich proteins that bind to a specific DNA. Alternatively, biotin offers precise detection, amplification, and/or immobilization of DNA substrates. In addition, the orthogonality of the biotin-streptavidin interaction allows researchers to use other labels in their experiments, without having to worry about cross-reactions.

Glen Research’s first biotin product was released in 1991, and our collection has grown substantially since then. Our newest product, 5’-Biotin II phosphoramidite (Figure 1), complements our traditional version I. These two 5’-biotin phosphoramidites differ slightly in terms of the linker, an all-carbon versus an ethylene glycol linker (Figure 1A-B). For those familiar with our amino-modifier offerings, the linkers in versions I and II are derived from amino-modifier C6 and amino-modifier 5, respectively (Figure 1C-D). The addition of 5’-Biotin II phosphoramidite allows researchers accustomed to using the amino-modifier 5 linker to continue to do so in their experiments. Moreover, this new version matches the standard 5’-biotin structure used by major oligonucleotide synthesis providers.

Figure 1

Figure 1. Structures of (A) 5’-Biotin (I), (B) 5’-Biotin II Phosphoramidite, (C) 5’-Amino Modifier C6 TFA, and (D) 5’-Amino Modifier 


The 5’-biotin phosphoramidites offer several advantages to customers:

  • Benefits from on-column automated incorporation, as opposed to post-synthetic conjugations, such as click chemistry or NHS ester couplings.
  • Contains a DMT-group that enables reverse phase (RP) cartridge, such as Glen-Paks, and HPLC purification techniques.
  • CAUTION: Even though this structure contains a DMT group, the 5’-Biotin II can only be added once to the 5’-terminus of an oligonucleotide. The DMT group in this structure protects one of the urea nitrogens in the biotin structure and does not enable oligonucleotide elongation, like a normal DMT-O would.

Recent Applications

With the surge of public awareness in disease detection, point-of-care (POC) diagnostic testing is becoming essential to the rapid analysis of patient analytes as it facilitates better diagnosis, monitoring, and management. POC diagnosis based on nucleic acid testing typically relies on nucleic acid amplification and detection in a single device. While PCR is a powerful tool and meets requirements of diagnostic tools, such as specificity, sensitivity, and rapidity, it requires numerous experimental steps, skilled technicians, and costly materials.
Low-cost tools are of tremendous interest and paper microfluidic devices offer a promising platform to translate isothermal amplification tests to POC diagnostics.2,3

The 5’-Biotin II structure was incorporated into oligonucleotides for paper-based devices to detect disease-associated DNA. A 5’-biotinylated reverse primer and a FAM-labeled forward primer were used in a paper-and-plastic device coupled to an isothermal recombinase polymerase amplification (RPA) reaction to detect malarial DNA.3 In this system, the RPA reaction produced a primary product labeled with a biotin tag from the reverse primer. The 5’-FAM forward probe recognized the primary product, resulting in a new secondary probe labeled with both biotin and FAM.  The secondary product was detected using lateral flow strips with streptavidin to capture the product on the test line and gold nanoparticles functionalized with anti-FAM to yield a color change in the presence of the target nucleic acid sequence (Figure 2). A similar fluorescence-based detection system used 5’-Biotin II labeled reverse primers to amplify and detect Chlamydia trachomatis.4


Figure 2

Figure 2. Workflow of point-of-care diagnostics using 5’-biotinylated reverse primers.

Not only is nucleic acid detection applicable to the field of diagnostics, but it can also be valuable in elucidating the role of certain nucleic acid sequences. Long noncoding RNAs (lncRNAs) play important roles in cellular development, chromatin structure, and gene regulation.5 Cross-linking and immunoprecipitation (CLIP) methods are typically used to study direct RNA-protein interactions in vivo, but they have limited utility in identifying new protein partners for a specific lncRNA. A novel technique called RNA-antisense purification coupled with mass spectrometry (RAP-MS) used UV-light to cross-link zero distance interacting RNA and protein partners followed by capture of the 5’-biotinylated RNA through hybridization on streptavidin beads. This method was used to understand the mechanism of Xist lncRNA in female mammals, where one X chromosome is transcriptionally silenced. A new protein partner of Xist lncRNA was identified: a large multidomain transcriptional coregulator protein (SHARP), which activated histone deacetylase HDAC3 and excluded Pol II across the X chromosome.

Recently, the strong streptavidin-biotin interaction was used to immobilize nucleic acid substrates. This was particularly helpful in the study of nitrogen mustard-induced interstrand cross-link (ICL) bypass by translesion DNA synthesis (TLS) polymerases. TLS is the bypass of a lesion during DNA replication and TLS polymerases play important roles in DNA damage tolerance. Bypass of a lesion avoids fork stalling and collapse, which can lead to cell death. This process must be tightly regulated as TLS is a major source of DNA damage-induced mutagenesis. Bezalel-Buch et al. immobilized a 93-mer ICL oligonucleotide substrate with terminal 5’-Biotin II on streptavidin beads and found the Rev1-Polζ complex faithfully inserted dCMP opposite the dG-ICL, yielding a full-length extension product, while other TLS polymerases inefficiently bypassed the induced ICL.6 This work has significant implications for disease-associated cells harboring Polζ mutations and their hypersensitivity to ICL-forming agents. 

In another study, the 5’-Biotin II structure was used to reduce noise in precise editing through preassembly of CRISPR ribonucleoproteins (RNPs) by S1m, an RNA aptamer with  a strong affinity for streptavidin. In this method, the conjugation of S1m to a sgRNA allowed for complexation between streptavidin-S1m-sgRNA-cas9 to form the RNP, termed S1mplex. In the presence of 5’-biotinylated single stranded donor template, accurate homology-directed repair (HDR) was induced at target sequences, confirmed by deep sequencing.7

The versatility of 5’-Biotin II phosphoramidite makes it an excellent addition to our product catalog. As always, we ensure this product meets our high-quality requirements and is optimized for your use. A 2-minute coupling time is recommended for 5’-Biotin II Phosphoramidite. 5’-Biotin II is slow to detritylate. If the final DMT-group is to be removed on the synthesizer, we recommend a second deblocking step. If the final DMT-group is to be left on for purification, treat the biotinylated oligonucleotide with TFA solution for 10 minutes. Again, only a single biotin can be incorporated at the 5’-end using this product.


  1. N. Green, Advances in Protein Chemistry, 1975, 29, 85-133.
  2. P. Maffert, S. Reverchon, W. Nasser, C. Rozand, and H. Abaibou, Eur J clin Microbiol Infect Dis, 2017, 36, 1717-1731.
  3. M. Cordray, R. Richards-Kortum, Malar J, 2015, 14, 472-479.
  4. J. Linnes, A. Fan, N, Rodriguez, B, Lemleux, H. Kong, C. Klapperich, RSC Adv., 2014, 4, 42245-42251.
  5. C. McHugh, and M. Guttman, Methods Mol Biol. 2018, 1649, 473-488. 
  6. R. Bezalel-Buch, Y. Cheun, U. Roy, O. Scharer, and P. Burgers, Nucleic Acids Research, 2020, 48, 8461-8473.
  7. J. Carlson-Stevermer, A. Abdeen, L. Kohlenberg, M. Goedland, K. Molugu, M. Lou, and K. Saha, Nature Communications, 2017, 8, 1711-1723.

Product Information

5'-Biotin Phosphoramidite (10-5950)

5’-Biotin II Phosphoramidite (10-1954)