In the Cappable-seq technique 3′-Desthiobiotin-GTP can be used to label the 5′ end of mRNA.


However in a commercial technical article on biotinylated-RNA affinity probes I encountered the following biotinylated cap, in which the biotin affinity tag is attached to the 2′-position of the ribose at the 5′ end of the dinucleotide.

Biotinylated cap

This indicates that a 2′-biotinylated GTP had been used, and will be incorporated into mRNA.

What could be the reason for this difference in tag position?

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    $\begingroup$ Welcome to SE Biology. Could you please provide some context to your question, which seems rather specialized. You show the structures of two different derivatives of biotin and GTP, which presumably are commercial products (one appears to be from a catalogue). Are you asking what they are used for? And what do you mean by a “typical standalone biotin-GTP?” And where have you encountered this RNA derivative? Help us to help you. $\endgroup$
    – David
    Mar 29, 2022 at 21:35
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    $\begingroup$ Thank you Mr. David for the advices. GTP is a very common structure in biology. If biotin is incubated, then the biotin is bound by the 3' carbon, as shows the second image. If N7 methylated guanosine is incubated with biotin, then the biotin will bound by the 2' carbon, as shows the first image. In this image, the m^7-biotin-GTP has been flipped horizontally and has been added to the phosphate groups, by GTase (RNA Guanylyltransferase). Why this difference in the bounded carbon? I hope to help you. Thanks $\endgroup$
    – DanK
    Mar 29, 2022 at 23:12
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    $\begingroup$ You haven't answered my questions. I know all about GTP and about biotin, and — as far as I am aware — that they never occur in Nature covalently bound to one another. The structures you show are clearly of commercial reagents. Where did you encounter these structures? Why do you want to know about them? Is this some sort of homework? Or are you new to some research laboratory? And I see this same question on Biostars. Please only post your questions in one place — cross-posting, as it is called, is not allowed on SE sites as it wastes people's time when a question is asked somewhere else. $\endgroup$
    – David
    Mar 30, 2022 at 13:18
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    $\begingroup$ @David regarding cross-posting - I don't see an issue with posting both here and at biostars, because they aren't affiliated with Stack Exchange, and are their own separate entity. Cross-posting to different SE sites is discouraged, but not if they post outside the network. Granted, both sites may share part of their user base, so some people will see the post twice, but that shouldn't be an issue. $\endgroup$
    – MattDMo
    Mar 31, 2022 at 0:03
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    $\begingroup$ You still do not provide the source of the diagrams. I can see that the second is from NEB and is the capping reagent described in the paper by Ettwiller et al. The second comes from some page I cannot locate on the Biosynthesis company website. It does not show a biotinylated RNA but a biotinylated dinucleotide cap. Presumably it is generated with some reagent that Biosynthesis sells, and for all I know this reagent is different from that NEB cells for patent reasons. It would probably be able to form 5'-3' bonds in RNA, but you need to find data on the reagent. $\endgroup$
    – David
    Mar 31, 2022 at 10:07

1 Answer 1


In the Cappable-seq technique, the aim is to biotinylate primary transcripts at the 5′-cap so that they can be separated by affinity columns, or with affinity beads, of the biotin-binding protein, streptavidin. It would not appear to matter if the biotin linker is attached at the 2′ or 3′ position of the ribose ring, and there may be non-scientific reasons (e.g. cost, availability) for the choice of the 3′ position.

The cap structure in the technical article is adapted from a paper by Bednarek et al. on the synthesis of a variety of such caps, for which one specific use was to pull down complexes of mRNA with eukaryotic initiation factor eIF4e. It turned out that having the biotin affinity tag in the 3′ position of the ribose ring impeded this interaction, to a greater extent than at the 2′ position. Hence the difference.

This is clear from the start of the results section of that paper, in which an additional advantage is also explained:

The affinity tag was attached at the 2′ position of m7G via an amide bond, which was possible due to the presence of the primary aliphatic amino group in the starting material—2′-amino-2′-deoxyguanosine—and in situ biotin activation with O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). The site for biotin moiety attachment was chosen in a way that ensures the least possible interference with eIF4E interaction, because the hydroxyl groups of 7-methylguanosine in the cap-eIF4E complex are free and solvent exposed [16] (electronic supplementary material, figure S1). The ability of the cap analogues conjugated with biotin to serve as substrates for RNA polymerase in an in vitro transcription reaction was of equally high importance. In this context, the additional advantage of introducing the tag at the 2′ position is that, similar to 2′-O-methylated dinucleotides, the resulting compound can only be incorporated into RNA in the correct orientation (and, as such, belongs to a class of the so-called Anti-Reverse Cap Analogs or ARCAs).


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