We now offer both single nucleotides and labeling kits for efficient preparation of Desthiobiotinylated long non-coding RNA (lncRNA) probes (Fig. 1, Tab. 1).
Desthiobiotinylation offers several advantages over Biotinylation due its significantly lower binding affinitiy to streptavidin[1]:
Figure 1: Enzymatic properties of Desthiobiotin-11-UTP (A) and Desthiobiotin-11-CTP (B) are comparable to their Biotin-labeled counterparts. 1400 nt RNA probes were generated by in vitro transcription using the corresponding HighYield T7 RNA Labeling Kit components (HighYield T7 Desthiobiotin11 RNA Labeling Kit (UTP-based) or HighYield T7 Desthiobiotin11 RNA Labeling Kit (CTP-based), respectively). Reaction conditions: 100 ng DNA template, increasing percentage of UTP/CTP substitution by (Desthio)biotinylated UTP/CTP (0 – 100%), 30 min at 37°C. Crude in vitro reaction mix was subsequently analyzed by agarose gel electrophoresis (EtBr-based visualization of product yield).
| Nucleotide | Cat. No. | Amount |
|---|---|---|
| Desthiobiotin | ||
| Desthiobiotin-11-UTP | NU-821-Desthiobio | 30 µl (10 mM) |
| Desthiobiotin-11-CTP | NU-831-Desthiobio | 30 µl (10 mM) |
| Desthiobiotin-11-ATP | NU-957-Desthiobio-S | 10 µl (1 mM) |
| NU-957-Desthiobio-L | 5 x 10 µl (1 mM) | |
| Desthiobiotin-11-GTP | NU-971-Desthiobio-S | 10 µl (1 mM) |
| NU-971-Desthiobio-L | 5 x 10 µl (1 mM) | |
| Biotin | ||
| Biotin-11-UTP | NU-821-BIOX | 30 µl (10 mM) |
| Biotin-11-UTP - high concentration | NU-821-BIOX-HC | 30 µl (75 mM) |
| Biotin-11-CTP | NU-831-BIOX | 30 µl (10 mM) |
| Biotin-11-ATP | NU-957-BIOX-S | 10 µl (1 mM) |
| NU-957-BIOX-L | 5 x 10 µl (1 mM) | |
| Biotin-11-GTP | NU-971-BIOX-S | 10 µl (1 mM) |
| NU-971-BIOX-L | 5 x 10 µl (1 mM) | |
| (Desthio)Biotin in vitro Transcription Kits | ||
| HighYield T7 Desthiobiotin11 RNA Labeling Kit (UTP-based) | RNT-101-Desthiobio | 30 reactions x 20 µl |
| HighYield T7 Desthiobiotin11 RNA Labeling Kit (CTP-based) | RNT-401-Desthiobio | 30 reactions x 20 µl |
| HighYield T7 Biotin11 RNA Labeling Kit (UTP-based) | RNT-101-BIOX | 30 reactions x 20 µl |
| HighYield T7 Biotin11 RNA Labeling Kit (CTP-based) | RNT-401-BIOX | 30 reactions x 20 µl |
Please note: Digoxigenin-labeled UTP, a corresponding HighYield T7 Digoxigenin RNA Labeling Kit and Biotin-modified nucleotides with various linker lengths and linker positions are available as well.
Just contact Lisa if you are interested in other Hapten-modified nucleotides!
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[1] Hirsch et al. (2002) Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Analytical Biochemistry 308:343.
[2] Mattick et al. (2023) Long non-coding RNAs: definition, functions, challenges and recommendations. Nature Reviews Molecular Cell Biology 24:430.
[3] Chen et al. (2017) Quantitative proteomics reveals that long non-coding RNA MALAT1 interacts with DBC1 to regulate p53 acetylation. Nucleic Acids Research 45(17):9947.
[4] Tichon et al. (2016) A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nature Communications 7:12209.
[5] Priyanka et al. (2021) The lncRNA HMS recruits RNA-binding protein HuR to stabilize the 3'-UTR of HOXC10 mRNA. Journal of Biological Chemistry 297(2):100997.
[6] Nishitsuji et al. (2016) Long noncoding RNA #32 contributes to antiviral responses by controlling interferon-stimulated gene expression. Proc. Natl. Acad. Sci. 113(37):10388.
