Photocrosslinker - labelling and cross linking of proteins and oligonucleotides
As the name suggests, photocrosslinkers are excited by light to produce a covalent bond between two molecules. The light induction is usually performed at wavelengths close to UV range (250-460 nm depending on the linker) and can be controlled precisely in time and space, e.g. only in certain tissue parts or at a defined developmental stage.
By binding the photocrosslinker to one end of an oligonucleotide, a complementary DNA section can be controlled with utmost precision. After photoinduction, a covalent bond is formed to the complementary strand. According to this, for example, a promoter region and the associated gene expression can be analysed, as well as the endogenous repair mechanisms which cells have developed in order to protect their DNA from UV damage.
As an alternative to conventional coupling strategies, photoreactive linkers also offer the possibility to bind functional groups to oligonucleotides.
Photocrosslinkers are also widely used to covalently link proteins and DNA.
DNA-protein-contacts are important switching points in cells, so accurate knowledge of the exact contact areas may allow modulation of such processes.
For effective cross-linking, we offer different photo-reactive groups that can be attached to the
5'-end of the oligonucleotide.
1. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label. Yang Y, Song H, He D, Zhang S, Dai S, Lin S, Meng R, Wang C, Chen PR; Nat Commun. (2016), 7:12299. doi: 10.1038/ncomms12299.
2. Photolyase-like Repair of Psoralen-Crosslinked Nucleic Acids. Stafforst T, Hilvert D; Angew. Chem. Int. Ed. (2011), 50, 9483 –9486.
3. Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide. Takasugi M, Guendouz A, Chassignol M, Decout JL, Lhomme J, Thuong NT, Hélène C; Proc Natl Acad Sci U S A. (1991), 88(13):5602-6.
4. UV crosslinking of proteins to nucleic acids. Chodosh LA; Curr Protoc Mol Biol. (2001), Chapter 12:Unit 12.5. doi: 10.1002/0471142727.
Oligonucleotides with the cytostatic drug gemcitabine dFdC
Gemcitabine (2´,2´-difluoro deoxycytidine, dFdC) is an analogue of the pyrimidine nucleoside deoxycytidine (dC). Gemcitabine differs from dC in two fluorine atoms (instead of two hydrogen atoms) at the 2´-position of the sugar.
The effect of the two additional fluorine atoms is shown in the inhibition of DNA synthesis.1,2 It is particularly interesting that after incorporation of gemcitabine, DNA can only be extended by one further nucleotide. Then, the synthesis interrupts and is blocked thus resulting the death of the cell. This process is called “masked chain termination” since the last nucleotide prevents the cytidine analogue dFdC from detection and degradation by exonucleases or DNA repair enzymes.3 Due to its activity as cytostatic drug, gemcitabine is preferably used in chemotherapy as an anti-tumor agent.3
biomers.net now offers dFdC for incorporation into oligonucleotides!
The internal coupling is possible at any desired position of the oligo. Also multiple couplings can be synthesised. Combinations with other modifications are possible on request.
1. 2',2'-Difluoro-deoxycytidine (gemcitabine) incorporation into RNA and DNA of tumour cell lines. Ruiz van Haperen VW, Veerman G, Vermorken JB, Peters GJ; Biochem Pharmacol. (1993); 46(4):762-6.
2. Quantification of gemcitabine incorporation into human DNA by LC/MS/MS as a surrogate measure for target engagement. Wickremsinhe ER, Lutzke BS, Jones BR, Schultz GA, Freeman AB, Pratt SE, Bones AM, Ackermann BL; Anal Chem. (2010);82(15):6576-83. doi: 10.1021/ac100984h.
3. DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications. Kelley MR; Academic Press (2012), 95-98.
4. Synthesis and restriction enzyme analysis of oligodeoxyribonucleotides containing the anti-cancer drug 2',2'-difluoro-2'-deoxycytidine. Richardson FC, Richardson KK, Kroin JS, Hertel LW; Nucleic Acids Res. (1992); 20(7): 1763–1768.
Oligonucleotide-Coenzyme A conjugates
Many fields of research require a specific linking of native proteins with building blocks, affecting the transport behavior or allow specific immobilization. Using small 'tags' which can be fused to the proteins, proteins can be combined almost arbitrarily without compromising their folding or function. A highly flexible system makes use of the selective linking of the so-called ybbR tags with coenzyme A.
We are pleased to be able to offer coenzyme A-modified oligos. This opens up many possibilities of DNA-protein chimeras.
An impressive example can be found at:
Protein–DNA Chimeras for Nano Assembly (Pippig et al., ACS Nano, 2014)
Ask...we like to discuss your individual project!
- Protein-DNA Chimeras for Nano Assembly. Pippig DA, Baumann F, Strackharn M, Aschenbrenner D, Gaub HE; ACS Nano (2014), 8 (7), pp 6551–6555.
- The Ribosome Modulates Nascent Protein Folding. Kaiser CM, Goldman DH, Chodera JD, Tinoco Jr. I, Bustamante C; Science (2011), 334(6063): 1723–1727. doi:10.1126/science.1209740.