HaloTag®-labelled oligonucleotides for functional protein analysis
A notable representative among protein tags is the HaloTag®, which is widely used in protein analysis.
- protein purification of recombinant proteins from bacterial cells or cell culture lysates
- trafficking of desired proteins
- protein localisation in living and fixed cells
- multiplex imaging using antibodies or GFP
- analysis of protein-protein interactions
With biomers.net, you can now label oligonucleotides with a HaloTag® ligand.
In this case, the oligonucleotide is labelled with a chloroalkane linker at the 5 'or 3' end.
The HaloTag® (see Picture 1 in green) is a 34 kDa-sized haloalkane dehalogenase (34 kDa), which acts as a hydrolase and has a modified active site.
The modified HaloTag® protein is fused to the protein of interest (see pic. 1 in red) by means of a suitable expression system (HaloTag® vector from Promega), so that the resulting fusion protein can be further analysed by different ligands.
When the ligand is bound to the HaloTag® enzyme, the halogen group of the linker is cleaved by a nucleophilic displacement mechanism and released so that a covalent and irreversible ester bond can be formed between the haloalkane and the amino acid residue aspartate in the enzyme. In the WT hydrolase, the alkyl-enzyme intermediate would then be hydrolysed by a histidine residue.
However, the catalytic effect is inhibited by an amino acid exchange in the active site of the haloalkane dehalogenase (histidine to phenylalanine) and a stable and covalent linkage between ligand and HaloTag® enzyme is ensured.
The HaloTag® protein binds highly specifically to chloroalkane, which is connected via a linker to an oligo (see pic. 1 in blue). If desired the oligo itself can be further modified, e.g. with a fluorescent dye for easy tracking. By exchanging the oligonucleotide ligand, the function as well as the properties of the fusion protein can be influenced (e.g. membrane permeability, fluorescence labeling, surface binding etc.).
Picture 1: Oligonucleotide ligand with a chloroalkane linker that covalently binds the HaloTag® fusion protein.
Picture 2: Reaction of haloalkane dehalogenase for covalent binding of HaloTag® fusion proteins and oligo ligand.
We offer 5'- or 3'-labeled oligonucleotides with a chloroalkane linker.
Various functional modifications on the oligo are possible on request (fluorescent dyes, thiol, biotin, etc.).
Just contact us!
1. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV; ACS Chem Biol. (2008), 3(6):373-82. doi: 10.1021/cb800025k.
2. HaloTag Technology: A Versatile Platform for Biomedical Applications. England CG, Luo H, Cai W; Bioconjugate Chem. (2015), 26, 975−986975 DOI: 10.1021/acs.bioconjchem.5b00191.
3. Histidine 289 is Essential for Hydrolysis of the Alkyl-enzyme Intermediate of Haloalkane Dehalogenase. Pries F, Kingma J, Krooshof GH, Jeronimus-Stratingh CM, Bruins AP, Janssen DB; The Journal of Biological Chemistry (1995), DOI: 10.1074/jbc.270.18.10405.
HaloTag® is a registered trademark of Promega Corporation. HaloTag® Ligand Technology is licensed from Promega Corporation.
Immobilisation on surfaces via Methacrylamide-modified oligonucleotides
Methacrylamide-modified, also known as acrydite-modified oligonucleotides are an important part and parcel of molecular biology research for many years. By polymerising of free acrylic acid monomers (formation of polyacrylamides) or by coupling with thiol, methacrylamide-modified oligonucleotides can be covalently bound to surfaces. By means of this fast and simple attachment to surfaces, desired DNA single strands can be immobilised, enriched, identified or purified.
A well-known example is the biochip technology (microarrays) in which the methacralymide-modified oligonucleotides are bound to a thiol-labelled glass surface and thus accessible to single-stranded DNA.
This results in a variety of possible applications:
- Monitoring of mRNA expression
- Sequencing of DNA
- Identification of SNP
- Detection of viruses, bacteria and other pathogens
biomers.net offers 5'- and 3'-methacrylamide-modified DNA oligonucleotides.
1. Immobilization of acrylamide-modified oligonucleotides by co-polymerization. Rehman FN, Audeh M, Abrams ES, Hammond PW, Kenney M, Boles TC; N. Acids Research. 27, (1998), Bd. 2, 649-655.
2. Mutation typing using electrophoresis and gel-immobilized Acrydite probes. Kenney M, Ray S, Boles TC; Biotechniques (1998), (3):516-21.
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.
Protein labelling with cyanobenzothiazole-modified oligonucleotides
By means of cyanobenzothiazole-labelled oligonucleotides, suitable proteins can be rapidly and efficiently labelled and prepared for detection in the cell without restricting or inhibiting their function. The reactivity and specificity of the cyanobenzothiazole oligonucleotide tag are primarily dependent on the sequence of the protein. In order to ensure a stable adduct and a fast and selective reaction of the cyanobenzothiazole-labelled oligo with a protein, an N-terminal cysteine on the protein is essential.
Picture 1: Reaction scheme of a cyanobenzothiazole-labelled oligonucleotide with a suitable protein carrying an N-terminal cysteine.
The current coupling of proteins and oligos via click chemistry cannot be applied intracellularly due to toxic copper ions. Cyanobenzothiazole, however, allows the coupling of a protein with an oligonucleotide also in vivo.
According to this, a specific labelling of the protein for the detection or analysis of the protein structure and function is ensured.
- Labelling and localisation of specific proteins
- Luminescence analyses in vivo
- Detection and quantification of intracellular, biochemical processes in real time
For efficient protein labeling we offer 5'-cyanobenzothiazole-labelled oligonucleotides.
1. A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice. Godinat A, Park HM, Miller SC, Cheng K, Hanahan D, Sanman LE, Bogyo M, Yu A, Nikitin GF, Stahl A, Dubikovskaya EA; ACS Chem Biol. (2013), 8(5): doi:10.1021/cb3007314.
2. Sequence-Specific 2-Cyanobenzothiazole Ligation. Ramil CP, An P, Yu Z, Lin Q; J Am Chem Soc. (2016), 138(17):5499-502. doi: 10.1021/jacs.6b00982.
Antisense oligonucleotides and aptamers with ibuprofen
A new approach to significantly increase lifetime of aptamers in cell is the coupling of small molecules such as ibuprofen to DNA oligonucleotides.
Enzymatic degradation by endo- and exonucleases, as well as liver and kidney clearance of comparatively small molecules like aptamers and antisense oligonucleotides (25-30 kDa) from circulation can limit lifetime of these possible drugs and thus even reduce their therapeutic potential.
Drugs like ibuprofen are able to bind albumin or other serum proteins, as well as ibuprofen conjugates (e.g. chemically modified oligonucleotides with ibuprofen). 3
Due to increased molecular weights of these complexes (antisense oligonucleotide / aptamer-ibuprofen-albumin) the clearance by liver and kidney is hindered and resistence against enzymatic nucleases (endo- and exonucleases) is also increased. 1,2
In this way, lifetime as well as intracellular distribution and thus the pharmacological availability of the modified antisense oligonucleotides or aptamers can be improved.2
At the target site, the oligonucleotide-ibuprofen-conjugate can disassociate from albumin carrier in order to get fully effective.2
Now biomers.net offers ibuprofen as 5´-and /or 3´-modification for DNA oligonucleotides.
1. Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics. Bruno, JG; Molecules (2015), 20, 6866-6887; doi:10.3390/molecules20046866.
2. A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches. Bruno, JG; Pharmaceuticals (2013), 6, 340-357; doi:10.3390/ph6030340.
3. Improving Antisense Oligonucleotide Binding to Human Serum Albumin: Dramatic Effect of Ibuprofen Conjugation. Manoharan M, Inamati GB, Lesnik EA, Sioufi NB, Freier SM; Chembiochem. (2002); 3(12):1257-60.
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
New at biomers.net: coenzyme A-modified DNA
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
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.