Modifications for various applications
Puromycin is one of the aminonucleoside antibiotics which can be isolated from streptomyces strain Streptomyces alboniger. Puromycin shows structural similarity with the base adenosine or N6-N6-dimethyladenosine. Its effect is based on the inhibition of protein biosynthesis by binding to the 50S subunit of the ribosome via a peptide bond and thus affecting the growth of prokaryotic and eukaryotic cells.1 As an analogue of an aminoacyl-tRNA, puromycin can bind to the ribosomal A-site and integrates into the growing polypeptide chain. This leads to immediate termination of chain elongation, the ribosomal subunit dissociates and finally the translation stops.2 After termination, puromycin is located at the C-terminus of the truncated, newly synthesized protein and inhibits continuation of synthesis.3
biomers.net offers puromycin as 3´-modification with a maximum length of 45 bases.
1. Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis. Nishimura M, Ikeda K, Sugiyama M; J Biosci Bioeng. (2006), 101(1):63-9.
2. Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae. Cary GA, Yoon SH, Torres CG, Wang K, Hays M, Ludlow C, Goodlett DR, Dudley AM; Yeast (2014), (5):167-78.
3. Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition. Starck SR, Roberts RW; RNA (2002), 8:890–903.
In molecular biology, acridinium esters are commonly used for selective labeling of proteins and nucleic acids1. The acridinium ester can be bound to the corresponding target molecule (DNA, protein) by a covalent linkage2. According to their highly sensitive chemiluminescence releasing in the presence of hydrogen peroxide, acridinium esters also can be used for clinical diagnostics. In contrast to radiolabeled DNA, acridinium ester linkages provide a sensitive, stable and above all safe detection method in immunoassays3.
biomers.net offers acridinium ester as 3'- or 5'-modification up to a length of 45 bases.
1. Acridinium ester chemiluminescence: pH dependent hydrolysis of reagents and flow injection analysis of hydrogen peroxide and glutamate. Stuever Kaltenbach M, Arnold MA; Microchimica Acta (1992), Volume 108, Issue 3-6, pp 205-219.
2. Acridinium ester-labelled DNA oligonucleotide probes. Septak M; J Biolumin Chemilumin. (1989), (1):351-6.
3. Acridinium esters as high-specific-activity labels in immunoassay. Weeks I, Beheshti I, McCapra F, Campbell AK, Woodhead JS; Clin Chem. (1983), (8):1474-9.
Modifications for the immobilisation on surfaces
Carboxymethylaniline (4-CMA) allows to immobilise coupled biomolecules to appropriate prepared surfaces. In this way, the binding of oligonucleotides to conductive surfaces (e.g. graphite) enables the detection of currents. By coupling, either to the 5 'or 3' end of oligonucleotides, the molecules are covalently bonded in the desired orientation to the surface.
- Diazonium-Protein Adducts for Graphite Electrode Microarrays Modification: Direct and Addressed Electrochemical Immobilization. Corgier BP, Marquette CA, Blum LJ; Journal of the American Chemical Society, (2005), 127, 18328-18332.
- A versatile method for direct and covalent immobilisation of DNA and proteins on biochips. Corgier BP, Laurent A, Perriat P, Blum LJ, Marquette CA; Angewandte Chemie International (2007), 46, 4108-4110.
- On-Chip Chemiluminescent Signal Enhancement using Nanostructured Gold-Modified Carbon Microarrays. Corgier BP, Li F, Blum LJ, Marquette CA; Langmuir (2007), 23(16), 8619-8623.
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.