Method For Site-specific Labeling Research Articles

Site-Specific Fluorescent Labeling of RNA Molecules by Specific Transcription Using Unnatural Base Pairs

Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine (v) and y function in transcription, and the substrates of y and 5-modified y bases can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Ribonucleoside 5'-triphosphates of 5-fluorophore-linked y bases were chemically synthesized from the nucleoside of y. These fluorescent substrates were site-specifically incorporated into RNA by transcription mediated by the s-y and v-y pairs. By using this fluorescent labeling method, specific positions of Raf-binding and theophylline-binding RNA aptamers were fluorescently labeled, and the specific binding to their target molecules was detected by their fluorescent intensities. This site-specific labeling method using an unnatural base pair system will be useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.

Transfer of 131I and fluoresceinyl sialic acid derivatives into the oligosaccharide chains of IgG: a new method for site-specific labeling of antibodies

Biochemical modifications of IgG can help to avoid damages caused by oxidation or reduction. Terminal groups of the saccharide structures, located in the Fc-portion of IgG molecules, were modified by enzymatic reactions. IgG was pretreated with sialidase, to cleave bound sialic acid, and with galactosyltransferase, to increase the number of acceptor sites for transfer reactions. Afterward, modified sialic acid derivatives were transferred enzymatically into the oligosaccharide chains of IgG. Labeling was possible with sialic acids modified in either position 5 or position 9. The usefulness of this method was demonstrated for radioactive and fluoresceinylated reagents, with yields up to 90% in 1 h. Immunological investigations have shown no influence on the immunoreactivity by the described modification of saccharide structures.

Engineering antibodies for imaging and therapy

Several advances made during the past year will probably facilitate the development of therapeutic antibodies. Most notably, significant progress has been made in the rapid isolation of high affinity human antibodies from phage display libraries and by immunization of transgenic mice. The therapeutic potential of bispecific antibody fragments and Fc-containing proteins has been greatly enhanced by improved production methods. The utility of radiolabeled antibody fragments has been improved by the development of site-specific labeling methods and by the advent of the ‘minibody’, an engineered fragment that has proved to be highly successful for tumor imaging in mice.