Labelling of biomolecules

As can be seen from Table 4.2, nucleic acids, proteins or heterogeneous complexes of one or both of these, have been the most commonly investigated biomolecules using single molecule fluorescence techniques. As no intrinsic naturally occurring fluorophores with suitable photophysical properties for single molecule experiments are found in either nucleic acids or polypetides (see Section 4.2.1), it is necessary to introduce such moieties into these biomolecules [5,24,25]. This can be done in three ways  

The method employed is dependent on whether the target molecule is a nucleic acid or polypeptide and in the case of proteins, the number of amino acids in the target. 

Both ribonucleic and deoxyribonucleic acid polymers are almost exclusively synthesized using the same solid phase phosphoramidite chemistry utilized in the 

As well as single molecule fluorescence studies, oligonucleotides labelled with fluorophores are used in many different applications, such as quantitative PCR. Consequently, labelled oligonucleotides are readily available from commercial manufacturers but the range of dyes available from each company varies. This laboratory has obtained labelled DNA oligonucleotides from both MWG-Biotech, UK and IBA, Germany and obtained excellent results from both. 

The development of automated soUd phase synthesis of nucleic acids and chemical strategies to incorporate fluorophores at any position along their length, together with the wide array of molecular biological techniques available for their modification has resulted in a system highly amenable to single molecule fluorescent studies. By contrast, proteins, to which we now turn our attention, are rarely synthesized in vitro and thus a different approach needs to be taken. 

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Gonsalves MST (2009) Fluorescent labeling of biomolecules with organic probes. Chem Rev 109 190-212... 

Patonay G, Salon J, Sowell J, Strekowski L (2004) Noncovalent labeling of biomolecules with red and near-infrared dyes. Molecules 9 40-49... 

None of the N2S2 or N3S systems described above is suitable for the postformed labeling of biomolecules (i.e. coupling a chelator to the biomolecule, followed by incorporation of the radiometal into the chelator) because the unfavorable kinetics of complexation or transchelation require relatively harsh conditions (strong acid/alkali, heat) which the biomolecule would not tolerate. In order to combine the advantages of in vivo stability in the amido systems with the better complexation... 

NCA a-[18F]Fluoro-substituted acetic and propionic acid esters have been obtained169 by aminopolyether (APE 2.2.2) supported nucleophilic exchange as shown in equation 76 and applied169 for NCA 18F-fluoroacylation of ethanol (equation 77) and of -butylamine (equation 78). The above reactions are relevant to 18F-labelling of biomolecules such as peptides170. 

Fig. 2 Structures of selected acyclic and macrocyclic BFCs useful for the radiometal labeling of biomolecules, including antibodies, antibody fragments, small peptides, and nonpeptide receptor ligands...

Steps toward high specific activity labeling of biomolecules for therapeutic apphcation preparation of precursor [ Re(H20)3(C0)3j and synthesis of tailor-made bifimctional ligand systems. Bioconjugate Chem 13 750-756... 

Wald J, Alberto R, Ortner K, Candreia L (2001) Aqueous one-pot synthesis of derivatized cyclo-pentadienyl-tricarbonyl complexes of Tc-99m with an in situ CO source application to a serotonergic receptor ligand. Angew Chem Int Edit 40 3062-3066 Wei LFi, Babich J, Zubieta J (2005) Bifunctional chelates with mixed aromatic and aliphatic amine donors for labeling of biomolecules with the [Tc(CO)3] and [Re(CO)3] -cores. Inorg Chim Acta 358 3691-3700... 

Bioorthogonal labelling of biomolecules New functional handles and ligation methods 130BC6439. 

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