In vitro selection techniques

One of the most interesting ribozymes isolated by in vitro selection techniques is a novel catalytic RNA that promoted the formation of a glycosidic bond... 

In addition to natural ribozymes several synthetic ribozymes with new activity have been obtained by in-vitro selection techniques. Starting with a pool of random RNA sequences, molecules with a desired activity can be isolated by successive cycles of activity selection, reverse transcription into DNA, and amplification by polymerase chain reaction [4a,b]. 

This year s nucleotide and nucleic acid literature has been dominated by interest in internucleoside linkages. A number of approaches to novel internucleoside linkages in dimers have been published in addition to stereoselective routes to phosphorothioate and methylphosphonate linkages. In some cases these studies have also extended to the oligonucleotide level. In addition a number of novel nucleotide analogues have been described. One of the most exciting areas in the field of nucleic acid chemistry is the application of in vitro selection techniques and these are reviewed for the first time. 

As discussed above, in vitro selection techniques have been used to... 

A DNA molecule which binds to a A -methylmesoporphyrin has been evolved by in vitro selection techniques. ... 

A new departure in the development of nucleic acid based catalysts was made three years ago by Breaker and Joyce, who reported the isolation of the first deoxyribozyme. The proof that DNA can also catalyze chemical reactions is not completely unexpected, especially since it was shown shortly after the development of the in vitro selection technique that single-stranded DNA molecules can also be selected to bind to a variety of ligands. Meanwhile, several catalytically active DNAs have been described, expanding the range of nucleic acid catalyzed reactions even further. 

An in vitro selection technique was apphed to evolve a collection of catalytically active family of RNA sequences, with 5-(4-pyridyhnethyl)-uridine 5 triphosphate ( UTP) being used to provide additional metal-RNA coordination sites (beyond the nitrogen-containing heterocycles available in the native RNA structure). The RNA is then incubated with the zerovalent metal complex, tris-(dibenzylideneace-tone) dipalladium(O) ((Pd2dba)3) in an aqueous medium, for 2h at ambient temperature. 

In vitro selection is a combinatorial approach in which functional molecules are selected from large libraries of randomized RNAs or DNAs by selection techniques that are suitable for the enrichment of a particular property such as the binding to a target molecule or a particular catalytic activity (Fig. 5). 

Selective capturing of the active nucleic acid sequences is a crucial factor in determining the success of an RNA catalyst in vitro selection. A convenient technique for partitioning active from inactive RNA sequences involves capture of biotin by streptaviclin. Biotin is a widely used affinity tag because of its strong binding (K 10-15) to streptavidin. Potential substrates are typically synthesized with... 

On the basis of these considerations, this technique has become a widely used selection method to identify new binding molecules [12-17], mimotopes [18-23], or to characterize protein ligand and/or protein protein interactions [24-37]. Moreover, attempts to optimize the selection process have resulted in the setting up of new biopanning procedures, which have been performed not only on purified targets [24-37], cells [38, 39], and tissues [40] (in vitro selection), but also on living animals [41] (in vivo selection). 

This technique has been mainly used to purge bone marrow from tumor cells in case of autologous transplantation (Nimgaonkar et al., 1996 Duerst et al., 1991), but it can be nicely applied also to in vitro selection of tumor cell phenotypic variants (Starkey et al., 1982 Tsuruoka et al., 1993). 

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