In vitro selection using modified or unnatural nucleotides.

In vitro selection is the process by which a pool of nucleic acids is enriched via iterative selection and amplification for those species that are capable of performing a particular task. Nucleic acids have been selected that bind to particular targets (aptamers), catalyze reactions (ribozymes or deoxyribozymes), or act as molecular switches (aptazymes). Similarly, nucleic acids have been found in nature that control gene expression upon binding an analyte (riboswitches). Instructions for carrying out in vitro selection experiments have been detailed elsewhere in this chapter (i.e. UNITS 9.3, 9.4, and 9.5). This unit augments these units by describing how modified nucleotides can potentially be incorporated into a selection. It is strongly recommended that the researcher be conversant with a “normal” in vitro selection experiment prior to attempting selections with modified nucleotides. A normal in vitro selection experiment is already fraught with problems and pitfalls, and the addition of modified nucleotides adds an extra level of difficulty. For simplicity, this unit focuses on in vitro selection using RNA pools; however, similar procedures can be used for DNA pools. CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer. NOTE: Experiments involving RNA require careful precautions to prevent contamination and degradation by RNases (see APPENDIX 2A). The water used to make all solutions and buffers should be RNase free or treated with diethylpyrocarbonate (DEPC; APPENDIX 2A). Use sterile, disposable plasticware where possible.