In Vitro Selection Using Modified or Unnatural Nucleotides

Abstract

Incorporation of modified nucleotides into in vitro RNA or DNA selections offer many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.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 NIH Public Access Author ManuscriptCurr Protoc Nucleic Acid Chem. Author manuscript; available in PMC 2015 March 26. Published in final edited form as:Curr Protoc Nucleic Acid Chem. ; 56: 9.6.1-9.6.33. doi:10.1002/0471142700.nc0906s56. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript buffers should be RNase free or treated with diethylpyrocarbonate (DEPC; APPENDIX 2A). Use sterile, disposable plasticware where possible. STRATEGIC PLANNING Advantages of Using Modified Nucleotides in In Vitro S...

Non-natural nucleic acids for synthetic biology

“…A wide variety of sidechains may be attached to this position, ranging from simple hydrophobic groups to extended sidechains terminated by polar groups. These modified nucleotides are tolerated by a variety of T7 RNA polymerases [35]. …”

Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker

“…Aptamers can be chemically synthesized quickly and relatively cheaply with minimal inter-batch variability, in contrast to the expensive and work-intensive biological systems needed to produce monoclonal antibodies. A variety of nucleotide modifications have been described to increase aptamer stability and affinity (14)(15)(16). In particular, SomaLogic Inc. has demonstrated the potential for simultaneous analysis of hundreds of blood proteins using multiplexed modified aptamer assays (15,17).…”

Trends in aptamer selection methods and applications

“…Incorporation of an unnatural nucleotide with the hydrophobic base 7-(2-thienyl) imidazole [4, 5-b] pyridine into the random library delivered a VEGF-165 specific aptamer with a K D of 0.65 pM. Further aspects of the employment of peptide/drugs like moieties or unnatural bases in the random libraries were discussed in detail by Knudsen et al 22 and Rohloff et al, 23 respectively. 2' chemical modification mainly covers 2′-fluoro-or 2′-amino-substituted pyrimidines or 2′-O-methyl nucleotides that enhance the nuclease resistance of the resulting aptamers.…”