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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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In vitro selection using modified or unnatural nucleotides.

Gwendolyn M Stovall¹, Robert S Bedenbaugh, Shruti Singh

¹The University of Texas at Austin, Austin, Texas; Altermune Technologies LLC, Austin, Texas.

Current Protocols in Nucleic Acid Chemistry

|January 22, 2015

Summary

Modified nucleotides enhance in vitro selection by increasing nucleic acid stability and library diversity. This guide details protocols for optimizing their use in RNA and DNA selections.

Keywords:

aptamer deoxyribozymes in vitro selection modified nucleotides ribozymes unnatural nucleotides

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Area of Science:

Molecular Biology
Biochemistry
Synthetic Biology

Background:

Modified nucleotides offer advantages like enhanced stability and expanded functionality in nucleic acid applications.
In vitro selection (SELEX) is a powerful method for isolating functional nucleic acids.

Purpose of the Study:

To provide comprehensive protocols for the use of modified nucleotides in in vitro selection.
To guide researchers in optimizing transcription, reverse transcription, and replication steps with modified nucleotides.

Main Methods:

Detailed protocols for evaluating and optimizing transcription reactions with modified nucleotides.
Methods for confirming modified nucleotide incorporation and assessing their fidelity during replication.
Protocols for evaluating modified nucleotide transcripts in reverse transcription and comparing different selection conditions.

Main Results:

Established methods for successful incorporation and evaluation of modified nucleotides in SELEX.
Demonstrated protocols for optimizing key steps in the selection process.
Provided a framework for comparing modified nucleotide pools and selection parameters.

Conclusions:

Modified nucleotides significantly improve the outcomes of in vitro selection processes.
The provided protocols enable robust and efficient selection of modified nucleic acids.
This unit serves as a valuable resource for researchers utilizing modified nucleotides in SELEX.