Because there are no known naturally occurring deoxyribozymes, most known deoxyribozyme sequences have been discovered through a high-throughput in vitro selection technique, similar to SELEX.
in vitro selection utilizes a "pool" of a large number of random DNA sequences (typically 10–10 unique strands) that can be screened for a specific catalytic activity. The pool is synthesized through solid phase synthesis such that each strand has two constant regions (primer binding sites for PCR amplification) flanking a random region of a certain length, typically 25–50 bases long. Thus the total number of unique strands, called the sequence space, is 4 where N denotes the number of bases in the random region. Because 4 ≈ 10, there is no practical reason to choose random regions of less than 25 bases in length, while going above this number of bases means that the total sequence space cannot be surveyed. However, since there are likely many potential candidates for a given catalytic reaction within the sequence space, random regions of 50 and even higher have successfully yielded catalytic deoxyribozymes.
The pool is first subjected to a selection step, during which the catalytic strands are separated from the non-catalytic strands. The exact separation method will depend on the reaction being catalyzed. As an example, the separation step for ribonucleotide cleavage often utilizes affinity chromatography, in which a biological tag attached to each DNA strand is removed from any catalytically active strands via cleavage of a ribonucleotide base. This allows the catalytic strands to be separated by a column that specifically binds the tag, since the non-active strands will remain bound to the column while the active strands (which no longer possess the tag) flow through. A common set-up for this is a biotin tag with a streptavidin affinity column. Gel electrophoresis based separation can also be used in which the change in molecular weight of strands upon the cleavage reaction is enough to cause a shift in the location of the reactive strands on the gel. After the selection step, the reactive pool is amplified via polymerase chain reaction (PCR) to regenerate and amplify the reactive strands, and the process is repeated until a pool of sufficient reactivity is obtained. Multiple rounds of selection are required because some non-catalytic strands will inevitably make it through any single selection step. Usually 4–10 rounds are required for unambiguous catalytic activity, though more rounds are often necessary for more stringent catalytic conditions. After a sufficient number of rounds, the final pool is sequenced and the individual strands are tested for their catalytic activity. The dynamics of the pool can be described through mathematical modeling, which shows how oligonucleotides undergo competitive binding with the targets and how the evolutionary outcome can be improved through fine tuning of parameters.
Deoxyribozymes obtained through in vitro selection will be optimized for the conditions during the selection, such as salt concentration, pH, and the presence of cofactors. Because of this, catalytic activity only in the presence of specific cofactors or other conditions can be achieved using positive selection steps, as well as negative selection steps against other undesired conditions.