Why use DNA over RNA?

Synthetic nucleic acid enzymes can be composed of RNA (ribozymes) or DNA (DNAzymes). The choice of using either biopolymer is mostly decided by personal preference. However, it is worth considering that the selection design will be different. For the selection of DNAzymes, the randomized single-stranded (ss) DNA library is obtained through solid-phase synthesis. This library is then subjected to a function-based selection step in which the active and non-active species are separated from one another. The functionally desired sequences are then amplified using polymerase chain reaction (PCR). Lastly, the dsDNA need to be denatured and the strand that contains the catalytically active domain retrieved for the next cycle of selection (Figure 1A).

The selection of ribozymes begins with the generation of a dsDNA library that was synthesized from an ssDNA pool using PCR. This preparation step is required for in vitro transcription that will yield the ssRNA library used for selection. Similar to the selection of DNAzymes, a function-based step is applied to isolate sequences that are capable of catalyzing the reaction of interest. These sequences are then reverse-transcribed into an ssDNA pool that will be amplified by PCR. The cycle then begins again except with an enriched library (Figure 1B). These additional preparation steps may not necessarily affect the outcome for isolating a ribozyme, but the process is certainly more laborious and tedious than DNAzyme selection. Furthermore, extra care needs to be taken into consideration when handling RNA. The ubiquitous presence of RNases may contaminate the RNA library and substantially reduce progression

Depending on the application and intended use of the NAE, the choice of using DNA or RNA is an important consideration and there are certain situations in which one may be better suited than the other. For example, there is a growing interest in using DNA over RNA for bioanalytical applications. From the perspective of stability, DNA is chemically more stable than RNA due to the lack of a 2′-OH. The presence of a 2′-OH enables RNA to undergo spontaneous hydrolysis. Consequently, oligonucleotides that are made of RNA will have limited shelf life. In addition, RNA is susceptible to ribonucleases that will readily degrade RNA sequences. Although loss of this 2′-OH has been speculated to restrict the functionality of DNA-based enzymes [24,25], a substantial number of DNAzymes have been isolated that are as effective as their RNA counterparts. After all, DNA and RNA are very similar chemically and both are capable of creating intricate tertiary structures through similar molecular interactions such as hydrogen bonding, charge-charge and π-stacking.