Abstract
Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.
Highlights
Catalytic DNAs or DNAzymes are single-stranded DNA molecules that can catalyse a chemical reaction
A pool of 1014 molecules, denoted L1 and made of 60-nt random domain flanked by two 20-nt primer-binding sites, was used for the in vitro selection experiment
The cleavage product was purified by denaturing polyacrylamide gel electrophoresis (dPAGE) and subjected to two polymerase chain reactions (PCR1 and PCR2)
Summary
Catalytic DNAs or DNAzymes are single-stranded DNA molecules that can catalyse a chemical reaction. They are isolated from a random-sequence DNA library using an established technique known as “in vitro selection” [1,2]. The transesterification reaction involving RNA is well known in biology as many protein enzymes and ribozymes rely on this chemistry to cleave RNA [11,12,13,14]. This creates a unique opportunity to compare catalytic abilities of DNA, RNA and protein for the same reaction. The PLOS ONE | DOI:10.1371/journal.pone.0126402 May 6, 2015
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