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 ma- trices, they do not function effectively in complex biological samples due to ubiquitous RN- ases 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-cleav- ing 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 bio- logical 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.