An Efficient Catalytic DNA that Cleaves L-RNA.

PloS one (2015-05-07)
Kha Tram, Jiaji Xia, Rachel Gysbers, Yingfu Li
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
Triethylamine, ≥99.5%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Triethylamine, ≥99%
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Piperidine, ReagentPlus®, 99%
Sigma-Aldrich
Triethylamine, puriss. p.a., ≥99.5% (GC)
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Dimethyl sulfate, ≥99.5%
Sigma-Aldrich
Triethylamine, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
SAFC
HEPES
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Dimethyl sulfide, ≥99%
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
Dimethyl sulfate, ≥99%
Sigma-Aldrich
Piperidine, ≥99.5%, purified by redistillation
Sigma-Aldrich
Dimethyl sulfide, anhydrous, ≥99.0%
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR