An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling
Shirley H J Mei1, Zhongjie Liu, John D Brennan
1Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada.
Journal of the American Chemical Society
|January 9, 2003
Summary
Researchers developed a novel DNA enzyme (DEC22-18) that combines RNA cleavage with real-time fluorescence signaling. A derived enzyme (DET22-18) exhibits high catalytic efficiency, enabling new biosensor applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Catalysis
Background:
- DNA enzymes, or deoxyribozymes, are catalytic single-stranded DNA molecules selected via in vitro selection.
- These molecules offer potential as versatile molecular tools in various applications.
Purpose of the Study:
- To create and characterize an autocatalytic DNA enzyme (DEC22-18) that links chemical catalysis with real-time fluorescence signaling.
- To develop a trans-acting DNA enzyme (DET22-18) from DEC22-18 with enhanced enzymatic properties.
Main Methods:
- In vitro selection was used to isolate DNA sequences with catalytic activity.
- Characterization involved assessing the catalytic rate (kcat) and substrate cleavage efficiency.
- A chimeric RNA/DNA substrate with a fluorophore-quencher pair was designed to synchronize cleavage with signaling.
Main Results:
- An autocatalytic DNA enzyme, DEC22-18, was created, integrating catalysis and fluorescence signaling.
- A trans-acting DNA enzyme, DET22-18, was developed with a kcat of approximately 7 min⁻¹, one of the highest reported for DNA enzymes.
- DET22-18 efficiently cleaves a specific RNA linkage in a substrate, triggering fluorescence signal changes.
Conclusions:
- The developed DNA enzyme DET22-18 demonstrates efficient catalytic activity and real-time signaling capabilities.
- Its stem-loop structure allows conjugation with DNA aptamers, paving the way for allosteric deoxyribozyme biosensors.
- These findings highlight the potential of engineered DNA enzymes as sophisticated molecular tools for sensing and diagnostics.
Related Concept Videos
00:43Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
01:53Bacterial Transcription
RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
01:29Coordination of Gene Expression Processes in Bacteria
The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...