Cycling probe technology with RNase H attached to an oligonucleotide

Area of Science:

• Molecular Biology
• Biotechnology
• Enzyme Engineering

Background:

• Ribonuclease H (RNase H) is crucial for nucleic acid manipulation.
• Streptavidin offers robust biotin-binding capabilities.
• Combining these functionalities could enhance enzymatic assays.

Purpose of the Study:

• To construct and characterize a streptavidin-RNase H fusion protein.
• To evaluate the fusion enzyme's performance in cycling probe technology (CPT).
• To determine if streptavidin binding enhances RNase H activity in CPT.

Main Methods:

• Cloning of Thermus thermophilus RNase H into a streptavidin expression vector.
• Expression and purification of the fusion protein in Escherichia coli.
• Biochemical characterization, including molecular weight, subunit analysis, and thermal stability.
• Activity assays using CPT with varying ratios of biotinylated probes and enzyme concentrations.
• Comparison of fusion enzyme with native RNase H activity.

Main Results:

• A functional streptavidin-RNase H fusion protein (128 kDa, tetrameric) was successfully produced and purified.
• The fusion enzyme exhibited both biotin-binding and RNase H catalytic activity, remaining stable at 65°C.
• At a 1:1 probe:enzyme ratio, the fusion enzyme was active in CPT, while native RNase H was not.
• Enhanced CPT efficiency was observed with the fusion enzyme using biotinylated probes, attributed to enhanced binding.

Conclusions:

• The streptavidin-RNase H fusion enzyme is a stable, active bifunctional protein.
• Fusion to streptavidin significantly enhances RNase H activity in CPT, especially at low probe concentrations.
• This engineered enzyme offers improved performance for nucleic acid detection assays utilizing CPT.