Nucleic Acid Engineering (2017 spring)
Research area
- Nucleic acid bioengineering
- Microarray technology
- Electrochemical diagnostics
- Nanobiotechnology
Current project
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HOME > Research > Research area > Nucleic acid bioengineering

 

 

1. Novel isothermal/quantitative nucleic acid amplification methods for ultra-high sensitive detection of biomolecules

 

The invention of the PCR technique has made a tremendous impact on the areas of biological research and diagnostics since it can be used to generate quantities of nucleic acids needed for detection and measurements. However, the need for a temperature cycling instrument limits applications of PCR in point-of-care testing (POCT) environments.

 

In order to overcome this limitation, we have developed a powerful isothermal amplification method (ICA) as part of a quantitative amplification technology (iTPA) that combines ICA and FRET cycling probe technology (CPT). By creating a rationally designed amplification mechanism, ICA amplifies target DNA highly enough to detect down to 100 copies under isothermal conditions.

 

To achieve the iTPA, dual amplification of both the target and the FRET probe is efficiently utilized to bring about high sensitivity. The observations made in this study clearly demonstrate that even a single copy level of C. trachomatis pathogen can be reliably quantified. Furthermore, combining a target and signaling probe amplification method (iTPA) with a colorimetric detection method (GCA), we have successfully developed a novel and efficient assay for the direct colorimetric diagnosis of C. trachomatis pathogen as low as 100 copies under isothermal conditions.

 

The methods do not require an expensive thermo-cycler or other instrumentation and are remarkably simple and convenient. The proposed approaches are thus thought to be promising for application in portable sensor systems such as micro-fluidic devices or POCT diagnostic kits.

 

 

 

2. Metal ion aptamer-based target detection and logic gate

 

In recent years, an intense interest has grown in the interactions of nucleic acids with metal ions. Examples of such

novel interactions include the specific binding of aptamers with metal ions and selective incorporation of metal ions

as cofactors to promote the catalytic activities of nucleic acid enzymes (deoxyribozymes or ribozymes).

 

In addition, it has been observed that certain metal ions like Hg2+, Ag+, Cu2+, Ni2+, and Co2+ specifically bind to nucleosides or ligandosides to form metal ion-mediated base pairs. We focus on the specific interactions of the mismatched base pairs (thymine-thymine (T-T) or cytosine-cytosine (C-C)) with the respective metal ions (Hg2+ or Ag+) and develop a new strategy in which a polymerase enzyme is controlled to accomplish an unnatural extension reaction even at the mismatched site of a primer with template DNA.

 

The validity of this novel concept was systematically demonstrated by using Hg2+ and Ag+ to intentionally trigger an unusual illusionary polymerase activity at respective T-T and C-C mismatched primers. By utilizing this concept, we have successfully constructed a molecular scale logic gate system that uses Hg2+ or Ag+ as inputs and DNA amplification as an output.

 

To the best of our knowledge, this is the first time that key logic gates, which use PCR amplification as an output and metal ions as triggers, have been described. We believe that this novel concept might serve as tools for the identification of metal ions (Hg2+ or Ag+) and also be extended to develop new single nucleotide polymorphism (SNP) genotyping strategy.

 

                 The significance of this work derives from the following features!!

 

- Being different from previous efforts in which interactions of nucleic acids with metal ions

were studied, the current investigation probed these interactions in combination with polymerase activity.

The work has led to the discovery that the illusionary activity of a polymerase can be intentionally triggered

by using Hg2+ and Ag+ ions via their interaction with the respective mismatched base pairs T-T and C-C.

This phenomenon results in an unusual polymerase amplification reaction.

 

- By utilizing this concept, a novel strategy to construct molecular scale logic gates was

developed by rationally designing primers and selecting the type of DNA polymerase employed.

The novel strategy is both simple and cost effective because it only requires incorporation of a single

mismatched base (T and C) at the 3 end of the primer and the use of metal ions (Hg2+ and Ag+) as inputs.

In contrast, previously reported strategies for the construction of logic gates, which operate by specific

metal ion regulation of the catalytic activity of deoxyribozymes, typically rely on complicated designs for

gate switching and frequently require expensive RNA or chimeric DNA as operational substrates.

These requirements significantly limit the utility of the older strategies.

 

 

 

3. DNAzyme

 

Artificial DNAzymes (also called deoxyribozyme or catalytic DNA) are widely used as signal transduction

components of biosensors because one DNAzyme molecule is able to catalyze many cycles of a specific reaction generating amplified readout signals.

 

Among the DNAzymes acting as amplifying labels, a binary peroxidase DNAzyme attracted special efforts, which is composed of two single-stranded guanine-rich DNA strands self-assembling into G-quadruplex structure with hemin. Due to its ability to catalyze the generation of colorimetric or chemiluminescence signals, the binary peroxidase DNAzyme has been employed in many strategies for the detection of nucleic acids, small molecules, and proteins.

 

Recently, we developed a novel ultrasensitive colorimetric detection method for nucleic acids. This method relies on a unique enzyme-mediated isothermal amplification strategy to realize triplex amplification effects on the readout signals: the target DNA recycling, the amplified generation of active DNAzymeMBs, and the DNAzyme-based signal amplification. This new isothermal colorimetric DNA detection method possesses ultra-high sensitivity with a detection limit down to 10 attomole range which was the lowest detection limit among the previous reports for peroxidase DNAzyme-based detection methods.