Cycle 1:

Design:

• Initial mining and alignment software for DNAzymes
• Expanding DNAzyme usage beyond the primary focus, in literature, of logical processing capabilities.

Build:

• Built the Strucgen module in PrAtZy software to generate a variety of DNAzymes by mutating sequences and selecting the ones with similar secondary structures

Test:

• Characterization of Strucgen module

Learn:

• Additional validation stem is needed to ensure a high level of accuracy in the prediction
• Need to estimate the energy values of of the new structure to ensure the stability of the sequence at a temperature of interest.

Cycle 2:

Design:

• Identified a problem of multiple bands of PCR gel and poor detection of ssDNA due to multiple freeze-thaw cycles and contamination from the surroundings

Build:

• Development of a different protocol for easy and effective storage and transport
• Generating a 300bp dsDNA template with the DNAzyme at the terminal to maintain stock using normal PCR and extracting DNAzyme using asymmetric PCR

Test:

• Newly built protocols were implemented and tested to see if the multiple bands were avoided and if the detection was robust while surviving the freeze-thaw cycles and contamination

Learn:

• Efficient to store DNAzyme as a double-stranded template
• Production of DNAzyme on a requirement basis

Cycle 3:

Design:

• Increase the RecLoop-RecSeq interaction which was extremely low due to the diluted solution
• Deployment of DNAzyme in such a way the occlusion of one or more sites is avoided

Build:

• Immobilize the DNAzyme on nitrocellulose paper to enable easy visualization, less but concentrated DNAzyme and direct exposure to RecSeq-containing solution

Test:

• Verification of the result to avoid occlusion problem
• Market analysis for cost efficiency of nitrocellulose paper-based test vs existing kits

Learn:

• Enables efficient ReqLoop-ReqSeq interactions
• Maintains substrate fluorescence
• About 20-30% cheaper to develop compared to existing methods