After individual testings of signal amplification systems, we conducted experiments on TO-DNA and CRISPR-Cas12a.
First, we conducted signal input testing for the two systems. The aim of this testing is to determine the lowest concentration of key strand/ssDNA required to trigger reaction.
Fig 2.7.1 TO-DNA signal input testing with different concentrations of key strand 1.
Fig 2.7.2 CRISPR-Cas12a signal input testing with different concentrations of ssDNA-15nt.
Both TO-DNA and CRISPR-Cas12a are successful in their individual
testing. The lower limit of key strand 1 is 1 nM, and the limit of
ssDNA-15nt is 5 nM.
Simulated Combination Testing:
First, we conducted signal input testing for the two systems. The aim of this testing is to determine whether the two systems are suitable to bind to DNAzyme.
Fig 2.7.3 TO-DNA simulated combination testing with complementary sequences.
Fig 2.7.4 CRISPR-Cas12a simulated combination testing with dsDNA.
From TO-DNA testing, we discovered that key strand sequence is too long to be paired with its complementary sequence. The difference in length results in early reaction, since the extended part of key strand will bind to TO-DNA before DNAzyme cleaves its substrate. Therefore, fluorescence will be emitted despite the absence of heavy metal ions.
From CRISPR-Cas12a testing, we determined that the Cas12a reaction will only be triggered by single strand sequence. Thus, we selected CRISPR-Cas12a as a prospective system for combination with DNAzymes.