The goal of Aptapaper is to take a protein biomarker found in a patient sample and convert it into a detectable signal. Our design consists of three main steps: proximity dependent ligation, nucleic acid sequence based amplification (NASBA), and cell-free expression. Proximity dependent ligation works by using two single-stranded DNA molecules with aptamer ends. These aptamer ends are single-stranded DNA molecules that are engineered to bind to either side of our target protein. Once the aptamers seek out and bind to our target protein, the single-stranded DNA molecules are brought into very close proximity. A DNA bridge, which is complementary to sequences on both single-stranded DNA molecules, binds to the two strands, holding them in place. T4 DNA ligase then ligates the two long DNA molecules so that they form one continuous DNA strand. This DNA strand encodes the alpha-fragment of the lacZ gene. The alpha fragment is used rather than the full lacZ gene to limit the length of the single stranded DNA probes; extremely long probes have not been tested in proximity dependent ligation reactions to our knowledge. In order to drive transcription of the lacZ alpha-fragment, one of the single-stranded DNA molecules contains a T7 promoter. The DNA is double-stranded in the promoter region, a requirement for T7 polymerase activity.
The lacZ alpha fragment is then transcribed, producing mRNA. This mRNA must be amplified by NASBA to detect lower amounts of protein biomarkers in patient samples. NASBA uses Avian Myeloblastosis Virus Reverse Transcriptase to reverse transcribe DNA from the mRNA template. Note, the primers used for reverse transcription contain T7 promoters so that the final double-stranded product is transcriptionally active. We then must free the DNA from the RNA:DNA hybrids, in order to produce double stranded DNA. To do this, we use RNAse H, which degrades the RNA in the RNA:DNA hybrids. AMV reverse transcriptase is then able to fill in the single-stranded DNA, making it double-stranded. The double-stranded DNA contains a T7 promoter, enabling it to express the lacZ alpha-fragment. It is important to note that this reaction can be performed at a constant temperature near 37oC so it does not require specialized lab equipment.
The alpha fragment that is produced complements with the lacZ delta M15 mutant, which is constitutively expressed in the same reaction mixture on a separate DNA template. When the two fragments come together, they form the functional lacZ enzyme, which is able to break down X-gal, producing a colorimetric output. Our entire system could be freeze dried onto paper, allowing it to be stored for long periods of time at room temperature.