In vitro selection and RT-PCR
To evaluate the performance of our mRNA and cDNA display method, we
performed a binding assay against anti-FLAG M2 antibody magnetic beads
under neutral pH condition as described in the Materials and Methods
section. For both methods, FLAG-random and 10aa-random conjugate samples
were mixed to a 1:10,000 molar ratio. After the binding step and
repetitive wash step, beads were recovered and directly subjected to
RT-PCR. To avoid the PCR bias via over amplification, we checked the
amplified product every 5 cycles (0 to 35 cycles) to determine the
appropriate cycle for double-strand DNA library synthesis,
(Supplementary Figure 3). Amplified DNA products from both display
methods became visual at the 15th cycle, and were saturated after 20
cycles. As the selection round progressed, band intensity of the 15th
cycle became stronger, implying the quantity of bead-bound conjugate
products had increased over the course of selection. We also observed a
non-specific band appearing around 200 bp, however this band remained
weak throughout multiple rounds, thus we proceeded with the RT-PCR
reaction. PCR was carried out for 20 cycles for the 1st and 2nd round
libraries and 16 cycles for the 3rd round for both display methods.