A new method design for conjugation of nucleic acid and protein biomolecules to the DNA-dot

Abstract

ntroduction: DNA-protein and DNA-DNA bioconjugation are great target-tracking paradigm. The innovative ability of DNA molecule as a natural nanomaterial gives special fluorescence and physicochemical properties for employing in molecular and cellular imaging. The effective development of bioconjugation in the targeted imaging is contingent upon the attachment of diagnostics nanoprobe to biomolecular signatures such as antibody and oligonucleotide. Methods: In this Ph.D. thesis, we have carried out the synthesis of bio-dot derived DNA synthesis and its characterization, the covalent linkage of DNA-dot to goat IgG, EGFR antibodies (five different concentrations), and antimiR-21, validation of DNAdot@antibody and DNA-dot@antimiR conjugations, and fluorescent imaging of EGFR targeted lung cancer cell in the EGFR positive and negative cell lines. Results: Following our results, the average size of DNA-dot was 4.5-5 nm which be conjugated to amine-rich antibodies and NH2 modified anti-miR-21 with PO4-1 on the DNA-dot surface. DNA-dots linkage to antibody and antimiR occur by P-N bonds. DNA-dots conjugation to antimiR was verified by FTIR and gel-electrophoresis. DNAdots conjugation to goat IgG was confirmed for the feasibility of fluorescent detection via indirect Dot-blot assay and SDS-PAGE. Regarding the usability of DNAdot@antibody constructs, DNA-dot@EGFR conjugates could recognize the lung cancer cell line with a sensitivity of 100% and specificity about 84-92% related to 0.0025 to 0.04 g 100µL-1 DNA-dot concentration. Discussion: Our demonstration could pave the way for practical applications. DNAdot@antibody and DNA-dot@antimiR conjugates are obtained potentially through a non-toxic, rapid, and efficient approach to cellular and molecular tracking.