Abstract 2583: DNA-linked gold nanoprobe assay: An efficient technology for direct and high-sensitive quantification of serum microRNAs in prostate cancer

Background: Serum-based biomarkers in conjunction with advanced biosensing techniques for early detection or screening of indolent vs. aggressive prostate cancer (PCa) can improve patient care and outcmomes. Recent discoveries have identified miRNAs (miRs) as promising biomarkers and opened potential avenues for earliest diagnosis and examining aggressiveness of PCa. However, quantitation of ultra-low levels of miRs in sera or tissue of patient remains challenging with traditional techniques. The present study describes the development and application of the DNA-linked gold nanoprobe (DNA-AuNPr)-based fluorescence assay for quantification of target miRs in serum with high sensitivity. Methodology: Thorough meta-analysis followed by global in-silico analyses were performed to evaluate the differential expression of miRs among PCa studies. Further, the identified miRs were validated using TaqMan assay in human and mouse PCa serum. Thereafter, we synthesized DNA-AuNPr specific to target miRs, by conjugating FAM-tagged DNA-probes to the PEG-coated gold nanoparticle (AuNP) via cross-linker chemistry and fully characterized with UV, DLS and TEM. Sensitivity and target specificity of nanoprobe-based assay were determined using the Synergy-H1 multimode reader. As a proof of principle, a real-time evolution of fluorescence signal upon enzymatic digestion of DNA-probe in DNA-RNA heteroduplexes was tested. Finally, we utilized human (PCa n=44, control n=43) and Pten conditional knockout mouse (PCa n=8, control n=8) sera for our training and blank validation sets to determine the diagnostic significance of miRs in PCa. Results: TaqMan assays detected a significantly higher level of both miR-21 and miR-375 in the PCa compared to normal control. Analyses of DNA-AuNPr shown uniform-sized, highly stable AuNP core tagged with multiple target-specific DNA-probes (~150±21 per NP). Additional assay validation indicated an excellent sequence specificity of DNA-AuNPr in discriminating the single-base mutation (mismatch) in miRs sequence. The uniqueness of proposed assay was its capability to quantitate miR targets in the range 50 pM to 10 nM with a lower detection limit of ~5 pM (0.3 fmol) in just ~4 h of total assay time and using ~10 μL of total RNA isolated from serum. Finally, the assay performance was validated in serum for both miR-21 and miR-375. The two miRs efficiently differentiated PCa from the healthy control in training sets (AUC=0.94) as well as in independent validation sets (AUC=0.90), which reconfirmed their diagnostic potential. Conclusion: We developed a direct (without converting target miR into cDNA), highly sensitive and technologically efficient miR-quantitation strategy for diagnosis of PCa. Citation Format: Prakash Kshirsagar, Satyanarayana Rachagani, Parthasarathy Seshacharyulu, Sakthivel Muniyan, Sunandini Sharma, Ram Mahato, Surinder K. Batra, Maneesh Jain. DNA-linked gold nanoprobe assay: An efficient technology for direct and high-sensitive quantification of serum microRNAs in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2583.