Background: There is an urgent need for a novel, non-invasive and serum-based biomarker in conjunction with a highly sensitive technique to differentiate indolent vs. aggressive prostate cancer (PCa). Emerging evidence suggests that several micro RNAs (miRs) are differentially expressed in tumor tissue and sera of PCa patients and can potentially serve as biomarkers for disease progression and aggressiveness. However, quantification of ultra-low levels of serum miRNAs remains difficult with conventional quantification methods. Hence, the present study was aimed at developing, optimizing and testing a novel fluorescence-based DNA-gold nanoprobe (DNA-AuNPr) ‘OFF-to-ON’ assay for direct, highly sensitive and precise quantification of miRNAs in serum samples.

Methodology: To identify miRs associated with disease progression, TaqMan assays were performed to analyze the differential expression of miRs in PCa tissues excised from PTEN conditional knockout mice and in human PCa patient serum samples along with their respective controls. We synthesized a DNA-AuNPr specific to miR-21 by conjugating FAM-tagged probe DNA on PEGylated Au nanoparticles (AuNPs). The nanoprobe was characterized using UV, DLS, TEM, zeta potential, ICP-MS and gel electrophoretic analyses. Sensitivity and specificity of the nanoprobe were determined using Biotek Synergy H1 multimode reader. As a proof-of-concept, a kinetic evolution of fluorescence via DSN-catalyzed digestion of probe DNA in heteroduplexes was tested in the PCa serum/tissue samples.

Results: TaqMan assays revealed significant upregulation of miR-21 in both mouse PCa tissue and human PCa patient serum samples. Detailed analyses of DNA-AuNPr displayed uniform spherical shape, 40 nm core size (ϕ), ζ-potential −35±3 mV, and 150±21 coupled probe DNAs/AuNP. Efficient quenching of tagged-fluorophore and clearly reduced electrophoretic mobility of DNA−AuNPr compare to bare AuNP validated successful conjugation. Our initial analyses determined 75 pM of probe DNA (≈11 nM AuNPs) as optimal concentrations for miRNA quantification. Our real-time fluorescence assay allowed absolute quantification of as little as 5 pM of synthetic (syn) miR-21 with a very good linear response curve (R2 = 0.99) of fold change vs. syn-miR-21 concentrations. Assay results also demonstrated excellent sequence specificity that was verified using seed region mutated syn-miR-21 as well as miR-1 mimic, resulting in the abolishment of fluorescence compare to miR-21. Finally, DSN-mediated kinetic release of FAM gave significantly higher signal output from PCa patient samples compared to normal controls.

Conclusion: Our efforts successfully established a DNA-AuNPr-based kinetic assay for direct (without converting miR to cDNA), highly sensitive, and specific quantification of miRs in PCa patient samples.

Citation Format: Prakash Kshirsagar, Satyanarayana Rachagani, Parthasarathy Seshacharyulu, Sakthivel Muniyan, Ram Mahato, Surinder K. Batra, Maneesh Jain. Advanced DNA-gold nanoprobe-based direct and sensitive quantification of novel microRNAs in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-331. doi:10.1158/1538-7445.AM2017-LB-331