Magnetic resonance and molecular beacon hybridization probes for imaging KRAS mRNA
Authors: Nariman Amirkhanov, Ivan Dimitrov, Kaijun Zhang, Mohan Aruva, John Lackey, Song Lai, Mathew Thakur, Eric Wickstrom
Journal: Clinical Cancer Research (2007)

A21  Magnetic resonance imaging (MRI) or near infrared fluorescence (NIRF) imaging of gene expression non-invasively with high sensitivity and specificity might enable genetic profiling of malignant cells at an early stage. We hypothesized that chelating Gd(III) to 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would enable MRI quantitation of pathogenic mRNAs in the cells of living subjects. Depending on the strong, specific hybridization of PNA, as well as its resistance to nucleases and proteases, we synthesized a 12-base complementary PNA to hybridize with G12D mutant KRAS mRNA in human pancreas cancer cells. Cellular uptake of PNA depended upon receptor-mediated endocytosis by overexpressed IGF1 receptor, guided by the binding of a C-terminal d(Cys-Ser-Lys-Cys) disulfide-bridged peptide analog of insulin-like growth factor 1 (IGF1). A comparable 64Cu hybridization probe has demonstrated sequence-specific PET imaging of G12D mutant KRAS mRNA in human pancreas cancer xenografts [Chakrabarti, et al. (2007) Cancer Biology & Therapy6(6): in press].  To increase the Gd(III) shift intensity of MRI hybridization probes, we extended a novel DO3An-poly(diamidopropanoate) (PDAPm) dendrimer from the N-terminus of KRAS PNA by solid phase synthesis. We investigated whether MRI contrast in phantom solutions was increased by accumulating multiple Gd(III) with each PNA hybridization probe, finding increased T1 relaxivity comparable to that of PAMAM dendrimers. Intravenous administration of ([111In]DO3A)8-PDAP3-KRAS PNA-d(Cys-Ser-Lys-Cys) into immunocompromised mice bearing human AsPC1 pancreas cancer xenografts, followed by scintigraphic imaging, yielded evidence of tumor accumulation. We then investigated whether a (Gd)8-PDAP1-KRAS PNA-d(Cys-Ser-Lys-Cys) provided detectable MRI shift intensity in AsPC1 pancreas cancer xenografts. Preferential contrast enhancement of in the active core of the tumor vs. contralateral normal muscle was observed at 24 h and 48 h after administration. Sequence controls in the IGF1 analog and in the PNA are necessary in order to determine receptor and mRNA specificity. Higher generations of PDAP dendrimers with 16 or more Gd-DO3A residues attached to KRAS PNA-d(Cys-Ser-Lys-Cys) hybridization probes might provide greater contrast for more sensitive detection.  We then considered fluorescence as an alternative to magnetic resonance. We designed a stemless molecular beacon with a G12D mutant KRAS peptide nucleic acid (PNA) hybridization sequence coupled to the disulfide-bridged d(Cys-Ser-Lys-Cys) IGF1 analog to mediate endocytosis by overexpressed IGF1 receptors. Near infrared 664 (NIR664) was used as a fluorophore, at the C-terminus of the PNA, with black hole quencher 2 (BHQ2) at the N-terminus. In principle, molecular beacon hybridization with G12D mutant KRAS mRNA should free the fluorophore from the quencher, generating a fluorescent signal only in the presence of the target RNA. We have observed unquenching of NIR664 in the presence of a G12D mutant KRAS RNA 20-mer at 37°C in PBS. Both fluorescence and absorbance melting curves at 1 µM of each strand displayed Tm of 82°C, comparable to Tm of our PNA SPECT and PET probes. Cellular and xenograft NIRF imaging with specific sequences and mismatch controls remain to be done. Supported by NCI CO27175 and NCI CA105008.