Fluorescence detection of KRAS2 mRNA hybridization in lung cancer cells with PNA-peptides containing an internal thiazole orange.

We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5' end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5' terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA.

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA.

INTRODUCTION: Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD: WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS: SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION: Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.

Consistent surgeon evaluations of three-dimensional rendering of PET/CT scans of the abdomen of a patient with a ductal pancreatic mass.

Two-dimensional (2D) positron emission tomography (PET) and computed tomography (CT) are used for diagnosis and evaluation of cancer patients, requiring surgeons to look through multiple planar images to comprehend the tumor and surrounding tissues. We hypothesized that experienced surgeons would consistently evaluate three-dimensional (3D) presentation of CT images overlaid with PET images when preparing for a procedure. We recruited six Jefferson surgeons to evaluate the accuracy, usefulness, and applicability of 3D renderings of the organs surrounding a malignant pancreas prior to surgery. PET/CT and contrast-enhanced CT abdominal scans of a patient with a ductal pancreatic mass were segmented into 3D surface renderings, followed by co-registration. Version A used only the PET/CT image, while version B used the contrast-enhanced CT scans co-registered with the PET images. The six surgeons answered 15 questions covering a) the ease of use and accuracy of models, b) how these models, with/without PET, changed their understanding of the tumor, and c) what are the best applications of the 3D visualization, on a scale of 1 to 5. The six evaluations revealed a statistically significant improvement from version A (score 3.6±0.5) to version B (score 4.4±0.4). A paired-samples t-test yielded t(14) = -8.964, p<0.001. Across the surgeon cohort, contrast-enhanced CT fused with PET provided a more lifelike presentation than standard CT, increasing the usefulness of the presentation. The experienced surgeons consistently reported positive reactions to 3D surface renderings of fused PET and contrast-enhanced CT scans of a pancreatic cancer and surrounding organs. Thus, the 3D presentation could be a useful preparative tool for surgeons prior to making the first incision. This result supports proceeding to a larger surgeon cohort, viewing prospective 3D images from multiple types of cancer.

Effects of hypoxanthine substitution in peptide nucleic acids targeting KRAS2 oncogenic mRNA molecules: theory and experiment.

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.

Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.