SDS-PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs.

SDS-PAGE is considered to be a universal method for size-based separation and analysis of proteins. In this study, we applied the principle of SDS-PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, - phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.

Imaging human pancreatic cancer xenografts by targeting mutant KRAS2 mRNA with [(111)In]DOTA(n)-poly(diamidopropanoyl)(m)-KRAS2 PNA-D(Cys-Ser-Lys-Cys) nanoparticles.

95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, (64)Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([(111)In]DOTA-AEEA)(n)-PDAP(m)-AEEA(2)-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [(111)In](n)-PDAP(m)-KRAS2 G12D probes increased from 3.1 +/- 0.2 at n = 2, m = 1, to 4.1 +/- 0.3 at n = 8, m = 3, to 6.2 +/- 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [(111)In](n)-PDAP(m)-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 +/- 0.6 at n = 2, m = 1, 2.6 +/- 0.9 at n = 8, m = 3, and 3.7 +/- 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [(111)In]DOTA control results. Simultaneous administration of nonradioactive Gd(n)-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [(111)In](8)KRAS2 G12 V probes 3-6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of (111)In uptake increased from 3.1 +/- 0.5 to 6.5 +/- 1.0, and the kidney/tumor ratio of (111)In uptake decreased by more than 5-fold from 174.8 +/- 17.5 to 30.8 +/- 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd(n)-KRAS2 G12 V probes, enhanced tumor uptake of [(111)In](n)KRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd(n)-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.

Design of (Gd-DO3A)n-polydiamidopropanoyl-peptide nucleic acid-D(Cys-Ser-Lys-Cys) magnetic resonance contrast agents.

We hypothesized that chelating Gd(III) to 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would provide a magnetic resonance genetic imaging agent capable of hybridization to a specific mRNA. Because of the low sensitivity of Gd(III) as an magnetic resonance imaging (MRI) contrast agent, a single Gd-DO3A complex per PNA hybridization agent could not provide enough contrast for detection of cancer gene mRNAs, even at thousands of mRNA copies per cell. To increase the Gd(III) shift intensity of MRI genetic imaging agents, we extended a novel DO3An-polydiamidopropanoyl (PDAPm) dendrimer, up to n = 16, from the N-terminus of KRAS PNA hybridization agents by solid phase synthesis. A C-terminal D(Cys-Ser-Lys-Cys) cyclized peptide analog of insulin-like growth factor 1 (IGF1) was included to enable receptor-mediated cellular uptake. Molecular dynamic simulation of the (Gd-DO3A-AEEA)16-PDAP4-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) genetic imaging nanoparticles in explicit water yielded a pair correlation function similar to that of PAMAM dendrimers, and a predicted structure in which the PDAP dendron did not sequester the PNA. Thermal melting measurements indicated that the size of the PDAP dendron included in the (DO3A-AEEA)n-PDAPm-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) probes (up to 16 Gd(III) cations per PNA) did not depress the melting temperatures (Tm) of the complementary PNA/RNA hybrid duplexes. The Gd(III) dendrimer PNA genetic imaging agents in phantom solutions displayed significantly greater T1 relaxivity per probe (r1 = 30.64 +/- 2.68 mM(-1) s(-1) for n = 2, r1 = 153.84 +/- 11.28 mM(-1) s(-1) for n = 8) than Gd-DTPA (r1 = 10.35 +/- 0.37 mM(-1) s(-1)), but less than that of (Gd-DO3A)32-PAMAM dendrimer (r1 = 771.84 +/- 20.48 mM(-1) s(-1)) (P < 0.05). Higher generations of PDAP dendrimers with 32 or more Gd-DO3A residues attached to PNA-D(Cys-Ser-Lys-Cys) genetic imaging agents might provide greater contrast for more sensitive detection.

External imaging of CCND1, MYC, and KRAS oncogene mRNAs with tumor-targeted radionuclide-PNA-peptide chimeras.

In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one-quarter of cancers in women aged 40-49 years, and one-fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5-year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a Tc-99m-chelator peptide on the N-terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc-99m-chelator PNA-peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu-64-chelator PNA-peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4-24 h after tail-vein administration of MYC or CCND1 Tc-99m-chelator PNA-peptides, but not after administration of mismatch controls. Similarly, human Panc-1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail-vein administration of a KRAS Tc-99m-chelator PNA-peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail-vein adminstration of a KRAS Cu-64-chelator PNA-peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might in the future provide additional genetic characterization of pre-invasive and invasive breast cancers.

Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability, and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases.

Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show approximately 3 degrees C drop per modification in melting temperature (Tm) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the R Nase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.