Practical synthesis of cytidine-5-carboxamide-modified nucleotide reagents.

Chemically-modified derivatives of cytidine, bearing a 5-(N-substituted-carboxamide) functional group, are new reagents for use in aptamer discovery via the SELEX process (Systematic Evolution of Ligands by EXponential enrichment). Herein, we disclose a practical synthesis of 5-(N-benzylcarboxamide)-2'-deoxycytidine, and the corresponding 5-(N-1-naphthylmethylcarboxamide)- and 5-(N-3-phenylpropylcarboxamide)-2'-deoxycytidine analogs, as both the suitably-protected 3'-O-cyanoethylphosphoramidite reagents (CEP; gram scale) and the 5'-O-triphosphate reagents (TPP; milligram-scale). The key step in the syntheses is a mild, palladium(0)-catalyzed carboxyamidation of an unprotected 5-iodo-cytidine. Use of the CEP reagents for solid-phase oligonucleotide synthesis was demonstrated and incorporation of the TPP reagents by KOD polymerase in a primer extension assay confirmed the utility of these reagents for SELEX. Finally, the carboxyamidation reaction was also used to prepare the nuclease-resistant sugar-variants: 5-(N-benzylcarboxamide)-2'-O-methyl-cytidine and 5-(N-3-phenylpropylcarboxamide)-2'-deoxy-2'-fluoro-cytidine.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

BACKGROUND: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Expanding the chemistry of DNA for in vitro selection.

Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.