Binding of a tetracationic meso-porphyrin to polyadenylic acid: a spectroscopic study

Binding of a tetracationic porphyrin (TMPyP4+) to poly(rA) has been studied in neutral buffered solution of low ionic strength in a wide range of molar phosphate-to-dye ratios (P/D) using absorption spectroscopy, polarized fluorescence and fluorimetric titration. Two competitive binding modes were identified: partial intercalation of porphyrin chromophores between adenine bases prevailing at P/D > 20 and its outside binding to poly(rA) backbone dominating at P/D < 6. Both of them were accompanied by enhancement of the porphyrin emission. Absence of the emission quenching near stoichiometric P/D ratios allowed us to assume that external binding occurs without the self-stacking of the porphyrin chromophores.

A cautionary (spectral) tail: red-shifted fluorescence by DAPI–DAPI interactions

The fluorescent dye DAPI is useful for its association with and consequent amplification of an ∼460 nm emission maximum upon binding to dsDNA. Labelling with higher DAPI concentrations is a technique used to reveal Pi polymers [polyphosphate (polyP)], with a red-shift to ∼520–550 nm fluorescence emission. DAPI–polyP emissions of ∼580 nm are also generated upon 415 nm excitation. Red-shifted DAPI emission has been associated with polyP and RNA and has more recently been reported with polyadenylic acid (polyA), specific inositol phosphates (IPs) and heparin. We find that amorphous calcium phosphate (ACP) also demonstrates red-shifted DAPI emission at high DAPI concentrations. This DAPI spectral shift has been attributed to DAPI–DAPI electrostatic interactions enabled by molecules with high negative charge density that increase the local DAPI concentration and favour DAPI molecular proximity, as observed by increasing the dye/phosphate ratio. Excitation of dry DAPI (∼360 nm) confirmed a red-shifted DAPI emission. Whereas enzymatic approaches to modify substrates can help define the nature of DAPI fluorescence signals, multiple approaches beyond red-shifted DAPI excitation/emission are advised before conclusions are drawn about DAPI substrate identification.

Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer

The aims of the study were:i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures,ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, andiii) to use next generation sequencing (NGS) technology to analyzeKRAS,BRAF, andNRASsomatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated inKRASand one patient with aBRAFmutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.