Robert H. E. Hudson

Fiber optic biosensor for fluorimetric detection of DNA hybridization

Abstract Single stranded deoxyribonucleic acid (ssDNA) thymidylic acid icosanucleotides (dT 20 ) were grown onto optical fibers. The fibers were first derivatized with γ-aminopropyltriethoxysilane (APTEs) onto which a spacer arm of 1,10 decanediol bis-succinate terminated with 5′-O-dimethoxytrityl-2′-deoxythymidine was covalently attached. The synthetic route used to grow the ssDNA was the well established solid-phase phosphoramidite methodology. The covalently immobilized oligormers were able to hybridize with available complementary ssDNA (cDNA) which was introduced into the local environmnet to forum double stranded DNA (dsDNA). This event was detected by the use of the fluorescent DNA stain ethidium bromide (EB). The sampling configuration utilized total internal reflection of optical radiation within the fiber, resulting in an intrinsic mode optical sensor. The non-optimized procedure used standard hybridization assay techniques to provide a detection limit of 86 ng ml −1 cDNA, a sensitivity of 83% fluorescence intensity increase per 100 ng ml −1 of cDNA initially present, with a hybridization analysis time of 46 min. The sensor has been observed to sustain activity after prolonged storage times (3 months) and harsh washing conditions (sonication).

Four‐pool modeling of proton exchange processes in biological systems in the presence of MRI–paramagnetic chemical exchange saturation transfer (PARACEST) agents

Signal loss due to magnetization transfer (MT) from the macromolecular protons of biological tissues is an important consideration for the in vivo detection of paramagnetic chemical exchange saturation transfer (PARACEST) agents. In this study, a four‐pool model is presented that is based on the modified Bloch equations and incorporates terms for the proton exchange processes that occur in biological systems in the presence of MRI‐PARACEST contrast agents. The effect of the exchangeable proton chemical shift and PARACEST agent concentration are modeled in the presence of macromolecule‐derived MT. Experimental validation of the model was performed at 9.4 Tesla using Eu3+‐DOTAM‐glycine (Gly)‐phenylalanine (Phe) in both aqueous solution and samples containing 10% bovine serum albumin (BSA). The model was then used to measure the agent‐bound‐water chemical shift and the transverse relaxation time of macromolecular protons of a sample of Vero (nonhuman primate) cells labeled with Eu3+‐DOTAM‐Gly‐Phe and a phantom containing mouse brain tissue and 7 mM Eu3+‐DOTAM‐Gly‐Phe. In the brain tissue phantom, a chemical shift map with standard deviation (SD) < 0.7 ppm and a T2 map with SD < 0.6 μs were obtained. The results demonstrate the feasibility of in vivo temperature measurement based on the bound‐water chemical shift of Eu3+‐DOTAM‐Gly‐Phe in combination with this four‐pool model despite the inherent MT effect. Magn Reson Med 60:1197–1206, 2008.

A sensitive PARACEST contrast agent for temperature MRI: Eu3+-DOTAM-glycine (Gly)-phenylalanine (Phe)

Tissue temperature is a fundamental physiological parameter that can provide insight into pathological processes. The purpose of this study was to develop and characterize a novel paramagnetic chemical exchange saturation transfer (CEST) agent suitable for in vivo temperature mapping at 9.4T. The CEST properties of the europium (Eu3+) complex of the DOTAM‐Glycine (Gly)‐Phenylalanine (Phe) ligand were studied in vitro at 9.4T as a function of temperature, pH, and agent concentration. The transfer of magnetization (CEST effect) from the bound water to bulk water pools was ∼75% greater for Eu3+‐DOTAM‐Gly‐Phe compared to Eu3+‐DOTAM‐Gly at physiologic temperature (38°C) and pH (7.0 pH units) when using power level sufficiently low for in vivo imaging. Unlike Eu3+‐DOTAM‐Gly, whose CEST effect decreased with increasing temperature in the physiologic range, the CEST effect of Eu3+‐DOTAM‐Gly‐Phe was optimal at body temperature. A strong linear dependence of the chemical shift of the bound water pool on temperature was observed (0.3 ppm/°C), which was insensitive to pH and agent concentration. Temperature maps with SDs < 1°C were acquired at 9.4T in phantoms containing: 1) phantom A, an aqueous solution of 10 mM Eu3+‐DOTAM‐Gly‐Phe; 2) phantom B, 5% bovine serum albumin (BSA) with 15 mM Eu3+‐DOTAM‐Gly‐Phe; and 3) phantom C, mouse brain tissue with 4 mM Eu3+‐DOTAM‐Gly‐Phe. The temperature sensitivity combined with the high CEST effect observed at low concentration using low saturation power (B1) suggests this compound may be a good choice for in vivo temperature mapping at 9.4T. Magn Reson Med 59:374–381, 2008.

A Remarkably Simple Protocol for the N-Formylation of Amino Acid Esters and Primary Amines

A simple, convenient, and wide scope protocol for the N-formylation of amino acid esters and primary amines has been developed utilizing only imidazole in warm DMF.

Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs

Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

Simultaneous in vivo pH and temperature mapping using a PARACEST‐MRI contrast agent

Altered tissue temperature and/or pH is a common feature in pathological conditions, where metabolic demand exceeds oxygen supply such as in tumors and following stroke. Therefore, in vivo tissue temperature and pH may become valuable biomarkers for disease detection and the monitoring of disease progression or treatment response in conditions with altered metabolic demand. In this study, pH is measured using the amide protons of a thulium (Tm3+) complex with a DOTAM‐Glycine‐Lysine (ligand: Tm3+‐DOTAM‐Gly‐Lys). The pH was uniquely determined from the linewidth of the asymmetry curve of the chemical exchange saturation transfer spectrum, independent of contrast agent concentration, or temperature for a given saturation pulse. pH maps with an inter‐pixel standard deviation of less than 0.1 pH units were obtained in 10 mM Tm3+‐DOTAM‐Gly‐Lys solutions with pH ranging from 6.0 to 8.0 pH units at 37°C. Temperature maps were simultaneously obtained using the chemical shift of the chemical exchange saturation transfer peak. Temperature and pH maps are demonstrated in the mouse leg (N = 3), where the mean and standard deviation for pH was 7.2 ± 0.2 pH unit and temperature was 37.4 ± 0.5°C. Magn Reson Med, 70:1016–1025, 2013.

Fluorescence and Hybridization Properties of Peptide Nucleic Acid Containing a Substituted Phenylpyrrolocytosine Designed to Engage Guanine with an Additional H-Bond

A new pyrrolocytosine derivative has been designed to selectively interact with guanine and has been evaluated in peptide nucleic acid where it imparts increased selective binding affinity for complementary oligonucleotides. The modified nucleobase also possesses an exceptionally high fluorescence quantum yield that is responsive to hybridization.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.

A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: design, synthesis and cellular uptake studies.

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimers disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

The use of Sonogashira coupling for the synthesis of modified uracil peptide nucleic acid

Palladium-catalyzed Sonogashira coupling has been shown to be compatible with PNA monomers as illustrated by the reaction of 5-iodouracil peptide nucleic acid monomer (IU-PNA) with several terminal alkynes. These reactions have been performed in the solution phase and with IU-PNA linked to an insoluble polymer support. The results presented herein show that while the isolated yields from the solution phase chemistry are modest (38–53%), the yields of the resin-bound coupling reactions are essentially quantitative, at the monomer level. A selection of alkynes was used to install various additional functionality on the uracil nucleobase. Examples of a hydroxyl, protected thiol and protected amino group are given. Further, an example of derivatization of a resin-bound oligomer with a single IU insert is given.