Terry J. Amiss

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein.

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.

Engineering and rapid selection of a low‐affinity glucose/galactose‐binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high‐affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D‐glucose/D‐galactose‐binding protein (GGBP) having low affinity to glucose. Wild‐type GGBP has an equilibrium dissociation constant of 0.2 μM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7–33 mM). To accomplish this a focused library was constructed using structure‐based site‐saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H‐bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site‐specifically at E149C with the fluorophore IANBD (N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)ethylene‐diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000‐fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H‐bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure‐based site‐saturation mutagenesis and periplasmic expression screening to discover low‐affinity GGBP mutants having potential utility for measuring glucose in humans.

Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

Background: Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call “ligand protection.” Method: In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. Results: Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. Conclusions: The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1–30 mM). This general strategy may also have broad utility for other protein-labeling applications.

Nucleic Acid Blotting Techniques

This chapter deals with basic concepts and techniques in nucleic acid blotting. Many of the techniques involved with Southern blotting and Northern blotting are similar. Negatively charged, purified nucleic acids from prokaryotic or eukaryotic cells are separated according to size by electrophoresis through an agarose gel matrix. The RNA or denatured DNA is subse– quently transferred and immobilized onto a membrane com– posed of nitrocellulose or nylon. The nucleic acids on the membrane are then hybridized to a specific labeled “probe,” which consists of homologous single-stranded nucleic acids that carry molecules, allowing detection and visualization of the hybridized probe. Hybridization between the immobilized nucleic acids and labeled probe allows detection of specific DNA or RNA sequences within a complex mixture of DNA or RNA. The specific method of detection and visualization is dependent on the nature of the labeled probe; radioactive probes enable autoradiographic detection, and probes labeled with enzymes facilitate chemiluminescent or colorimetric detection. Nucleic acid blotting yields valuable information pertaining to gene integrity and copy number (Southern blot) and provides a means of analyzing gene expression and mRNA size (Northern blot). These methods can be used to character– ize tissues and cultured cells in the laboratory and often provide valuable information for clinical evaluation of patient samples.

Abstract A38: Optimization of whole-genome amplification for analysis of single cells using next-generation sequencing

The accurate analysis of both genotypic variation in tumors and tumor evolution due to selective pressure is important in determining treatment options in the clinic. Next generation sequencing (NGS) of single cells from solid tumors and circulating tumor cells has the potential to dramatically increase the amount of genotypic detail obtained from these samples. For the analysis of single cells the challenge is to employ whole genome amplification (WGA) to obtain enough DNA for analysis while minimizing amplification bias and maintaining copy accuracy. We are developing tools for single cell sequencing to improve these parameters while also increasing sample throughput. Initially, three commercially available WGA methods were tested on 5-10 genome quantities of HCT-15 genomic DNA. Using the Ion Torrent AmpliSeq Cancer Hotspot Panel, the amplification accuracy, amplicon coverage and DNA yield were determined. When compared to controls significant differences were observed in the three WGA methods ability to accurately amplify DNA. The most accurate kit produced a specificity and sensitivity of 98.5% and 100% respectively. This protocol also produced a substantial DNA yield of 25 µgs and although, the average amplicon coverage indicated some bias, greater than 99% of the amplicons had at least 100-fold coverage. This WGA kit was then tested on single cells from a human breast cancer; an ER/PR/Her2 negative spindle cell metaplastic carcinoma. To obtain single cells the tumor was grown in a PDX mouse model and index sorted using a BD FACSAria II flow cytometer. The WGA produced an average DNA yield of 2.7± 0.8 µg, with 100-fold amplicon coverage of 92±7%. For the variants identified, the amplification bias differed by as much as 3,000 reads/variant. We are now taking steps to improve WGA of single cells and will report on these efforts in detail. To date when monitoring variant allele frequency and coverage depth in the triple negative tumor, KRAS and MET mutations were identified in single cells, as well as in the bulk sample. Interestingly, a TP53 mutation was identified only in single cells. This data supports the utility of WGA and single cell analysis to identify mutations, some having clinically relevant role for targeted therapy. Citation Format: Terry J. Amiss, Frances Tong, Eileen Snowden, Richard Kelly, Rainer Blaesius, Nick Herrmann, Friedrich Hahn, Warren Porter, Mitchell Ferguson, Chen Chang, Daphne Clancy, Stewart Jurgensen. Optimization of whole-genome amplification for analysis of single cells using next-generation sequencing. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A38.