Joonyul Kim

The Arabidopsis LUT1 locus encodes a member of the cytochrome P450 family that is required for carotenoid ε-ring hydroxylation activity

Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid β-and ε-hydroxylases catalyze the formation of lutein from α-carotene (β,ε-carotene). In contrast to the well studied β-hydroxylases that have been cloned and characterized from many organisms, the ε-hydroxylase has only been genetically defined by the lut1 mutation in Arabidopsis. We have isolated the LUT1 gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases, LUT1 encodes a cytochrome P450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of LUT1, lut1-3, into the β-hydroxylase 1/β-hydroxylase 2 (b1 b2) double-mutant background, in which both Arabidopsis β-hydroxylases are disrupted, yielded a genotype (lut1-3 b1 b2) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated β-rings were still produced in lut1-3 b1 b2, suggesting that a fourth unknown carotenoid β-hydroxylase exists in vivo that is structurally unrelated to β-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional β-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron β-hydroxylases.

Quantitation of Femtomolar Protein Levels via Direct Readout with the Electrochemical Proximity Assay

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.

The Evolution and Function of Carotenoid Hydroxylases in Arabidopsis

To gain insight into the evolution of xanthophyll synthesis in Arabidopsis thaliana, we analyzed two pairs of duplicated carotenoid hydroxylase enzymes in Arabidopsis thaliana: the cytochrome P450 enzymes CYP97A3 and CYP97C1, and non-heme di-iron enzymes, BCH1 and BCH2. Hydroxylated carotenes did not accumulate in a quadruple mutant for these four genes, demonstrating that they encode the full complement of carotenoid hydroxylases in A. thaliana. We were thus able to infer definitively the activity of each enzyme in vivo based on the phenotypes of selected double and triple mutant genotypes. The CYP97 and BCH gene pairs are primarily responsible for hydroxylation of alpha- and beta-carotenes, respectively, but exhibit some overlapping activities, most notably in hydroxylation of the beta-ring of alpha-carotene. Surprisingly, triple mutants containing only CYP97C1 or CYP97A3 activity produced 74 and 6% of the wild-type lutein level, indicating that CYP97C1 can efficiently hydroxylate both the beta- and epsilon-rings of alpha-carotene and that CYP97A3 also has low activity toward the epsilon-ring of alpha-carotene. The modes of functional divergence for the gene pairs appear distinct, with the CYP97 duplicates being strongly co-expressed but encoding enzymes with different in vivo substrates, while the BCH duplicates encode isozymes that show significant expression divergence in reproductive organs. By integrating the evolutionary history and substrate specificities of each extant enzyme with the phenotypic responses of various mutant genotypes to high light stress, we propose two likely scenarios for the evolution of alpha-xanthophyll biosynthesis in plants from ancestral organisms.

Isothermal DNA amplification in bioanalysis: strategies and applications

Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.

A Reusable Electrochemical Proximity Assay for Highly Selective, Real-Time Protein Quantitation in Biological Matrices

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)—as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Improvement of sensitivity and dynamic range in proximity ligation assays by asymmetric connector hybridization.

The proximity ligation assay (PLA) is one of the most sensitive and simple protein assays developed to date, yet a major limitation is the relatively narrow dynamic range compared to other assays such as enzyme-linked immunosorbent assays. In this work, the dynamic range of PLA was improved by 2 orders of magnitude and the sensitivity was improved by a factor of 1.57. To accomplish this, asymmetric DNA hybridization was used to reduce the probability of target-independent, background ligation. An experimental model of the aptamer-target-connector complex (apt(A)-T-apt(B)-C(20,PLA)) in PLA was developed to study the effects of asymmetry in aptamer-connector hybridization. Connector base pairing was varied from the PLA standard of 20 total bases (C(20)) to an asymmetric combination with 15 total bases (C(15)). The results of this model suggested that weakening the affinity of one side of the connector to one aptamer would significantly reduce target-independent ligation (background) without greatly affecting target-dependent ligation (signal). These predictions were confirmed using PLA with asymmetric connectors for detection of human thrombin. This novel, asymmetric PLA approach should impact any previously developed PLA method (using aptamers or antibodies) by reducing target-independent ligation events, thus generally improving the sensitivity and dynamic range of the assay.

Direct hydrogel encapsulation of pluripotent stem cells enables ontomimetic differentiation and growth of engineered human heart tissues.

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue.

Creating biocompatible oil-water interfaces without synthesis: direct interactions between primary amines and carboxylated perfluorocarbon surfactants.

Currently, one of the most prominent methods used to impart biocompatibility to aqueous-in-oil droplets is to synthesize a triblock copolymer surfactant composed of perfluoropolyether and polyether blocks. The resulting surfactants (EA surfactant, KryJeffa, etc.) allow generation of highly biocompatible droplet surfaces while maintaining the heat stability of the starting material. However, production of these surfactants requires expertise in synthetic organic chemistry, creating a barrier to widespread adoption in the field. Herein, we describe a simple alternative to synthetic modification of surfactants to impart biocompatibility. We have observed that aqueous-in-oil droplet surfaces can be made biocompatible and heat stable by merely exploiting binding interactions between polyetherdiamine additives in the aqueous phase and carboxylated perfluorocarbon surfactants in the oil phase. Droplets formed under these conditions are shown to possess biocompatible surfaces capable of supporting picoliter-scale protein assays, droplet polymerase chain reaction (PCR), and droplet DNA amplification with isothermal recombinase polymerase amplification (RPA). Droplets formed with polyetherdiamine aqueous additives are stable enough to withstand temperature cycling during PCR (30-40 cycles at 60-94 °C) while maintaining biocompatibility, and the reaction efficiency of RPA is shown to be similar to that with a covalently modified surfactant (KryJeffa). The binding interaction was confirmed with various methods, including FT-IR spectroscopy, NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and fluorescence microscopy. Overall, our results suggest that, by simply introducing a commercially-available, polyetherdiamine additive (Jeffamine ED-900) to the aqueous phase, researchers can avoid synthetic methods in generating biocompatible droplet surfaces capable of supporting DNA and protein analysis at the subnanoliter scale.

Protein quantification using controlled DNA melting transitions in bivalent probe assemblies.

Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.

Quantifying aptamer–protein binding via thermofluorimetric analysis

Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (-dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument-even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding.