Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Non-contiguous protein recombination

Swapping sequence elements among related proteins can produce chimeric proteins with novel behaviors and improved properties such as enhanced stability. Although homologous mutations are much more conservative than random mutations, chimeras of distantly-related proteins have a low probability of retaining fold and function. Here, I introduce a new tool for protein recombination that identifies structural blocks that can be swapped among homologous proteins with minimal disruption. This non-contiguous recombination approach enables design of chimeras and libraries of chimeras with less disruption than can be achieved by swapping blocks of sequence. Less disruption means that one can generate libraries with higher fractions of functional enzymes and enables recombination of more distant homologs.

Using this new tool I design and construct many functional chimeric cellulases. I illustrate the structurally conservative nature of this recombination by creating a functional prokaryotic-eukaryotic chimera and solving its structure. I also show how non-contiguous recombination can be used to efficiently identify stabilizing mutations that have been incorporated into homologs in nature.

I. Rigid body penetration into brittle materials. II. Phase change effect on shock wave propagation

Part I.

We have developed a technique for measuring the depth time history of rigid body penetration into brittle materials (hard rocks and concretes) under a deceleration of ~ 10⁵ g. The technique includes bar-coded projectile, sabot-projectile separation, detection and recording systems. Because the technique can give very dense data on penetration depth time history, penetration velocity can be deduced. Error analysis shows that the technique has a small intrinsic error of ~ 3-4 % in time during penetration, and 0.3 to 0.7 mm in penetration depth. A series of 4140 steel projectile penetration into G-mixture mortar targets have been conducted using the Caltech 40 mm gas/ powder gun in the velocity range of 100 to 500 m/s.

We report, for the first time, the whole depth-time history of rigid body penetration into brittle materials (the G-mixture mortar) under 10⁵ g deceleration. Based on the experimental results, including penetration depth time history, damage of recovered target and projectile materials and theoretical analysis, we find:

1. Target materials are damaged via compacting in the region in front of a projectile and via brittle radial and lateral crack propagation in the region surrounding the penetration path. The results suggest that expected cracks in front of penetrators may be stopped by a comminuted region that is induced by wave propagation. Aggregate erosion on the projectile lateral surface is < 20% of the final penetration depth. This result suggests that the effect of lateral friction on the penetration process can be ignored.

2. Final penetration depth, P_max, is linearly scaled with initial projectile energy per unit cross-section area, e_s , when targets are intact after impact. Based on the experimental data on the mortar targets, the relation is P_max(mm) 1.15e_s (J/mm² ) + 16.39.

3. Estimation of the energy needed to create an unit penetration volume suggests that the average pressure acting on the target material during penetration is ~ 10 to 20 times higher than the unconfined strength of target materials under quasi-static loading, and 3 to 4 times higher than the possible highest pressure due to friction and material strength and its rate dependence. In addition, the experimental data show that the interaction between cracks and the target free surface significantly affects the penetration process.

4. Based on the fact that the penetration duration, t_max, increases slowly with e_s and does not depend on projectile radius approximately, the dependence of t_max on projectile length is suggested to be described by t_max(μs) = 2.08e_s (J/mm² + 349.0 x m/(πR²), in which m is the projectile mass in grams and R is the projectile radius in mm. The prediction from this relation is in reasonable agreement with the experimental data for different projectile lengths.

5. Deduced penetration velocity time histories suggest that whole penetration history is divided into three stages: (1) An initial stage in which the projectile velocity change is small due to very small contact area between the projectile and target materials; (2) A steady penetration stage in which projectile velocity continues to decrease smoothly; (3) A penetration stop stage in which projectile deceleration jumps up when velocities are close to a critical value of ~ 35 m/s.

6. Deduced averaged deceleration, a, in the steady penetration stage for projectiles with same dimensions is found to be a(g) = 192.4v + 1.89 x 10⁴, where v is initial projectile velocity in m/s. The average pressure acting on target materials during penetration is estimated to be very comparable to shock wave pressure.

7. A similarity of penetration process is found to be described by a relation between normalized penetration depth, P/P_max, and normalized penetration time, t/t_max, as P/P_max = f(t/t_max, where f is a function of t/t_max. After f(t/t_max is determined using experimental data for projectiles with 150 mm length, the penetration depth time history for projectiles with 100 mm length predicted by this relation is in good agreement with experimental data. This similarity also predicts that average deceleration increases with decreasing projectile length, that is verified by the experimental data.

8. Based on the penetration process analysis and the present data, a first principle model for rigid body penetration is suggested. The model incorporates the models for contact area between projectile and target materials, friction coefficient, penetration stop criterion, and normal stress on the projectile surface. The most important assumptions used in the model are: (1) The penetration process can be treated as a series of impact events, therefore, pressure normal to projectile surface is estimated using the Hugoniot relation of target material; (2) The necessary condition for penetration is that the pressure acting on target materials is not lower than the Hugoniot elastic limit; (3) The friction force on projectile lateral surface can be ignored due to cavitation during penetration. All the parameters involved in the model are determined based on independent experimental data. The penetration depth time histories predicted from the model are in good agreement with the experimental data.

9. Based on planar impact and previous quasi-static experimental data, the strain rate dependence of the mortar compressive strength is described by σ_f/σ⁰_f = exp(0.0905(log(έ/έ_0) ^1.14, in the strain rate range of 10^-7/s to 10³/s (σ⁰_f and έ are reference compressive strength and strain rate, respectively). The non-dispersive Hugoniot elastic wave in the G-mixture has an amplitude of ~ 0.14 GPa and a velocity of ~ 4.3 km/s.

Part II.

Stress wave profiles in vitreous GeO₂ were measured using piezoresistance gauges in the pressure range of 5 to 18 GPa under planar plate and spherical projectile impact. Experimental data show that the response of vitreous GeO₂ to planar shock loading can be divided into three stages: (1) A ramp elastic precursor has peak amplitude of 4 GPa and peak particle velocity of 333 m/s. Wave velocity decreases from initial longitudinal elastic wave velocity of 3.5 km/s to 2.9 km/s at 4 GPa; (2) A ramp wave with amplitude of 2.11 GPa follows the precursor when peak loading pressure is 8.4 GPa. Wave velocity drops to the value below bulk wave velocity in this stage; (3) A shock wave achieving final shock state forms when peak pressure is > 6 GPa. The Hugoniot relation is D = 0.917 + 1.711u (km/s) using present data and the data of Jackson and Ahrens [1979] when shock wave pressure is between 6 and 40 GPa for ρ₀ = 3.655 gj cm³ . Based on the present data, the phase change from 4-fold to 6-fold coordination of Ge⁺⁴ with O^-2 in vitreous GeO₂ occurs in the pressure range of 4 to 15 ± 1 GPa under planar shock loading. Comparison of the shock loading data for fused SiO₂ to that on vitreous GeO₂ demonstrates that transformation to the rutile structure in both media are similar. The Hugoniots of vitreous GeO₂ and fused SiO₂ are found to coincide approximately if pressure in fused SiO₂ is scaled by the ratio of fused SiO₂to vitreous GeO₂ density. This result, as well as the same structure, provides the basis for considering vitreous Ge0₂ as an analogous material to fused SiO₂ under shock loading. Experimental results from the spherical projectile impact demonstrate: (1) The supported elastic shock in fused SiO₂ decays less rapidly than a linear elastic wave when elastic wave stress amplitude is higher than 4 GPa. The supported elastic shock in vitreous GeO₂ decays faster than a linear elastic wave; (2) In vitreous GeO₂ , unsupported shock waves decays with peak pressure in the phase transition range (4-15 GPa) with propagation distance, x, as α 1/x^-3.35 , close to the prediction of Chen et al. [1998]. Based on a simple analysis on spherical wave propagation, we find that the different decay rates of a spherical elastic wave in fused SiO₂ and vitreous GeO₂ is predictable on the base of the compressibility variation with stress under one-dimensional strain condition in the two materials.

A biochemical and structural characterization of Drosophila neuroglian

A number of cell-cell interactions in the nervous system are mediated by immunoglobulin gene superfamily members. For example, neuroglian, a homophilic neural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C- 2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats. Neuroglian shares this domain organization and significant sequence identity with Ll, a murine neural adhesion molecule that could be a functional homologue. Here I report the crystal structure of a proteolytic fragment containing the first two FnIII repeats of neuroglian (NgFn 1,2) at 2.0Å. The interpretation of photomicrographs of rotary shadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five neuroglian Fn III domains, is also discussed.

The structure of NgFn 1,2 consists of two roughly cylindrical β-barrel structural motifs arranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined by the cylinder axes. The folding topology of each domain is identical to that previously observed for single FnIII domains from tenascin and fibronectin. The domains of NgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the longest dimension of the fragment. Assuming this relative orientation is a general property of tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other proteins are modeled as thin straight rods with two domain zig-zag repeats. When combined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4 and CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 Å in diameter) that extends up to 370Å from the cell surface.

In photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible rod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean total length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 Å, consistent with a head-to-tail packing of neuroglian's C2-type domains.

Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.

To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.

Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.

A mathematical study of finite-amplitude rock-folding

The problem of the finite-amplitude folding of an isolated, linearly viscous layer under compression and imbedded in a medium of lower viscosity is treated theoretically by using a variational method to derive finite difference equations which are solved on a digital computer. The problem depends on a single physical parameter, the ratio of the fold wavelength, L, to the "dominant wavelength" of the infinitesimal-amplitude treatment, L_d. Therefore, the natural range of physical parameters is covered by the computation of three folds, with L/L_d = 0, 1, and 4.6, up to a maximum dip of 90°.

Significant differences in fold shape are found among the three folds; folds with higher L/L_d have sharper crests. Folds with L/L_d = 0 and L/L_d = 1 become fan folds at high amplitude. A description of the shape in terms of a harmonic analysis of inclination as a function of arc length shows this systematic variation with L/L_d and is relatively insensitive to the initial shape of the layer. This method of shape description is proposed as a convenient way of measuring the shape of natural folds.

The infinitesimal-amplitude treatment does not predict fold-shape development satisfactorily beyond a limb-dip of 5°. A proposed extension of the treatment continues the wavelength-selection mechanism of the infinitesimal treatment up to a limb-dip of 15°; after this stage the wavelength-selection mechanism no longer operates and fold shape is mainly determined by L/L_d and limb-dip.

Strain-rates and finite strains in the medium are calculated f or all stages of the L/L_d = 1 and L/L_d = 4.6 folds. At limb-dips greater than 45° the planes of maximum flattening and maximum flattening rat e show the characteristic orientation and fanning of axial-plane cleavage.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes

Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean nuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form independent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the Holliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.

Phase behavior of complex superprotonic solid acids

Superprotonic phase transitions and thermal behaviors of three complex solid acid systems are presented, namely Rb3H(SO4)2-RbHSO4 system, Rb3H(SeO4)2-Cs3H(SeO4)2 solid solution system, and Cs6(H2SO4)3(H1.5PO4)4. These material systems present a rich set of phase transition characteristics that set them apart from other, simpler solid acids. A.C. impedance spectroscopy, high-temperature X-ray powder diffraction, and thermal analysis, as well as other characterization techniques, were employed to investigate the phase behavior of these systems.

Rb3H(SO4)2 is an atypical member of the M3H(XO4)2 class of compounds (M = alkali metal or NH4+ and X = S or Se) in that a transition to a high-conductivity state involves disproportionation into two phases rather than a simple polymorphic transition [1]. In the present work, investigations of the Rb3H(SO4)2-RbHSO4 system have revealed the disproportionation products to be Rb2SO4 and the previously unknown compound Rb5H3(SO4)4. The new compound becomes stable at a temperature between 25 and 140 °C and is isostructural to a recently reported trigonal phase with space group P3̅m of Cs5H3(SO4)4 [2]. At 185 °C the compound undergoes an apparently polymorphic transformation with a heat of transition of 23.8 kJ/mol and a slight additional increase in conductivity.

The compounds Rb3H(SeO4)2 and Cs3H(SeO4)2, though not isomorphous at ambient temperatures, are quintessential examples of superprotonic materials. Both adopt monoclinic structures at ambient temperatures and ultimately transform to a trigonal (R3̅m) superprotonic structure at slightly elevated temperatures, 178 and 183 °C, respectively. The compounds are completely miscible above the superprotonic transition and show extensive solubility below it. Beyond a careful determination of the phase boundaries, we find a remarkable 40-fold increase in the superprotonic conductivity in intermediate compositions rich in Rb as compared to either end-member.

The compound Cs6(H2SO4)3(H1.5PO4)4 is unusual amongst solid acid compounds in that it has a complex cubic structure at ambient temperature and apparently transforms to a simpler cubic structure of the CsCl-type (isostructural with CsH2PO4) at its transition temperature of 100-120 °C [3]. Here it is found that, depending on the level of humidification, the superprotonic transition of this material is superimposed with a decomposition reaction, which involves both exsolution of (liquid) acid and loss of H2O. This reaction can be suppressed by application of sufficiently high humidity, in which case Cs6(H2SO4)3(H1.5PO4)4 undergoes a true superprotonic transition. It is proposed that, under conditions of low humidity, the decomposition/dehydration reaction transforms the compound to Cs6(H2-0.5xSO4)3(H1.5PO4)4-x, also of the CsCl structure type at the temperatures of interest, but with a smaller unit cell. With increasing temperature, the decomposition/dehydration proceeds to greater and greater extent and unit cell of the solid phase decreases. This is identified to be the source of the apparent negative thermal expansion behavior.

References

[1] L.A. Cowan, R.M. Morcos, N. Hatada, A. Navrotsky, S.M. Haile, Solid State Ionics 179 (2008) (9-10) 305.

[2] M. Sakashita, H. Fujihisa, K.I. Suzuki, S. Hayashi, K. Honda, Solid State Ionics 178 (2007) (21-22) 1262.

[3] C.R.I. Chisholm, Superprotonic Phase Transitions in Solid Acids: Parameters affecting the presence and stability of superprotonic transitions in the MHnXO4 family of compounds (X=S, Se, P, As; M=Li, Na, K, NH4, Rb, Cs), Materials Science, California Institute of Technology, Pasadena, California (2003).

Developing peptide based capture agents for diagnostics and therapeutics

Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.

Multiplexed DNA-mediated electrochemistry

The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of π-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the π-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA π-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.

First, Methylene Blue (MB′) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB′ was reduced through DNA-mediated charge transport. The redox signal intensity for MB′-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB′ on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.

Next, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening π-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA. Further gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.

Finally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Binding site size limitations of imidazole-pyrrole polyamides for recognition in the minor groove of DNA

The discovery that the three ring polyamide Im-Py-Py-Dp containing imidazole (Im) and pyrrole (Py) carboxamides binds the DNA sequence 5'-(A,T)G(A,T)C(A,T)-3' as an antiparallel dimer offers a new model for the design of ligands for specific recognition of sequences in the minor groove containing both G,C and A,T base pairs. In Chapter 2, experiments are described in which the sequential addition of five N- methylpyrrolecarboxamides to the imidazole-pyrrole polyamide Im-Py-Py-Dp affords a series of six homologous polyamides, Im-(Py)_2-7-Dp, that differ in the size of their binding site, apparent first order binding affinity, and sequence specificity. These results demonstrate that DNA sequences up to nine base pairs in length can be specifically recognized by imidazole-pyrrole polyamides containing three to seven rings by 2:1 polyamide-DNA complex formation in the minor groove. Recognition of a nine base pair site defines the new lower limit of the binding site size that can be recognized by polyamides containing exclusively imidazole and pyrrolecarboxamides. The results of this study should provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

In Chapter 3 the design and synthesis of the hairpin polyamide Im-Py-Im-Py-γ-Im- Py-Im-Py-Dp is described. Quantitative DNase I footprint titration experiments reveal that Im-Py-Im-Py-γ-Im-Py-Im-Py-Dp binds six base pair 5'-(A,T)GCGC(A,T)-3' sequences with 30-fold higher affinity than the unlinked polyamide Im-Py-Im-Py-Dp. The hairpin polyamide does not discriminate between A•T and T•A at the first and sixth positions of the binding site as three sites 5'-TGCGCT-3', 5'-TGCGCA-3', and 5 'AGCGCT- 3' are bound with similar affinity. However, Im-Py-Im-Py-γ-Im-Py-Im-PyDp is specific for and discriminates between G•C and C•G base pairs in the 5'-GCGC-3' core as evidenced by lower affinities for the mismatched sites 5'-AACGCA-3', 5'- TGCGTT-3', 5'-TGCGGT-3', and 5'-ACCGCT-3'.

In Chapter 4, experiments are described in which a kinetically stable hexa-aza Schiff base La³⁺ complex is covalently attached to a Tat(49-72) peptide which has been shown to bind the HIV-1 TAR RNA sequence. Although these metallo-peptides cleave TAR site-specifically in the hexanucleotide loop to afford products consistent with hydrolysis, a series of control experiments suggests that the observed cleavage is not caused by a sequence-specifically bound Tat(49-72)-La(L)³⁺ peptide.

Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.