Membrane-Embedded Channel of Bacteriophage phi29 DNA Packaging Motor for Single Molecule Sensing and Nanomedicine

A dissertation submitted to the Graduate School of the University of Cincinnati

in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Energy, Environmental, Biological and Medical Engineering of the College of Engineering & Applied Science April 2012

By

Jia Geng Bachelor of Science, Nanjing University 2004

Committee Chair: Jing-Huei Lee, Ph.D.

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I. ABSTRACT

Linear double-stranded DNA (dsDNA) viruses package its genome into a preformed procapsid fueled by the energy from ATP hydrolysis. The bacteriophage phi29 motor has a truncated cone shaped protein component, named connector, with a central channel of 3.6 nm at its narrowest part. The connector protein has been successfully inserted into an artificial lipid bilayer membrane, and the channel exhibited robust capability under various salt and pH conditions as revealed by single channel studies.

This channel is suitable for extremely precise assessment of the transportation of small molecules, such as ions, DNA and RNA.

There is an urgent need to development a highly sensitive detection system, for the applications in the area of pathogen detection, disease diagnosis, environmental monitoring, etc. The current challenges and limitations of these technologies are the sensitivity and accuracy issues arising from background noise and nonspecific reactions.

The property of phi29 motor channel has been studied at various conditions, and was incorporated into lipid membrane. The motor channel exercised a one-way traffic property during the process of dsDNA translocation with a valve mechanism. In addition, the opening and closure of the channel also exhibit reversible and controllable. A modified version of the connector channel is founded to have a smaller channel size, which is able to detect the ssDNA and ssRNA. These findings have important implications since this artificial membrane-embedded channel would allow detailed investigations into the mechanisms of viral motor operation, as well as future applications for therapeutic molecule packaging, delivery, single molecule sensing and drug screening.

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and best regards to Professor Peixuan Guo, my PhD advisor at University of Cincinnati, for his support and guidance during my PhD study. His devotion to research also inspired me to explorer the frontier of science.

I also greatly appreciate the Dr. Jing-Huei Lee, Ph.D. for chairing my committee during my stay at University of Kentucky. His advices and informative instructions are very indispensable to my study. It is my great honor to have Dr. Dr. Chong Ahn, Dr. Jarek

Meller and Dr Marepalli Rao in my committee, and their insightful suggestions and help are greatly appreciated.

The research project are also greatly supported by our supporters: Dr. Carlo

Montemagno and Dr. David Wendell from UC College of Engineering for single channel recording, Dr. Jacob Schmidt from UCLA and Dr. Liqun Gu from the University of

Missouri for α-haemolysin proteins; Dr. Rong Zhang from UC for Q-PCR analysis; Dr.

Nicola Stonehouse from University of Leeds , UK for recombinant connectors; Dr. Jarek

Meller from CCHMC and Andrew Herr from UC for connector amphiphilicity analysis; Dr.

Chong Ahn and Dr. Joon Sub Shim from UC for MEMS fabrication, and Dr. Xing-Jie

Liang, Dr. Jinghong Li and Dr. Haichen Wu from China for valuable discussion and suggestions.

I would like to sincerely thank many previous and current members in Dr. Guo’s group for the support and help in various aspects. Dr. Feng Xiao, Dr. Taejin Lee, Dr. Oana

Coban, Dr. Faqing Yuan, Dr. Wenjuan Wang, Dr. Peng Jing, Dr. Anne Vonderheide and

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Dr. Jing Liu for their previous research and instructions; Current members, especially Dr.

Dan Shu, Dr. Hui Zhang, Dr. Farzin Haque, Dr. Randall Reif, Dr. Zhanxi Hao, Yi Shu,

Huaming Fang, Chad Schwartz, Le Zhang and Daniel Binzel for their continuing encouragement and valuable advices. I would also sincerely thank other current colleagues: Dr. Brent Hallahan, Fengmei Pi, Hui Li, Nayeem Hossain, Shaoying Wang and Zhengyi Zhao, as well as work-study students in Dr. Guo’s lab. It has been great pleasure to work in the team.

I would like to acknowledge many people on the faculty and staff of the Biomedical

Engineering program at University of Cincinnati, and College o Pharmacy at University of for their help, assistance in various ways during my course of studies.

This work won’t be possible without the personal and practical support of numerous people. Thus my sincere gratitude goes to my parents, all my friends, and my companions for their love and support over the last few years.

This work was supported by the National Institutes of Health grants R01 EB012135,

R01 EB003730, GM059944, and NIH Nanomedicine Development Center: Phi29 DNA

Packaging Motor for Nanomedicine through the NIH Roadmap for Medical Research to

Peixuan Guo (PN2 EY 018230).

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Table of Contents

page

Abstract ...... iii

Acknowledgements ...... v

List of Tables ...... xii

List of Figures ...... xiii

Chapter 1 GENERAL INTRODUCTION AND LITERATURE REVIEW ...... 1

Introduction ...... 2

Structure of phi29 DNA Packaging Motor ...... 3

Packaging and assembly of DNA viruses ...... 3

Structural components: procapsid, scaffolding and connector ...... 3

Nonstructural components: pRNA and packaging enzyme gp16 ...... 7

Packaging substrate: Genomic DNA-gp3 ...... 10

Fiber (gp8.5), neck and tail (gp9, gp11-12) proteins ...... 11

Mechanism of the phi29 DNA packaging motor ...... 11

Single-molecule studies of DNA packaging motors ...... 11

Symmetry argument: pentamer or hexamer ...... 14

ATP consumption translated to force generation by the DNA packaging motor 17

Applications of Phi29 DNA Packaging Motor ...... 18

Applications of the phi29 DNA packaging motor to nanotechnology ...... 19

Applications of the phi29 connector to nanotechnology ...... 19

Applications of the phi29 pRNA to nanotechnology...... 24

Perspectives ...... 29

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Chapter 2. Reconstitution of connector into lipid bilayer for dsDNA translocation ...... 31

Introduction ...... 31

Materials And Methods ...... 33

C-His tagged connector construction ...... 33

FITC labeling of the connector protein ...... 33

Incorporation of the connector protein into giant liposome...... 34

Separation and filtration of the connector-containing proteoliposome ...... 34

Lipid bilayer electrophysiological measurements ...... 35

Insertion of the connector into lipid bylayer ...... 35

Passive translocation of dsDNA driven by electric field ...... 36

Results and Discussion ...... 37

Reconstitution of the connector into liposomes ...... 37

Connector channel insertion into planar lipid membranes ...... 38

Conductivity of the channels ...... 38

Translocation of double-stranded DNA ...... 39

Comparison of the phi29 connector channel with α-haemolysin ...... 40

Applications ...... 40

Limitations ...... 41

Conclusion ...... 42

Chapter 3. Reversible and controllable gating of the phi29 connector channel ...... 46

Introduction ...... 47

Materials and Methods ...... 50

Materials ...... 51

Regineering, expression and purification of phi29 connector ...... 52

Electrophysiological measurements ...... 53

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Probe the connector conformational change by antibody ...... 53

Probing the connector conformational change by Nickel-NTA-Nanogold ...... 53

Results ...... 53

Step-wise conformational change gating by high-voltage ...... 53

Gated channel reopening by voltage dropping ...... 54

Protein binding to the connector’s C-terminal induced discrete conformational

changes with one-third step-wise reduction ...... 57

Ni-NTA Nanogold binding to the connector’s C-terminal induced discrete

conformational changes with one-third step-wise ...... 57

Gating behavior of connectors with the internal flexible loop cleavage or N-terminal

flexible segment removal ...... 60

Effect of C-terminal flexible loop segment in the conformational change ...... 61

Discussion ...... 62

Conclusions ...... 68

Chapter 4. Reengineered connector for single-stranded nucleic acids sensing ...... 69

Introduction ...... 69

Materials and methods ...... 71

Materials ...... 71

Cloning and purification of the phi29 connector protein ...... 71

Incorporation of the connector protein into giant liposomes ...... 71

Insertion of the connector protein into preformed lipid bilayer...... 72

Electrophysiological measurements ...... 72

Purification of the DNA/RNA used in the experiment ...... 73

Translocation experiments of DNA and RNA ...... 73

Results ...... 74

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Narrowing channel size by reengineering the phi29 DNA packaging motor ..... 74

Sensing of single-stranded DNA by the modified channels ...... 75

Sensing of single-stranded RNA by the modified channels ...... 75

Two-way translocation of single-stranded nucleotides by the modified channels 76

Stability of the narrowed channel in various salt, pH and voltage condition ...... 76

Discussion ...... 77

Conclusion ...... 79

Chapter 5. Single-molecule assay of phi29 pRNA and connector protein interaction ...... 84

Introduction ...... 84

Materials and methods ...... 86

Preparation of lipid monolayer on quartz slide ...... 86

Preparation of Cy5 labeled RNA ...... 86

C-His Connector Conjugation to Ni-NTA Lipid bilayer ...... 87

Binding Cy5 RNA to the connector bound to Ni lipid monolayer ...... 87

Results ...... 88

Immobilization of connector via monolayer ...... 88

Interaction with pRNA ...... 88

Single molecule imaging of immobilized Cy Aa’ pRNA ...... 88

Single molecule counting of pRNA with connector ...... 89

Discussion ...... 90

Conclusion ...... 91

Chapter 6. Unfinished Work, and Prospects ...... 98

Binding of connector protein to KB cell and induction of cell apoptosis ...... 98

Specific interaction of connector/sapc-dops complex to cells ...... 98

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Cell membrane embodied connector channel by electrophysiological measurement 99

BIBLIOGRAPHY ...... 104

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LIST OF TABLES

Table Page

5.1 Lipid in chloroform / C-His tagged gp10 connector in Hepes ...... 93

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LIST OF FIGURES

Figure Page

1.1 Negative stain electron images of phi29 virions ...... 5

1.2 Structure of pRNA and Hexamer Formation...... 9

1.3 Packaging of neutral DNA analogues by optical tweezers ...... 13

1.4 Illustration of hexmeric RNA and Cryo-EM images showing pRNA forms hexamer 15

1.5 Dual-view imaging showing procapsids with both Cy3-pRNA I and Cy5-pRNA II. .. 16

1.6 Connector nanoparticle assembly ...... 21

1.7 Images of giant liposomes reconstituting connector, and the translocation

of double-stranded DNA through connector channels in lipid bilayer ...... 23

1.8 Method for the construction of pRNA nanoparticles by loop/loop interaction ...... 25

1.9 Illustration of constructing pRNA multimers...... 26

1.10 pNA as building blocks for nanotechnology ...... 15

1.11 pRNA multimers and Specific GFP knockdown assay of chimeric

pRNA/siRNA (GFP) in KB cells...... 27

2.1 Connector protein of phi29 DNA packaging motor and its reengineering ...... 43

2.2 Conductance assay of the current traces of connector channel ...... 44

3.1 Illustration of the phi29 connector channel structure ...... 49

3.2 Stepwise gating of the connector channel induced by high voltage ...... 55

3.3 Controllable closing and re-opening of the C-His connector channel ...... 56

3.4 Changes of channel conductivity induced by binding to C-terminal ...... 58

3.5 Rationale for mutant connector design ...... 59

3.6 Collection of all current traces under repeated ramping

and the comparison between mutants ...... 62

4.1 Conductance of the gp10C-His/Δloop connector channel ...... 80

4.2 Sensing of ssRNA and ssDNA by the modified channel ...... 81

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4.3 Two way translocation of ssRNA and ssDNA through the narrowed channel ...... 82

4.4 Probe the length of single-stranded nucleotides from translocation fingerprints ..... 83

5.1 Structure of phage phi29 DNA packaging motor and design of

single molecule experiment...... 94

5.2 Immobilization of connector via monolayer and interaction with

pRNA by microscopy imaging...... 95

5.3 Single molecule imaging of immobilized Cy Aa’ pRNA ...... 96

5.4 Statistics of control experiments ...... 97

6.1 Microscopy image of FITC labeled Cstrep connector incubated with

KB Cell in the existence of lipofectamine ...... 100

6.2 FITC-connector /SapC-DOPS complex ...... 101

6.3 FITC-connector /SapC-DOPS complex merged view...... 102

6.4 Scheme of reconstituting connector into a single cell membrane, and

detection of trans-cell-membrane potential ...... 103

6.5 Current steps across the cell membrane after proteoliposome added...... 103

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CHAPTER 1 GENERAL INTRODUCTION AND LITERATURE REVIEW

This chapter was published in Nano LIFE, 2010, Vol 1&2, 54-62.

JIA GENG and ANNE P.VONDERHEIDE

Nanobiotechnology entails the use of nano-sized materials to build structures that can be applied in both biotechnology and medicine. In one vein of this field of study, scientists seek to mimic the wide variety of nanomachines and macromolecular structures that exist in nature and to replicate them in both structure and function. As a most intriguing example, the bacterial virus phi29 uses a self-contained nanomotor to package its DNA after replication. The 30-nm nanomotor contains 12 copies of a protein

(gp10) which together form a 3.6-nm central channel through which the genomic DNA passes into the procapsid during viral assembly and exits during infection. This connector has been recently reengineered and embedded it into a lipid bilayer, creating a system with tremendous application for DNA detection and characterization through electrophysiological measurement. A second component of the phi29 bacteriophage is an ATP-binding pRNA that forms a hexameric ring to gears the motor. The pRNA has been utilized to construct nanoparticles of dimers, trimers, hexamers and patterned superstructures via the interaction of the two interlocking loops. Such structures constructed via bottom-up assembly have been used in the delivery of drugs, siRNA, ribozymes, and genes to specific cells, both in vitro and in vivo. This review will summarize current studies on the structure, function, and mechanism of the phi29 DNA

1 packaging motor, as well as address the applications of these motor components in the field of nanobiotechnology.

Keywords: bacteriophage phi29, nanomotor, nanotechnology.

Introduction

Living organisms accomplish movement, both microscopic and macroscopic, through the employment of molecular motors. For example, the movement of myosin results in muscle contraction; one of the functions of kinesin is the transport of cargo within the cell. As scientists have elucidated the structure and function of these natural motors, they have aspired to integrate these machines into synthetic devices.(1-5)

In double-stranded DNA viruses, a DNA packaging motor is used in the viral replication process. Once the genome and viral proteins are synthesized in the host cell, the packaging motor is involved in the assembly of the component parts into mature virions

(6, 7). This entails the movement of the newly synthesized DNA into a preformed protein shell called a procapsid which serves to protect it(6, 7) This is entropically unfavorable due to the extreme compact nature of the DNA arrangement within the capsid (the DNA is packaged to near-crystalline density (~400 mg/ml)) (6, 8-10), and thus is powered by the energy converted from ATP hydrolysis (11), (12), (11), (10)

The many facets of the phi29 DNA packaging motor have been extensively studied due to several unique characteristics. Not only does the actual motor of phi29 hold promise in bionanotechnology, its component parts have been found individually to be useful in this area. Further, the phi29 DNA packaging motors is one of the strongest biomotors discovered to date. This manuscript will review the module parts of the phi29 bacteriophage, as well as their applications in nanotechnology and gene delivery.

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Structure of phi29 DNA Packaging Motor

Packaging and assembly of DNA viruses

The phi29 DNA packaging motor has been extensively used as a model to study the fundamental mechanism that transduces chemical energy from ATP hydrolysis into the physico-mechanical motion of DNA.(6, 13) When ATP is added as an energy source, approximately 90 percent of the added DNA can be packaged into the procapsid. Once the DNA is packaged by the in vitro assembly system, a DNA-filled capsid can be converted into an infectious phi29 virion upon addition of the appropriate purified proteins, such as tail protein (gp9), neck protein (gp11 and gp12) and morphogenic factor (gp13). (Figure 1)

Structural components: procapsid, scaffolding and connector

Phage procapsids are composed of three types of structural proteins: the capsid protein, the scaffolding protein, and the connector protein.(10, 14) Reconstruction of cryo-electron microscopy (cryo-EM) images showed the three-dimensional structure of the phi29 procapsid (15, 16) as an icosahedral, consisting of 235 copies of the major capsid protein (gp8), 180 copies of scaffolding protein (gp7), and 12 copies of the connector protein (gp10).(15, 16)

Capsid Protein (gp8)

Capsid proteins are the major structural components of viral protein shells. The protein shells protect the viral genomes from degradation by ubiquitous nucleases. The capsid protein in the phi29 bacteriophage is gp8.

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Scaffolding Protein (gp7)

Scaffolding proteins are structural proteins that are required for correct procapsid assembly; however, they are released at either the initiation of or during DNA packaging, and are not part of the mature virion. The exact function of the scaffolding proteins is not yet defined, however, it is clear that they are not required for the procapsid to function. It is theorized that they may form a core structure around which capsid proteins assemble (17-21). They may also serve as chaparones with the task of promoting the correct folding of the capsid proteins. Other functions may include mediating a capsid protein/connector protein interaction, excluding cellular proteins from the inside of the procapsid, or facilitating the early stages of DNA entrance into the procapsid (9, 22).

Assembly of the procapsid is not a sequential assembly pathway, but rather requires the coexistence of the scaffolding proteins, the major capsid proteins, and the connector proteins at specific concentration levels. During this process, the scaffolding protein links the major capsid protein and the connector (24). The three components interact so quickly that no clear intermediates can be observed.

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Fig. 1.1. Negative stain electron images of phi29 virions (A).the purified recombinant antireceptor gp12 oligomer by STEM (Scanning Transmission Electron Microscopy) (B), the schematic of the structure of phi29 virion (left inset), the tobacco mosaic virus control (the bar), and the closeup view of the antireceptor (right inset). Adapted from

Ref. (23) with permission)

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Connector Protein (gp10)

The procapsid attaches to the connector (gp10), which plays a role in DNA translocation. It is also involved in the formation of the procapsid and is the base on which the procapsid is assembled. Studies of several bacteriophages (T4, Lambda, T3,

P22, and phi29) revealed that each of their connectors actually performed several tasks including assisting in the formation of the procapsid and DNA packaging, as well as binding the tail proteins upon maturation (9, 25-30). Even though the connectors of T4,

Lambda, P22, and phi29 lack similarity in amino acid sequence, they have been found to share a common 3D structure. All have been shown to contain 1.) a penetrating region at the center, 2.) a twelve-fold domain composed of twelve identical morphological units perpendicular to the axis of the virion particle (the axis direction is parallel to the direction of DNA packaging into the procapsid), and 3.) a narrower domain with a cylindrical shape along the same axis (31, 32).

The three dimensional structure of the phi29 connector has been determined by the employment of several techniques including atomic force microscopy (33), TEM (34),

Cryo-EM (35), immunoelectron microscopy (36), and x-ray crystallography (37-39).

Immunoelectron microscopy showed that the phi29 connector is divided into three regions termed the narrow end, the central area, and the wide end (36). The wide end is inserted in the procapsid and the narrow end extends out of the shell (35, 40, 41). Four domains were found in the phi29 connector: the procapsid domain which is essential for correct procapsid formation (42, 43); the tail domain which is responsible for the binding of the collar and tail proteins (44); the DNA-binding domain that is important for the

6 recognition of DNA at the initial stage of packaging (45); and the RNA-binding domain for specific pRNA binding (46-48).

Several studies indicate that the connector is responsible for procapsid binding of pRNA

(49-51). This has led to the conclusion that the affinity of pRNA to the connector is much stronger than to the capsid, although the possibility of transient interaction between the pRNA and capsid protein cannot be excluded.

Nonstructural components: pRNA and packaging enzyme gp16 pRNA

In 1987, it was discovered that the packaging of phi29 DNA was sensitive to treatment with RNase A or T1. Subsequent investigation revealed the presence of a DNase- resistant 120-base nucleic acid (packaging RNA, pRNA). Incubation with EDTA released the pRNA from the procapsids; conversely, the presence of Mg+2 allowed the binding of pRNA to RNA-free procapsids. Further, the essentiality of the pRNA was proved in experiments in which pRNA-free procapsids were found inactive in DNA packaging; packaging activity was restored after its addition.

Although nonstructural, RNA was discovered as one of the components of the phi29 bacteriorphage that is vital to the DNA packaging function. (46). Two domains of pRNA have been identified; the first is termed the procapsid-binding domain and it is located in the central region of the pRNA and binds to the N-terminus of the connector(51, 52).

The second is the DNA translocation domain and it is located at the 5’/3’ paired ends and binds to gp16 (53, 54). Prior to packaging, the pRNA binds to the connector on the procapsid for the motor to function (51, 55); upon completion of packaging and the addition of gp9 (tail protein) (56), the pRNA leaves the capsid (56) and is not present in

7 the mature virion (46). The pRNA also serves to bind ATP (57) and stimulate the

ATPase activity of gp16 (48, 53, 58). It has been shown that pRNA can be redesigned by a two-module architecture approach, resulting in full activity in DNA packaging and assembly of infectious phi29 virions in vitro(59). Six copies of pRNA molecules are necessary for DNA packaging. The pRNAs form dimers which are the building blocks of a pRNA hexamer conformation (Figure 2). The six phi29 pRNAs form a ring through intermolecular base-pairing between the right loop (bases 42-45) and the left loop

(bases 82-85) (Figure 2C), as observed by transmission electron microscopy with nanogold-labeled pRNA (60, 61). Cryo-AFM has also been used to directly visualize the tertiary structure of pRNA monomers, native and covalently linked dimers, and native trimers.(5) Still, cryo-EM image reconstruction remains challenging for RNA due to its sensitivity to RNase degradation during sample preparation as well as its structural flexibility. A three-base bulge (C18C19A20) located in the DNA translocating domain

(Figure 2A) is critical for DNA translocation.

Similar but distinct pRNAs have been found through phylogenetic analyses of other phages. Although few conserved bases were found, their observed secondary structures are quite similar (62). Nonetheless, if phi29 pRNA is replaced by other phages’ pRNA, DNA packaging will not occur(62).

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Fig. 1.2. Structure of pRNA and Hexamer Formation. A. Sequence and predicted secondary structure of wild-type pRNA. Two domains, the connector binding domain and the DNA translocation domain, are marked with bold lines, and the four bases in the upper and lower loops responsible for inter-RNA interactions are boxed and in bold type. (B and C) Diagrams depicting the formation of a pRNA hexameric ring by upper loop sequence I, 5′-AACC, and lower loop sequence i′, 3′-UUGG, interaction. (Adapted from Ref. (63) with permission)

Packaging enzyme gp16

The gp16 packaging enzyme contains both Walker A- and B-type consensus ATP- binding sequences; therefore, it likely functions as an ATPase during DNA packaging.(12) The A-type sequence of gp16 contains “basic-hydrophobic region-G-X2-

G-X-G-K-S-X7-hydrophobic” amino acids. It has been shown that gp16 can bind and

9 hydrolyze ATP,(11, 57, 58, 64, 65) but the role of gp16 in the phi29 DNA packaging motor is still unclear. This is due to the fact that several inherent properties of this packaging enzyme have hindered attempts to refine the understanding of its functions and attributes. These include its hydrophobicity, low solubility, and self-aggregation.

For this reason, much contradictory data has been produced regarding ATPase activity, binding location, and the stoichiometry of gp16.(11, 48, 66) It has been shown that gp16 binds to the pRNA-containing procapsid more strongly than to the pRNA-free procapsid and the pRNA’s 5’/3’ paired region is the domain for such binding (53, 54). It has also been observed that gp16 binds DNA without sequence specificity, but specifically to pRNA,(53) which implies that the binding properties of gp16 are important determining factors for the biological activity of gp16.

Packaging substrate: Genomic DNA-gp3

The phi29 genome is made up of a 19,285 bp linear dsDNA to which a viral-encoded terminal protein, gp3 is covalently bonded to both the 5’ ends via a linkage between its serine residue 232 and the 5’ dAMP of the DNA (67). The gp3 functions as a “primer” in the initiation of DNA replication (67), and as an enhancer for DNA packaging (68). Only one copy of DNA-gp3 is required for the assembly of a virion (69-71).

In vitro studies have revealed that both gaps and single-stranded (ss)DNA will stall or block the DNA packaging, while nicks can be tolerated by the phi29 packaging motor.

Additionally of interest is the fact that the viral motor packages only its own genomic

DNA; it will not package nonspecific DNA. Few investigations have reported on the conformational requirements of the DNA substrates that can be packaged. Natural DNA in solution is generally present in the supercoiled or relaxed form, which suggests that

10 the helical nature of the B-form DNA is the preferred conformation. Since the phi29 genomic DNA and terminal protein gp3 are covalently linked together, it is treated as a single complex and therefore, does not require either terminase activity to cut concatemeric DNA into a single copy, as in λ, P2, P4, T3 and T7, or a “headful” mechanism, as observed in T4, P22 and P1. Full-length phi29 genomic DNA can be synthesized in vitro (67, 69, 72-75).

Fiber (gp8.5), neck and tail (gp9, gp11-12) proteins

Fiber and tail proteins are not involved in viral DNA packaging in the phi29 system. The head fibers (gp8.5) branch out from the apical regions of the viral head (76-78) and may serve as a stabilizer for the procapsid. The tail proteins penetrate the host cell wall for viral DNA delivery. Once the genomic DNA is packaged into the procapsid, six copies of lower collar (gp11), twelve appendages (gp12), and ten copies of tail knob protein (gp9) are assembled onto the DNA-filled procapsid in a single morphogenetic pathway, yielding a mature virion (42, 79-81). For neck and tail (gp11-12) assembly, a morphogenetic factor (gp13) is also required (42, 69). The morphogenetic factor gp13 may also assist in the retention of the genome inside the capsid (82).

Mechanism of the phi29 DNA packaging motor

Single-molecule studies of DNA packaging motors

Recent advances in single-molecule microscopy permit the direct counting of motor components and the observation of motion events. This allows the analysis of individual motor components, as opposed to averaging the measurements from a massive population of homogenous molecules that are either motionless or in synchronous motion.(83, 84) Determining the stoichiometry of motor components that are actively

11 involved in the motor function will make it possible to elucidate the properties of bionanomotors and other biological machines, and it will also help in the design of nanodevices.

The use of optical tweezers in studies of motor mechanism

The use of optical tweezers, or laser traps, is a technique that exploits the force generated from laser radiation pressure to catch or trap a small particle and the method therefore offers a direct way to measure packaging force and speed (40, 85, 86). A partially packaged motor complex had a microsphere attached via unpackaged DNA.

This microsphere was caught in an optical trap and tethered to a second bead. Upon the introduction of ATP and initiation of packaging, the two beads moved closer together. The amount of DNA tension can be monitored, and from this, the bead displacement can be calculated; various measurements of packaging dynamics are then possible, including an examination of the presence of packaging “slips” and

“pauses”, where irregularities occur in packaging speed.(87) (See Figure. 3)

When the force from the phi29 DNA packaging motor was measured, it was found that the motor can package DNA against an internal force of 57 pN, implying that the phi29 motor is the most powerful motor ever constructed to date.(40) The result also indicates that the viral capsid containing the tightly packed DNA should be able to withstand substantial internal pressure. Using this method, it was possible to determine the speed of phi29 DNA packaging, which was initially around 100 bases per second, gradually slowing as the capsid was filled (40, 85, 86).

The optical tweezer has also been applied to measure ATP hydrolysis during phi29

DNA packaging (88). It was found that the phi29 motor packages DNA at increments of

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10 bp, with four 2.5 bp substeps for each ATP hydrolysis. A coordinative mechanism in phi29 DNA packaging was suggested according to the results (88).

Direct observation of motor motion through fluorescence imaging

Fig. 1.3. Packaging of neutral DNA analogues by optical tweezers (Adapted from Ref.

(87) with permission)

The motion of DNA during its translocation into the procapsid has been tracked by fluorescent imaging. (89). A fluorescent bead is attached to the genomic DNA and this amplifies the signal in order that it may be directly observed by a fluorescence microscope. Its motion can be diagrammed in 3-dimensions, along the x, y and z axes.

The DNA packaging can also be stalled by the addition of non-hydrolysable γ-S-ATP, and then restarted by the addition of ATP, and this starting and stopping can be observed in real time by fluorescence microscopy. As the DNA molecules are packaged into the procapsid, the fluorescent bead shows a gradual reduction in swing range.

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Once the DNA is completely packaged, the motion stops, and the fluorescent dot appears as a zero distance change from the reference origin under the CCD camera.

3.2 Symmetry argument: pentamer or hexamer

In addition, pRNA with fluorescence-labeling technology has been successfully adapted to the single-molecule fluorescence technique for stoichiometry studies, as described above, to resolve whether there are five or six copies of pRNA in an intact motor.(89,

90) As described earlier, the phi29 DNA packaging motor contains a six-fold (12- subunit) connector attached to a five-fold symmetrical capsid shell. The pRNA molecules form a ring and have been found to participate in the motor motion. However, whether pRNA stoichiometry is 6-fold (a hexamer) or 5-fold (a pentamer) is under debate. Scientists that support the hexamer formation argue that pRNA binds to the connector (12 subunits and six-fold symmetry), while the pentamer supporters claim pRNA binds to the procapsid shell, (five-fold symmetry) (91). This important piece of information regarding the symmetry of the ring is very important for elucidating the motor mechanism. (See Figure. 4)

Two labs independently reported six copies of pRNA formed a hexamer configuration when incorporated into the phi29 motor (63, 92).Crosslinking experiments revealed that pRNA does not bind to the capsid proteins, but to the connector (50). Additional study provided solid evidence that the pRNA interacted with the N-terminus of the connector protein gp10, specifically, the three basic amino acids at its N-terminus (93). Under the condition in which two of these three amino acids were mutated, pRNA binding to the

DNA packaging motor was nonexistent. Further, recent single molecule counting using

14 the very sensitive dual view total internal reflection fluorescence microscopy revealed that each DNA packaging motor contained six copies of pRNA (61, 89).(Figure 5)

Fig. 1.4. Left: Illustration of hexmeric RNA. Right: Cryo-EM images showing that the pRNA forms hexamer (A) and pentamer (B) from two different laboratories using the similar approaches (Adapted from Ref. (41) and (91) with permission)

A recent novel study based on the use of pRNA interlocking loops investigated the configuration necessary to accomplish DNA packaging. Here, two inactive pRNAs, such as pRNA Cd' and Dc' were mixed in a 1:1 molar ratio and this resulted in full production of infectious virions, indicating that the stoichiometry of the pRNA must be a multiple of two. The situation in which three inactive pRNAs, Ab’, Be’ and Ea’ became fully active when mixed together suggested that the number of pRNAs in the DNA packaging complex is a multiple of three. That the common multiple of two and three is six leads to the hexamer conclusion. Further, single molecule imaging in this report revealed that the ring was formed by either a pure dimer or by pure trimer alone. Again, this data

15 strongly supports the argument that the ring is a common multiple of two and three, hence a hexamer.

Fig. 1.5. Dual-view imaging of procapsids containing both Cy3-pRNA I and Cy5-pRNA

II. (A) pRNA dimer constructed with Cy3-pRNA I and Cy5-pRNA II. (B) Typical fluorescence image of procapsids bound with dual-labeled pRNA dimers. The yellow color spots are the combination of green and red, representing coexistence of Cy3- pRNA I (green) and Cy5-pRNA II (red) on one procapsid. (C) Fluorescence intensity versus time to show photobleaching steps of procapsids reconstituted with the dimer, elucidated in the inset showing procapsids with a pRNA hexamer composed of three

Cy3-pRNA I and three Cy5-pRNA II. (Adapted from Ref. (89) with permission)

16

Although these results taken together may seem straightforward and undisputable,

Cryo-EM experiments have fueled the debate. While some results indicated a hexameric pRNA ring (41), others reported a pRNA pentamer ring on the procapsid (91,

94, 95). These conflicting results may be the result of the fact that RNA is sensitive to

RNase degradation during the sample preparation and because cryo-EM is based on averaging, RNA degradation might result in an underestimation of stoichiometry.

Furthermore, pRNA is relatively flexible and therefore, the pRNA arrangement might not be perfectly symmetrical. Either situation could promote the underestimation of pRNA molecules in the ring.

Those in support of the pRNA pentamer formation further theorize that pRNA hexamers could be formed initially, but after binding to procapsids, one of the pRNAs was dissociated from the procapsids leaving five pRNAs still bound (91). However, the argument of one pRNA molecule leaving after procapsid binding was not supported by the finding that covalently linked pRNA dimers are active in DNA packaging (96).

3.3 ATP consumption translated to force generation by the DNA packaging motor

Molecular motors need energy to function and most use chemical energy from the hydrolysis of ATP. The active sites on motor proteins bind ATP molecules and catalyze the decomposition to ADP and inorganic phosphate (Pi), thereby releasing a significant quantity of energy, which in turn leads to conformational change in the motor protein, ultimately resulting in motor movement. This catalytic process repeats with another ATP molecule so that the motor protein can continue the movement. DNA packaging is entropically unfavorable, since the DNA arrangement within the capsid is extremely

17 compact, and the packaged DNA undergoes an approximately 30- to 100-fold decrease in volume compared to that before packaging.(10) ATP hydrolysis provides the driving force for viral DNA-packaging motors. ATP is believed to be consumed for the initiation of DNA packaging and translocation, with all components of the packaging system, including pRNA, procapsid, gp16, and DNA-gp3, involved in the generation of maximal

ATPase activity.

Applications of Phi29 DNA Packaging Motor

In nanotechnology, a nanomachine is defined as a mechanical or electromechanical device with nanometer scale dimensions.(97, 98) The nanomachines found in living systems include motors, arrays, pumps, membrance cores and valves and these are constructed from \protein, DNA, and RNA. Remarkable efforts have been exerted to understand and develop such nanomachines for their potential applications in various related fields.(99, 100)

In a separate approach, bottom-up assembly is utilized to construct nanostructures from building blocks of DNA(101-104) RNA,(5, 105, 106) and protein(93, 107-109). This has definitive advantages over chemically synthesized materials because biomolecules allow for further modification, including site-directed modification or specific conjugation with defined stoichiometry. Further, self-assembly may be a feasible construction path.(5, 102-106) In addition, high precision in replication and manipulation offers strong advantages for applications of tissue engineering, cell scaffolding, drug delivery, sensors, imaging, and nanomedicine.

18

Applications of the phi29 DNA packaging motor to nanotechnology

The phi29 DNA packaging motor itself as well as its individual motor components can be used in a variety of nanotechnological applications. First and foremost is the incorporation of the packaging motor into nanomachines. To date, the motor particles have been attached to the nanopores of Anodic Aluminum Oxide (AAO) membranes.

The attachment is either to the aldehyde-silanized inner surface, or by centrifugation.

The 40 nm pores of the membrane were capable of separating empty procapsids from

DNA-filled procapsids. The nanoporous AAO membranes could therefore be utilized for future interfacing of the phi29 motor with artificial nanostructures (110).

Incorporation of the phi29 DNA packaging motor may also prove beneficial in gene therapy. It is known that this nanomotor uses ATP hydrolysis to provide the energy necessary to accomplish the translocation of dsDNA. If an antibody labeled connector and pRNA were fused to a targeted cell membrane, this would allow the construction of an intact active motor. Furthermore, the N-terminal and C-terminal of the connector can be fused with cell membrane penetrating peptide such as TAT, polyarginine or penetratin, which will help the connector insert into the cell membrane. By using the energy generated from ATP hydrolysis, the motor may introduce functional genes into the cells with the goal of rectifying genetic deficiencies or translocating therapeutic siRNAs into cancer or viral-infected cells to induce apoptosis.

Applications of the phi29 connector to nanotechnology

The phi 29 connector has been artificially constructed through various means, including

N- or C-terminal truncations, extensions, or insertions of extra amino acids.(111, 112)

The addition of a cleavable His- or Strep-tag onto either the N- or C-terminus of the

19 connector facilitated protein purifications to near homogeneity. Neither connector nor procapsid assembly was affected by truncations/extensions up to 14 residues at the N- terminus and up to 25 residues at the C-terminus.(112) However, if the Arg-Lys-Arg residues were deleted from the N-terminal of the connector, it could not bind pRNA, as the deleted residues are essential this binding. A 25-residue deletion and/or 14-residue extension at the C-terminus of gp10 did not affect procapsid assembly. A 42-amino acid extension at the N-terminus did not interfere with the procapsid assembly but significantly decreased the DNA packaging efficiency. (112)

4.2.1 Connector arrays and ellipsoid nanoparticle

Phi29 connectors have been constructed in vitro into an array format(33, 60, 93, 113).

The final assembly has potential in the construction of nanostructures. The employment of a supporting lipid monolayer allowed the construction of a uniform and highly ordered single layer array of connectors (60).

Interestingly, the addition of pRNA to this system resulted in the conversion of tetragonal arrays into larger decagonal structures. That this conformational shift was caused by the pRNA attachment to the connector was confirmed by RNase digestion.

This conformational shift can potentially be used as a source of force for biomotor motion. Further, both the connector array and the decagon can be used as templates to build patterned suprastructures in nanotechnology. (Figure 6 top)

A 24 × 30 nm homogeneous ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered connector was also constructed recently, as shown in figure 6 bottom.

20

Fig. 1.6. Top: Multilayer versus single layer sheet arrays of phi29 motor. Bottom: 24 ×

30 nm ellipsoid nanoparticles containing 84 subunits or 7 dodecamers of the connector.

(Adapted from Ref. (60)and (117) with permission).

21

4.2.2. Membrane-adapted phi29 motor protein nanopores and DNA translocation

The connector protein was incorporated into lipid bilayer membrane via a two-step strategy (114), firstly by reconstitution into proteoliposome, and then by membrane fusion between the connector-containing liposome with the artificial lipid bilayer. The connector acts like other ion channels or membrane proteins but with a much wider channel (Figure 7 top). The insertion of each connector produced a steady current change corresponding to the insertion of an individual channel. This is the first viral portal protein incorporated into a lipid layer, and Q-PCR experiments confirmed the translocation of dsDNA through the nanopore. The linear 5.5-kilobase plasmid DNA,

141-bp DNA and 35-bp DNA were able to be translocated through the channel upon an applied transmembrane potential of ±75 mV, while circular plasmid DNA could not pass through the channel. The translocation of each DNA produced a blockade in the current through the channel, and single molecule translocation behavior can be characterized by the dwell time and blockade percentage (Figure 7 bottom).

The properties of the phi29 connector were also investigated (115), showing a robustness at different salt concentrations and buffer pH, without demonstrating gating properties. These discoveries provide a system for future electrophysiological studies of the phi29 DNA packaging motor. Furthermore, the connector is a biological nanopore that is extremely reproducible and easily engineered, making it suitable for future biomedical and nanotechnological applications.

22

Fig. 1.7. Images of giant liposomes containing connector, and translocation of dsDNA through connector channels in a BLM. (Adapted from Ref. (114) with permission)

23

4.2.3 The DNA-packaging motor as a DNA-sequencing apparatus or molecular sorter

The development of a nanopore-based DNA sequencing device would have tremendous application in the area of genome mapping(116). As designed, the phi29

DNA-packaging process involves movement of the DNA through the pRNA bound connector channel Therefore, theoretically, the channel can be modified to accept chemical or electrical signals and may subsequently possess the ability to recognize a single base pair, based on the signals generated through the interaction of the bases of the DNA with a pore.

Applications of the phi29 pRNA to nanotechnology

Phi29 pRNA has two functional domains: the procapsid binding domain and the DNA translocation domain, which is located at the 5’/3’ paired ends. The two domains fold independently of each other.(Figure 8) There exist complementary loop sequences at the procapsid binding domain and these allow for the formation of pRNA multimers(119)(Figure 9). The pRNA dimers, trimers, hexamers and arrays have been observed by transmission electron microscope with nanogold-labeled pRNA (60) and cryo-EM.(5, 105) These pRNA nanoparticles can be used as the building blocks for the construction of suprastructures.(Figure 10)

The unique formation of multimers and patterned superstructures via loop-loop interactions (63, 92, 105, 120, 121) make pRNA a promising component not only for nanomachine fabrication but also for gene delivery. Several RNA-based therapeutic approaches using small interfering RNA (siRNA) (122-125), ribozymes (126-129) and anti-sense RNA (130) have been shown to downregulate specific gene expression in

24 cancerous or viral-infected cells. Although the methods for gene silencing with high efficacy and specificity have been achieved in vitro, the effective delivery of RNA to specific cells in vivo remains challenging. Further, the therapeutic applications of siRNA have been hindered by the lack of an efficient, safe, and nonimmunogenic in vivo delivery system to target specific cells. (Example see Fig. 11)

Because multimer formation is based on complementary loop sequences at the procapsid binding domain, replacement of or insertion into the 5’/3’helical domain does not interfere with multimer formation. (120) Thus, end conjugation of pRNA with a receptor-binding RNA aptamer, small interfering RNA (siRNA), or ribozyme may not disturb multimer formation or interfere with the function of inserted

Fig. 1.8. Method for the construction of pRNA nanoparticles by loop/loop interaction.

(Adapted from Ref. (118) with permission)

25

Fig. 1.9. Illustration of constructing pRNA multimers. A. pRNA chimeric monomer with

3’-end annealed oligo which conjugate the detection molecule and chemical ligand at the same time. B. a. Twin dimer formed through palindrome sequence; b. Dimer formation through loop-loop interaction. C. Tetrameric RNA chimera. D. pRNA trimer. E. pRNA hexamer. (Adapted from Ref. (119) with permission)

26

Fig. 1.10. pNA as building blocks for nanotechnology. A and B: A mixture of two complementary twins, A−b’ and B−a’, assembled into two distinct supramolecular structures. A.Two complementary twins were able to form a stable tetramer (double- twins) by assembling into a circular structure. B. Concatemers of alternating twins formed when a twin interacted with two rather than one complementary twin. C. Atomic force microscopy (AFM) showing arrays of pRNA. (Adapted from Ref. (5) with permission)

Fig. 1.11. Top: Illustration of constructing pRNA multimers and polyacrylamide gel showing pRNA multimers in native (upper panel) and denatured (lower panel) condition.

Bottom: Specific GFP knockdown assay of chimeric pRNA/siRNA (GFP) in KB cells.

(Adapted from Ref. (119) with permission)

27 moieties. (131-134) Therapeutic siRNA and receptor-binding RNA aptamers have been engineered into individual phi29 pRNA molecules and the pRNAs harboring the therapeutic molecule were subsequently fabricated into trimers. The polyvalent pRNA complex can deliver up to six kinds of therapeutics to specific cells, as demonstrated in breast cancer, leukemia, lung cancer and prostate cancer cell lines(134) as well as hepatitis B virus infected cells.(135) A pRNA-based vector was designed to carry hammerhead ribozymes that cleaved the hepatitis B virus (HBV) polyA signal (135).

Another pRNA/ribozyme (survivin) chimera which targeted the anti-apoptosis factor survivin was shown to suppress survivin expression and initiate apoptosis (133). Anti- cancer drugs could also be attached to a pRNA subunit to enhance the therapeutic effect or overcome the drug resistance by combination therapy (135).

Not only can pRNA multimers carry multiple therapeutic loads, they also have the advantage of size. The particles of sizes of 20 to 40 nm will provide an opportunity for repeated administration and treatment of chronic diseases. This will avoid the problems of the short half-life of smaller molecules encountered in vivo, due to short retention times and the fact that these smaller molecules are unable to be delivered to cells of molecules larger than 100 nm. Using such protein-free, controllable nanoscale RNA particles as therapeutic delivery reagents additionally avoids antibiotic response.

A final advantage results from the ability of the pRNA nanoparticles to target specific cells. Incubation of the chimeric pRNA complex containing receptor-binding aptamers or folate resulted in cell binding and transport of the chimeric pRNA/siRNA, pRNA/ribozyme, or drugs into cells, consequently modulating programmed cell death.(131, 133, 134) Target delivery and specificity were brought about by engineering

28 a subunit in the complex to include cell receptor-binding ligands for receptor-mediated endocytosis. The conjugation of pRNA with folic acid molecules may help target certain kinds of cancer cells that have folate receptors highly expressed on the cell surface.(132) Another subunit can carry components to facilitate endosome disruption for the release of therapeutic molecules. The efficiency of this procedure was confirmed in animal trials.(134) The application of in vitro SELEX (136, 137) to screen RNA aptamers which bind to specific targets has become a powerful tool for selecting RNA molecules specific to cell surface receptors. To facilitate independent folding, a poly U or poly A linker might be placed between the pRNA and the aptamer.

Perspectives

Specific motor parts of the phi29 bacteriophage, as well as the overall motor, have great potential for use in nanotechnological applications. These include the targeted delivery of therapeutic agents to cells, precise single-molecule sequencing techniques, and direct attachment of various motor parts to nanodevices.

The properties of the connector can be modified by engineering either the N- or C- terminus of the gp10 protein through deletion, extension, insertion, or tagging. The engineered connectors can then be used as building blocks for the bottom-up assembly of single- or multiple-layered arrays. Their incorporation into lipid bilayers demonstrates a system with tremendous potential for dsDNA sequencing or single molecule sensing.

The ability of phi29 pRNA to form dimers, trimers, and hexamers can potentially be utilized to construct polyvalent gene/drug delivery vectors or self-assembling arrays.

Small packaging RNA can be used to construct nanoparticles as polyvalent vehicles for the delivery of multiple therapeutics, such as siRNA or drug molecules, with specific cell

29 targeting for the treatment of cancers, viral infections, and genetic diseases. The pRNA arrays can be useful as building pieces in nanotechnology for tissue engineering.

Acknowledgments

We sincerely thank Dr. Peixuan Guo for advice in preparing this review. The work was supported by the National Institutes of Health through the NIH Roadmap for Medical

Research (PN2 EY 018230).

30

CHAPTER 2 RECONSTITUTION OF CONNECTOR INTO LIPID BILAYER FOR

dsDNA TRANSLOCATION

Introduction

Nanotechnology has been utilized in nature since the beginning of life to process fundamental biological process. By studying their mechanism and developing artificial nanomachines, we will be able to understand more about biological entities and influence the behavior vice versa. Bacteriophages such as phi29 are the classical examples of natural nanomachines. They use nanomotors to package their viral genomes into a preformed nanometer dimension capsid. This delicate biological nanomatchine can carry several tasks during the different stages of a phage’s life cycle, such as controlled assembly of a delicate maching packaging the viral genome, and inject the viral genome during the stage of infection into the host cell.

As one of the most well studied virus, different components of phi29 DNA packaging motor has been studied extensively during the past decades. A key component of bacteriophage phi29, like many other double-stranded DNA (dsDNA), is a truncated cone shaped portal protein (Fig 2.1a). The connector of phi29 DNA packaging consists of twelve subunits, and forms a central channel with a width of 3.6 to 6.0 nm (Fig 2.1b).

During the stage of DNA packaging and stage of viral infection, the viral genome will enter and exit this channel respectively (Fig 2.1c). Traditional physical methods to study this protein channel include cryo electron microscopy (EM), x-ray crystallography, atomic force microscopy (AFM), etc, but none of these methods could provide real-time

31 dynamic information of the channels. Biological characterization such as DNA packaging assay, viral assembly assay are able to verify the package and release of viral genome, but real-time single-molecule studies are still desired to study the dynamics of genome translocation process.

Native ion channels only allow the transport of small molecules such as water and ion, and most biological channel are not wide enough for the transport of large molecules such as DNA. Biological nanopores, such as α-hemolysin (138-142) or MsPA

(143) have been investigated for the applications of DNA transport and characterization.

However, due to the smaller diameter of the α-hemolysin channel, it only allows the transport of single-stranded DNA. Synthetic nanopores (144-147) are prepared by electron beams drilling holes on silicon substrate, but the diameter and shape of synthetic nanopore are relative difficult to control.

Due to the portal nature of phi29 connector protein channel, it’s interesting to investigate the channel conductance and DNA transport capability. With modifying the classical patch clamp instruments and combining the lipid bilayer membrane technique, a design for assessment of the connector channel by electrophysiological measurement had been designed. However, there are several obstacles to achieve this goal. First, to incorporate a non-native membrane protein into lipid bilayer; Second, to assay and confirm the transport ability for different types of DNA.

A two-step strategy was designed to study the connector channel in vitro. The first step is to incorporate the connector protein into liposome, and the second step is to insert the connector channel into a preformed lipid bilayer by membrane fusion with the connector-containing proteoliposome(Figure 1.7E). By creating a highly conductive

32 channel in the lipid membrane, the passive transport of DNA was assayed and confirmed.

MATERIALS AND METHODS

Phospholipid Lipid 1,2-diphytanoyl-sn glycerol-3-phosphocholine (DPhPC) was purchased from the company of Avanti Polar Lipids (Alabaster, AL). The fluroscent labeled phospholipid N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-

Glycero-3-Phosphoethanolamine, Triethylammonium Salt (NBD-PE) were purchase from Molecular Probes. Fluoro TagTM Fluorescein isothiocyanate (FITC) conjugation kit was purchase from Sigma-Aldrich. n-Decane and hexane were purchase from Fisher.

C-His tagged connector construction

Connector protein mutant with 14 histidine (His) tagged to C-terminus was used in this study. The cloning, expression and purification of these C-His connector has been reported in the recent publication in Guo lab (112). After the transformation of plasmid vectors into E. Coli, conector protein was expressed. His-affinity chromatography was used for the His-–tagged connector protein. 10% SDS-PAGe was used for the analysis of the purified connector protein. (Figure 2.1f)

FITC labeling of the connector protein

A Fluoro TagTM FITC conjugation kit from Sigma-Aldrich was used to label the connector protein, and the labeling procedure of the kit was followed. First the buffer containing the connector protein was exchanged to a sodium carbonate / bicarbonate buffer by column chromatography for optima labeling efficiency. Under gentle stirring, the connector in sodium carbonate / bicarbonate buffer was added with FITC solution

33 dropwise and incubated for two hours (under continuous stirring). Column chromatography from sigma-aldrich was used to remove the free unlabeled FITC, and then PBS buffer was used to elute the labeled connector protein. The purity of the connector was examined by 10% SDS-PAGE electrophoresis. UV-Vis spectrophotometer was used to measure the molar ratio of fluorescein to protein for the calculation of labeling efficiency.

Incorporation of the connector protein into giant liposome

A classical dyhydration-rehydration method was used to reconstitute the connector protein into liposome vesicles. A total volume of 1 ml lipid was mixed with NBD-PE with a 100:1 ratio in a glass vial with aTeflon seal. The solvent chloroform was evaporated away by a gentle flow of nitrogen gas to avoid oxidation. 2 ml of 250 mM sucrose in 1 M

NaCl buffer was used to hydrate the dried lipid layer to form vesicles overnight. After formation of the vesicle, bright field microscopy can be used to observe the form vesicles. Fluorescent labeled lipid can be used to prepare the fluorescent labeled lipid vesicle. (Figure 1.7A). To prepare the proteoliposome containing connector protein, connector protein was added during the rehydration step. The final molar ration of lipid to connector was 75:1 (for fluorescent imaging), and the ratio of from 4000:1 - 16000:1 for lipid bilayer insertion experiments.

Separation and filtration of the connector-containing proteoliposome

In order to remove the free connector protein from the proteoliposome, sucrose gradient sedimentation (5–20% linear) in TMS buffer (50 mM Tris, 100 mM NaCl , pH

8.0, 10 mM MgCl2). 0.1 mL of raw connector-containing proteoliposome was loaded to the top of 5 mL gradient. The sample on the gradient was spun for thirty minutes at

34

27,000 rpm (Beckman L-80 ultracentrifuge, SW55 rotor) at 20°C. Then the sample was collected and 10% SDS-PAGE was use to analyze the fractions (Fig 1.7c). Filtration method was also used to remove the free connector. A cellulose acetate membrane

(0.45 µm pore diameter, Life Science Products, Inc.) was used to filtered the proteoliposome. 500 µL volume of the prepared connector-containing liposome vesicles filtrated. After spinning for 15 minutes under 3000×g, the liquid retained was less than two hundred microliter, and the filtration process was repeated 5 times. The final samples were collected from the top of the filter, and then fluorescence microscopy was used for imaging (Fig. 1.7B).

Lipid bilayer electrophysiological measurements

The electrophysiological detection and data acquisition system consist of the following parts: A pair of Ag/AgCl electrodes (pellet, 1.5X3.0 mm, Warner Instruments

LLC) was connected to electrical wires via Ag wire (70 mm length, Warner Instruments

LLC). Another end of the wires was connected to a 1 mm pin (Warner Instruments LLC), which is used to be inserted directly into the headstage. The measured current across the lipid bilayer was then sent a patch clamp amplifier (Axopatch 200B). An Axon

DigiData 1440 analog-digital converter (Digidata 1440A, Molecular Devices) were used to digitalize the signal, which is then stored in computer by the PClamp 9.1 software

(Axon Instruments).

Insertion of the connector into lipid bilayer

BLM Cells (Chamber for Lipid Bilayers) (BCH-1, Eastern Scientific LLC) were used to form horizontal lipid bilayer membrane. A Teflon partition with 0.2 mm hole

(Eastern scientific LLC) was attached to the bottom of the BLM cell by grease, and then

35 put on a Mini dish (Eastern Scientific LLC) to form a chamber with two compartments.

The chamber, headstage and custom made insulation box were put onto a bilayer workstation active table (BILAYER WKSTN ACTIVE TBLE 120, Warner Instruments LLC) to minimize the effect of noise and vibration during the recording.

A pre-painting with 0.5 µL 5% hexane solution to the aperture was done to help the better formation of lipid bilayer. The top compartments had a working volume of 0.2 L, and the bottom chamber had a working volume of 2 L. After assemble the chambers with prepared partition, working buffer solution was added to the two compartments, and two electrodes were put into the two compartments respectively. A painting technique with air bubbles by pipette was used to form a thin lipid bilayer around the aperture, until a lipid bilayer think enough had been achieved.

The connector-containing proteoliposome was extruded through a 400 nM membrane filter to form small vesicles. After diluted to 10 times, 1-2 µL SUV containing the connector was added to the buffer in top compartment. The cross-membrane holding potential used in this study was ± 30 mV, ±45 mV and ± 75 mV. An applied ramping voltage -100 mV to +100 mV was also used to measure the channel conductance.

Passive translocation of dsDNA driven by electric field

DNA was premixed in the buffer in the compartments, or added after the channel insertion into lipid bilayer. A certain voltage was applied to drive the transport of the

DNA.

36

RESULTS AND DISCUSSION

Reconstitution of the connector into liposomes

To facilitate the interaction of connector protein with lipid bilayer, 14 histidine were added to each subunit of the connector protein. An analysis of the connector surface was shown in fig 2.1a with acidic, basic and other amino acids. Three color of red, blue and white were used for representation respectively. This hydrophilic-hydrophobic- hydrophilic layer structure allows a higher possibility of insert the connector into lipid bilayer. The modified connector had a larger molecular weight as shown in the SDS-

PAGE in Fig 2.1F.

The diameter of the prepared giant liposome ranges up to 50 µm (Fig 1.7a). After sedimentation and filtration, unlabeled free dye and connector protein were removed.

From the SDS-PAGE gel containing the samples in direction of sedimentation in Fig

1.7C, it was noted that the connectors in liposome appeared in the bottom of the gradient, while free connectors showed in the upper fractions. Filtration studies (Fig

1.7b) also shows the different before and after filtration. However, these methods couldn’t distinguish the difference between connector attachment to liposome membrane surface, or inserted into the lipid membrane.

Fluorescent labeled liposome showed a clear ring outside the membrane (Fig 1.2A, left). In another control experiment, FITC labeled connector with unlabeled liposome showed a similar ring (Fig 1.2A, middle). In the control experiment of simple mixture of formed liposome with FITC connector, it is seen that there were no such a ring (Fig 1.2A, right).

37

Connector channel insertion into planar lipid membranes

Single channel recording experiments had the advantage of detecting extremely lower intensity of electrical current. Since lipid bilayer is a highly insulation materials, it would stop the current and ion transport between the two compartments once formed. If the connector channel was inserted into the membrane, a electrical signal should be able to be detected. In two control experiments of adding samples in the buffer, the lipid bilayer kept intact (Fig 2.2A). This indicated that without liposome, the connector would not be able to be integrated into lipid bilayer. After addition of connector containing proteoliposome under -45 mV, continuous stepwise current drops were observed (Fig

2.2b). The current jump for one pore insertion was 69.9 pA, and simultaneous insertion of two pores was occasionally observed. It is also observed that the conductance of the channel was independent with the polarity of charge applied (Fig 2.2c). The current jump were similar under -30 mV and +30 mV.

All of the above experiments results indicated that a pore in the lipid membrane had been formed along with the addition of connector channel. Combined with the control experiments, only proteoliposome could create the insertion of such nanopore.

Conductivity of the channels

Conductance was used to describe the ability of channel for conducting an electric current, which is defined as G = I/V (siemens). In the buffer of 0.5 M Sodium chloride, 5 mM Tris and pH 7.9, the peak of one connector insertion step is 65 pA. A minor peak of

130 pA was observed, which represented the simultaneous insertion of two channels. In an I-V relationship plot (Fig 2.2e), it is clearly seen that a linear relationship between trans-membrane current and applied potential.

38

In a trial with scanning potential applied across the membrane, the slope of the current traces was proportional to the number of connectors in the channel (Fig 2.2d).

For example, the slope of one pore was 1.59 nS, and the slope of two pores was 3.40 nS, and the slope of three pores was 4.98 nS.

Translocation of double-stranded DNA

It was also interesting to find that with addition of DNA added to the chamber, there were additional spikes observed on one polarity of the trace. To confirm this result, both circular DNA and linear dsDNA were use for translocation test. In the control experiment without the presence of DNA (Fig 1.7middle paned), it is seen that there was no spikes.

A similar result was observed for the 5.5 kb circular plasmid DNA (Cx43), except for few non-specific events. However, the linear dsDNA showed a series of blockade event during translocation (top right of Fig 1.7 lower panel). After the treatment of DNaseI, a steady increase of events number were observed in both the circular plasmid DNA group, and the linear DNA group. Since the DNaseI could break the plasmid DNA and linear DNA into small fragments, this suggested that the increase of the spike events were induced by the increase number of DNA copies by digestion. All these results confirmed that the dsDNA was translocated through the channel.

Several parameters were used to characterize the DNA translocation event: Dwell time and blockade percentage were the most important two. The dwell time described the time of a DNA needed to going through channel, and the blockade percentage described the current of a event temporally reduce the current going through an intact channel.

Quantitative PCR (Q-PCR) was also used to quantify the numbers of DNA passage

39 through the channel.

Comparison of the phi29 connector channel with α-haemolysin

Reconstitution into planar lipid bilayer has been applied for ion channels, and is only capable of translocation ions but not larger molecules such as DNA. α-haemolysin was used for the electronic detection of a DNA molecule passing through it, and this is the start of the nanopore sequencing field (148), but it only allows the transport of single- stranded DNA due the limit of channel size 1.5 nm (138). Phi29 connector channel is the first viral protein channel that is reconstituted into lipid bilayer (114). Unlike α- haemolysin which can form protein channel by self-assembly, the phi29 connector channels needs to be engineered for better reconstitution result. Additional step of reconstitution into liposome is also required.

Applications

The phi29 connector based nanopore-lipid platform can be in various aspects, and falls into two major category: Study of the portal channel property, and sensing of small molecules. We have measured the conductivity of the connector channel in various pH, ion concentration, and ion types, the discovered the robust property of the channel (114,

115). The channel conductance has an almost perfect linear relationship with the ion concentration, and is stable between ph2 to pH 12. We also discovered that the transport of dsDNA through the channel has a one-way traffic property (149), that is the dsDNA can only travel from N-terminus (narrow end) to C-terminus (wider end). This observation by electrophysiological measurement coincides with the phenomena of genome transport during DNA packaging. The real-time conformational change of the connector channel has also been observed on this platform (150). Real-time

40 controllable and reversible opening and close of the connector channel upon environmental stimulus are observed and confirmed. Another version of the channel with lysine-234 mutated to cysteine were used detect the chemical molecules with the

SH group, including ethane (57 Da), thymine (167 Da), and benzene (105 Da). These chemicals were discriminated upon interaction with the cysteine residues. (151).

Other potential applications include drug screening, DNA/RNA base pair discrimination and sequencing, channel-size modification by molecule clone engineering, and coordination of moieties into the wall of channel for additional sensing purpose.

Limitations

Several problems are still needed to be addressed for future applications of connector / lipid nanopore system: Stability of the connector in the membrane, sampling frequency of the instrumentation recording, and translocation speed reduction for a better resolution for small molecule sensing. Lipid is relatively fragile compared with polymer or solid state materials, thus a better hybridization method could be developed to make the nanopore more robust either by modification of the lipid component, or immobilization onto substrate. This will open the industrial application of the connector nanopore system. Another limitation is the electrical recording sensitivity is constrained by the sampling frequency, which is 20 kHz (5 µs) interval in the current experiment setup. A more sensitive recording method is desired for the better discrimination of DNA or RNA during translocation at single nucleotide level.

41

CONCLUSION

In conclusion, the GP10 connector protein of bacteriophage phi29 was modified to increase the hydrophilicity at the C-terminus, and then incorporated into artificial lipid bilayer to form a nanopore with large conductivity. The conductance of the channel was measure to be 3.2 nS in 1 M NaCl with TMS. The capability of double-stranded DNA transport through the channel was also invested by both circular plasmid DNA and linear dsDNA. Further experiment of quantitative PCR confirmed that dsDNA can be translocated passively through connector channel driven by an external applied voltage.

This system developed in the study opens a new door for the study of viral portal channel and the mechanism of viral genome packaging and infection, as well as future application in nanotechnology and nanomedicine for drug delivery, drug screening and

DNA sequencing due to its extraordinary stability and ability for molecular engineering and chemical modification.

42

Fig 2.1 Connector protein of phi29 DNA packaging motor and its reengineering. a. Side view of the connector. b. Top view of the channe; c. Illustration of the components of phi29 DNA packaging motor. d. TEM image of connector protein. e.

TEM image of the assembled connector protein array. f. SDS-PAGE gel showing the connector protein before and after c-terminus modification. (Adapted from Ref (114) with permission)

43

Fig 2.2 Conductance assay of the current traces of connector channel. A.

Control experiment showing the current traces of adding connector protein only (top) and adding liposome only (bottom). No current changes or jump were observed. B.

Current traces showing a recording of continuous insertion of multiple connector proteins. Top insert: Current jump of 69.9 pA (one channel). Bottom insert: Current jump of 130.9 pA (two channel inserted simultaneously). C. Current traces and jump under

44 positive and negative applied potential: Top, +30 mV. Bottom, -30 mV. D. Histogram of current jumps. E. Relationship between channel current and applied trans-membrane potential. F. Current recording traces under scanning potential (-100 mV to +100 mV).

Traces with different slopes showing the different numbers of connectors in the membrane. G. Current traces showing the traces through one connector channel, with the presence of dsDNA in cis chamber. (Adapted from Ref (114) with permission)

45

CHAPTER 3. REVERSIBLE AND CONTROLLABLE GATING OF THE phi29

CONNECTOR CHANNEL

This chapter was published in Biomaterials, 2011, Vol 32, 8234-8242.

Jia Geng, Huaming Fang, Farzin Haque, Le Zhang and Peixuan Guo*

ABSTRACT

The channel of the viral DNA packaging motor allows dsDNA to enter the protein procapsid shell during maturation and to exit during infection. We recently showed that the bacteriophage phi29 DNA packaging motor exercises a one-way traffic property using a channel as a valve for dsDNA translocation. This raises a question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel inward.

We proposed that DNA forward or reverse travel is controlled by conformational changes of the channel. Here we reported our direct observation that the channel indeed exercises conformational changes by single channel recording at a single- molecule level. The changes were induced by high electrical voltage, or by affinity binding to the C-terminal wider-end located within the capsid. Novel enough, the conformational change of the purified connector channel exhibited three discrete gating steps, with a size reduction of 32% for each step. We investigated the role of the terminal and internal loop of the channel in gating by different mutants. The stepwise conformational change of the channel was also reversible and controllable, making it an ideal nano-valve for constructing a nanomachine with potential applications in

46 nanobiotechnology and nanomedicine.

KEYWORDS: Nanotechnology; bionanotechnology; DNA packaging; viral motor; nanopore; single molecule sensing.

1. INTRODUCTION

Linear double-stranded DNA viruses package their genomic DNA into a preformed protein shell called the procapsid (6, 8). This DNA encapsulation task is an intriguing step of the viral replication cycle that is accomplished by nanomotors using

ATP as energy (12, 85, 152-154). The ingenious design of viral DNA packaging motors and the novel mechanism of action has provoked a broad range of interest among scientists in virology, molecular biology, structure biology, nanotechnology, biophysics, biomaterials, nanomedicine, RNA biochemistry, and therapeutics. In bacteriophage phi29, the nanomotor consists of a protein channel, DNA packaging ATPase gp16, and a ring composed of six pRNA (packaging RNA (46)) to gear the motor (46, 63, 92) using one ATP to package 2(12) or 2.5(88) base pairs of DNA. The protein hub of this motor is a truncated cone shaped connector (15, 34, 155), which contains a 3.6 nm wide central channel that allows the 19.3 kb dsDNA genome to enter during maturation and to exit during the infection process (Fig 1A-C). The defined phi29 DNA packaging motor, constructed 24 years ago (11), is one of the strongest biomotors (40) assembled in vitro. Elucidation of the mechanism of motor action will impact areas of biology, engineering, medicine, and various other nanotechnological fields. The novelty and ingenious design of such machines have inspired the development of biomimetics. In

47 vitro, the biomimetic motor could be integrated into synthetic nanodevices (89, 100, 114,

156, 157). In vivo, the artificial nanomotors could be used to load drugs, deliver

DNA/RNA, pump ions, transport cargos, or drive the motion of components in the heart, eye or other sensing organs in the body. Although the protein sequence of each subunit of the connector holds little sequence homology, and each subunit varies in size (155,

158-160), the connector of many viruses display significant morphological similarity

(161). The structure of the phi29 connector has been previously solved by X-ray crystallography (15, 155, 162), which shows that it is composed of twelve protein subunits which form a ring with a wider end of 13.8 nm outer diameter buried within the viral procapsid and an extruding narrow end of 6.6 nm. The central channel is 6.0 nm at the wider end and 3.6 nm at the narrow end (Fig 1C).

Many phenomena concerning procapsid expansion during the life cycle related to

DNA packaging of bacteriophage have been reported (163-170). DNA packaging significantly involves the connector. It has also been reported that the connector is a vital component in the regulation of procapsid shape and size (171, 172). Based on logical analysis, it is reasonable to believe that procapsid expansion is linked to the connector conformational change. However, direct evidence of a conformational change of the connector has never been reported.

48

Figure 3.1. Illustration of the phi29 connector channel structure. (A) Side view without showing the C- and N- terminal fragment (155); (B) Side view and (C) bottom view with complete protein sequence (93); (D) Model of phi29 DNA packaging motor within the procapsid; (E) Illustration of dsDNA translocation through a connector channel reconstituted in an artificial lipid bilayer for the measurement of conductance.

We have recently inserted the reengineered connector into a lipid bilayer (114,

115) (Fig 1E). The translocations of ions and dsDNA through the channel demonstrate the potential to use the connector for DNA sensing and fingerprinting at the single- molecule level. We also demonstrated that the phi29 motor channel serves as a valve and exercises a one-way DNA traffic mechanism (149). The direction of DNA trafficking

49 is from the N-terminal narrower end to the C-terminal wider end. This raises a question of how dsDNA is ejected during infection if the channel only allows dsDNA to travel one way. It has been proposed that the motor connector adopts a conformational change after DNA packaging is completed (165). Such a conformational change renders the channel to allow the dsDNA to come out of the viral procapsid. Here we report that the connector channel indeed exercises conformational change during gating or motor function stimulated by a variety of factors. The conformational change exhibited three discrete identical steps, with each step reducing the size of the channel by one third.

Gating phenomenon has been observed in various protein pores or ion channels

(173) and plays a key role in regulating ion transportation through a membrane. Ion channels may be classified by the nature of their gating (174), such as voltage-gated

(175), ligand-gated (176), stretch-gated(177), or other gating(174). Voltage-gated ion channels are activated by changes in electrical potential difference near the channel, while the ligand-gated ion channels are opened or closed in response to the binding of a chemical messenger (i.e., a ligand). There is also recent progress on synthetic channels, which are sensitive to the environmental stimuli, such as temperature (178), voltage

(179), pH (180, 181), or their combination (182); but they have a gating mechanism different from protein channels. Here we report a real-time direct observation of the gating of the phi29 DNA packaging nanomotor connector protein channel. It is also interesting to find that this viral protein channel gating can be induced by both voltage and ligand binding, which is similar to the other ion channels.

2. MATERIALS AND METHODS

50

2.1 Materials

The phospholipid, 1,2-diphytanoyl-sn-glycerol-3-phosphocholine (DPhPC)

(Avanti Polar Lipids, Alabaster, AL), Nickel-NTA nanogold (1.8 nm; Nanoprobes), n- decane (Fisher), chloroform (TEDIA) were used as instructed by the vendor. All other reagents were from Sigma, if not specified. The construction and purification of phi29 C- terminal tagged connectors have been reported previously (112, 114).

2.2 Regineering, expression and purification of phi29 connector

The construction of the plasmid harboring the gene coding for gp10, the over- expression of gp10 and the purification of phi29 connector have been reported recently(93, 183). The deletion of the tunnel loop (N229-N246) of gp10 was performed by two-step PCR. First, primer pair F1-R1 and primer pair F2-R2 were used to amplify the DNA sequence coding for gp10 (1-228) region and gp10 (247-309) respectively. In the second round of PCR, F1 and R2 was used as primer pairs to link and amplify the

PCR product in the first round. The second PCR product was digested with NdeI/XhoI and ligated into NdeI/XhoI sites of the vector pET-21 a(+) (Novagen). The deletion of the

N-terminal (1-14) of gp10 was performed by PCR. The sequence coding for gp10 (15-

309) region was amplified by a pair of primers. The forward primer contained NdeI restriction site; the reverse primer contained XhoI restriction site and a 6-histidine affinity tag. The PCR product was digested with NdeI/XhoI and ligated into NdeI/XhoI sites of the vector pET-21 a(+) (Novagen).

The connector mutants constructed were expressed, and then were purified with

Nickel affinity chromatography (184). Cells were resuspended with His Binding Buffer

(15% glycerol, 0.5 M NaCl, 5mM Imidazole, 10mM ATP, 50mM Na2HPO4 – NaH2PO4,

51 pH 8.0), and the cleared lysate was loaded onto a His•Bind® Resin Column (Novagen) and washed with His Washing Buffer (50 mM Na2HPO4 – NaH2PO4, 15% glycerol, 0.5 M

NaCl, 50 mM Imidazole, 10 mM ATP, pH 8.0). The His-tagged connector was eluted by

His Elution Buffer (50 mM Na2HPO4 – NaH2PO4, 15% glycerol, 0.5 M NaCl, 0.5 M

Imidazole, 50 mM ATP, pH 8.0).

2.3 Electrophysiological measurements

A bilayer was formed on a thin Teflon film partition (aperture 200 m in diameter) which separates a bilayer lipid membrane (BLM) chamber into a cis- and trans-chamber

(compartment). The cis-chamber refers to the grounded compartment to which the connector reconstituted liposome are added. Connector channel insertion into the bilayer has been described previously using vesicle fusion (114). Briefly, connector reconstituted liposomes were prepared using a dehydration-rehydration method and further extruded to form unilamellar liposomes. The reconstituted liposomes were further diluted by 10-fold using the conducting buffer before applying to BLM chambers.

The final concentration of added protein was 5-50 µg/mL.

For electrophysiological measurements, both compartments in the BLM chamber were filled with a conducting buffer (1 M NaCl, 5 mM Tris, pH 7.8, if not specified). A pair of Ag/AgCl electrodes connected directly to both compartments were used to measure the current traces across the bilayer lipid membrane. The current trace was recorded using an Axopatch 200B patch clamp amplifier coupled with the Axon DigiData

1322A analog-digital converter (Axon Instruments) or the BLM workstation (Warner

Instruments). All voltages reported were those of the trans-compartment. Data was low band-pass filtered at a frequency of 5 kHz or 1 KHz and acquired at a sampling

52 frequency of 10 KHz. The PClamp 9.1 software (Axon Instruments) was used to collect the data, and the software Origin Pro 8.0 was used for data analysis.

2.4 Probing connector conformational change using antibody

C-His connector and rabbit polyclonal antibodies (where) specific for His tag were added to the bottom chamber and measured under a voltage of -75 mV at the bottom chamber (a buffer containing 150 mM NaCl at pH 7.8).

2.5 Probing connector conformational change using Nickel-NTA-Nanogold

Conductance measurements were carried out under asymmetric ionic conditions

(185). The chamber without nanogold contained 1 M NaCl, 20 mM Tris (pH 7.6). For the chamber that contained nanogold, the NaCl concentration was reduced to 150 mM to avoid the high salt effect that might interfere with the binding of nanogold to the His- tagged connecter. DNA was premixed in both chambers with the conducting buffer.

After forming the bilayer, a final concentration of 150 pM Ni-NTA nanogold (1.8 nm;

Nanoprobes) was added to the chamber, and incubated for 5-10 min to ensure an even particle distribution before the connector-reconstituted liposomes were added to the chamber.

3. RESULTS

3.1 Discrete one-third step-wise conformational change during high-voltage gating

The connector protein embedded in the lipid bilayer was stable and displayed a uniform conductance under a wide range of experimental conditions including different salt concentrations and varying pH (115), and has a uniform conductance in each of the

53 conditions. At a constant holding potential (-75 mV), the channel conductance is uniform

(Fig 2A). However, when a higher potential (-150 mV or +200 mV) was applied, a 3-step channel size reduction was often observed immediately thereafter (Fig 2C and D), which is very similar to the gating behaviors of membrane proteins. In the given experiment conduction (1 M NaCl with 5 mM Na2HPO4 buffer, pH 12), the step size of a single C-

His connector is around 190 pA at + 75 mV (Fig 2A), and – 190 pA at -75 mV (Fig 2B).

The channel was stable over minutes, and even hours without any blockade or vibration.

When the voltage was switched to -150 mV, the current through the channel doubled in accordance with the voltage increase from -190 pA to -380 pA. Immediately after that, the same channel closed after a three-step reduction in 5 seconds (Fig 2C). Each reduction step size was 120 pA, accounting for 31% of the entire channel. This 3-step channel gating can be triggered by the high voltage despite the buffer conditions. It is interesting to note that after the 3-step reduction, there was still a residual current of 20 pA opening, equal to ~5.3% of one channel. Similar results were observed at higher potentials of 200 mV (Fig 2D).

3.2 Reopening of the gated channel by voltage dropping

It was also observed that the gating of the C-His connector protein channel was reversible. When a transmembrane voltage of 0 pA was applied for 10 to 30 seconds, the gated channel was observed to re-open (Fig 3). After the channel shutting down was triggered by higher voltage (-100 mV in Fig 3A, and -150 mV in Fig 3B), a lower voltage was applied to allow the connector protein to recover. It was found that the conductance of the channels restored to their original state within 10 seconds.

54

Figure 3.2. Stepwise gating of C-His connector channel triggered by high voltage.

(A) The channel was stable at +75 mV and (B) at -75 mV after insertion into a bilayer.

(C) Immediately after a high voltage (-150 mV) was applied, the channel gated in 3 discrete steps. (D) Similar events happened at + 200 mV.

55

Figure 3.3. Controllable and switchable closing and re-opening of the connector channel. (A) The C-His channel with three steps of gating at high voltage (-100 mV or -

150 mV) was re-opened after the voltage was reduced to 0 mV. (B) The channel closed at -150 mV was re-opened to its original size at a lower voltage of -75 mV.

56

3.3 Discrete one-third step-wise conformational change of connector induced by protein binding to the C-terminal

A C-terminal His tagged connector and rabbit polyclonal antibody (Ab) specific against His tag were used to measure the conductivity under a voltage of -75 mV with the negative pole placed at the bottom chamber (the –trans side). Since the channel exercises a one-way traffic mechanism, orientation of the connector was determined by their capability to translocate dsDNAs which was placed in only one, either top or bottom of the two chambers via the polarity of applied electrical current (see ref. (149) for details). Addition of the Ab to the bottom chamber (Fig 4B) resulted in six discrete

31% current reduction steps for two connectors, indicating binding events induced by interaction of one Ab molecule with one of the two connector channels. As a negative control, two connectors were inserted into BLM with the His-tagged C-terminal exposed to the bottom chamber. DNA was premixed with conducting buffer before the insertion of the connector channels. When the Ab was added to the top chamber, no binding events were observed (Fig 4A). The results indicate a three-step conformational change of connector after the anti-His antibody bound to the His tag at the C-terminal.

3.4 Discrete one-third step-wise conformational change of the connector channel induced by Ni-NTA Nanogold binding to the C-terminal

A 1.8-nm Ni-NTA nanogold particle was used to bind to the His-tag at the C- terminal of the connector. The binding was promoted via a Ni-NTA/His-tag interaction.

The nanogold was premixed with the conducting buffer (asymmetric conditions; see

Methods) at only one side of the chamber (the trans- side). In the presence of a single

57

Figure 3.4. Changes of channel size by binding to C-terminal. (A) Negative control since the antibody was placed in the cis- chamber opposite to the C-terminal. (B) Six discrete steps of changes for two C-His tagged connector channels induced by anti-His tag antibodies. (C) Three discrete steps of changes for one C-His tagged connector channels induced by Ni-NTA nanogold binding (1.8 nm).

or multiple connector channels, the nanogold only bound to a connector with its His- tagged C-terminal end oriented towards the trans-chamber to the bottom, and did not bind to connectors with the N-terminal facing the trans–side. Clear discrete stepwise closing of the channels with a corresponding decrease in conductance was observed when a single connector with the appropriate orientation for nanogold binding was present (Fig 4C). Binding of each nanogold particle resulted in ~31% reduction in

58

Figure 3.5. Rationale for mutant connector design. (A) Structure of one protein subunit of phi29 connector showing the channel loops and the C-terminal(93); (B) SDS-

PAGE of different mutants connectors used in this report.

channel current. Such observations enabled the direct counting of the number of nanogold particles bound to each connector. The step size of each current reduction, resulting from the binding of one nanogold particle, was nearly identical, even in the case of membranes with multiple channels (data not shown).

59

3.5 Gating behavior of mutant connector with the internal 18-amio acid flexible loop or the N-terminal 14-amino acid removal

Phi29 connector protein contains three flexible fragments, the N-terminal fragment amino acid (aa) 1-14, the internal fragment aa 229-246, and the C-terminal fragment aa

287-309 (Fig 5A) (15, 155). The structures of these three fragments were not included in the crystal structure due to their flexible nature. The flexibility of the connector channel is expected to play a critical role in motor motion and DNA translocation, instead of channel structure construction. To investigate whether these three loops were involved in channel gating, three connector mutations, gp10-C-His/NΔ1-14, gp10-C-His/Δ229-

246, and gp10-C-Strep/CΔ25 were constructed, with the deletion of the loop aa 1-14,

229-246, and 285-309, respectively (Fig 5B). The step-wise gating was observed under both the higher holding potentials and under a ramping voltage (from -150 mV to +150 mV, or from -200 mV to +200 mV). Figure 6A and 6B shows the events happened on a gp10-C-His/NΔ1-14 connector channel, which has a similar channel conductance with the C-His connector. Gating occurred immediately when the ramping began at -150 mV, as shown by the step-wise reductions in the slope of the current trace (Fig 6B). The slope restored to its original state around -50 mV, indicating that the channel conductance re-opened completely. Complete gating happens at a positive potential higher than +110 mV. Such a ramping voltage was applied periodically to test the possibility of the channel gating and re-opening (Fig 6C and E). It was found that both

C-His (Fig 6D) and gp10-C-His/NΔ1-14 can be closed completely after several ramping circles, while the gp10-C-His/Δ229-246 were much less likely to be gated completely

(Fig 6E). The current trace through gp10-C-His/Δ229-246 was still linear (Fig 6F) under

60

120 ramping cycles after 3 hours, indicating the amazing stability of the channel conductance and significant differences between the channels with/without intact internal loops.

3.6 The effect of the C-terminal region in regulating the conformational change

The aforementioned results suggest that phi29 connector implemented a conformational change with three discrete steps. It was proposed that these three steps of conformational change is regulated by the interaction of DNA with the connector, and the binding or contact of components to the C-terminal would result in the similar discrete steps of conformational change. This proposal was supported by a conductance assay using mutant connector conjugated with a His-tag to its C-terminal and incubated with anti-his tag antibody or nanogold coated with Ni-NTA. (Fig 4). It was hypothesized that the translocation of DNA into the procapsid or the internal pressure of the fully packaged DNA within the procapsid might lead to the contact of DNA with the

C-terminal flexible domain, inducing a conformation changes with two subsequence functions: 1) to prevent the dsDNA from exit; and 2) to prepare a new channel configuration to facilitate the injection of DNA during the host cell infection process. To further confirm this hypothesis, a connector mutant with the removal of a 25-amino acid segment at each C-terminal of the 12 subunits was constructed. Similarly, a strep-tag was conjugated to the C-terminal fused to amino acid #287. Conductance assay of the mutant connector by electrical ramping revealed that three-discrete steps of conformational changes were also observed (Fig 6G). However, the discrete steps of

32% conductance change were not observed in the presence of streptavidin.

61

Figure 3.6. Summation and superimposition of all current trace collections and

comparison between mutants. (A, C, E) Superimposition of multiple current traces

under continuous repeated ramping voltages (2.2 mV/s) of individual membrane-

connector complexes. (A-B) Representation of gating and reopening of a C-His/NΔ14

connector channel under ramping voltage (-150 mV to +150 mV). The C-His (C-D), the

C-His/Δloop (E-F), and the C-Strep/CΔ25 (G) connectors under the ramping voltage

were also presented.

4. DISCUSSION

Viral DNA packaging has been investigated extensively in many viral systems,

but the actual mechanism remains elusive. During the last several decades, many

models have been proposed to interpret the mechanism of motor action. These include

1) Gyrase-driven supercoiled and relaxation (153, 186, 187); 2) Force of osmotic

pressure (188); 3) Ratchet mechanism (189) ; 4) Brownian motion (190); 5) Five-

fold/six-fold mismatch connector rotating thread (191); 6) Supercoiled DNA wrapping

(192); 7) Sequential action of motor components (56, 88); 8) Electro-dipole within

62 central channel (155); and, 9) Connector contraction hypothesis (193). All these models are very intriguing, but none of them have been supported by conclusive experimental data; or in other cases, validated in one viral system but disproved in another. The five- fold/six-fold mismatch connector rotating thread model (191) has been popular for more than 30 years, since this model could bring about a new mechanical motor prototype.

Even within the last several years, numerous laboratories, including our own, have persevered to search, interpret, match, link, and even design a five-fold ring to adapt findings to this fascinating and extraordinary model (23, 88, 88, 194, 195). Unfortunately, recent studies in many viral DNA packaging motors reveal that the stoichiometry of motor components is not an odd number but actually an even number (61, 89, 196, 197).

In 1998, Guo and co-workers first proposed and revealed that the mechanism of DNA packaging is simply via a mechanism similar to the hexameric AAA+ ATPase that translocate dsDNA during DNA replication and repair (63). The finding of even-number structures is consistent with the mechanism of many other well-studied DNA tracking motors (198-201) and the AAA+ ATPase family. Most recent publications (59, 184, 195,

202, 203) all support our long-term findings (61, 89, 96) that pRNA dimer is indeed the building block for hexameric structure (96). X-ray crystallography also confirmed that pRNA forms a dimer in solution and the dimer forms a tetramer in the absence of the procapsid (195), supporting the theory that the dimer is indeed the building block of the hexamer and the sequential action in hexamer assembly is 2 → 4 → 6. Furthermore, explicit results conclude that the connector does not rotate during the DNA translocation process (204, 205). Recently, Guo and coworkers proposed a “Pushing through a one- way valve model” (6, 149). In this model, the connector remains as non-rotating valve to

63 allow DNA travel one way towards the procapsid; DNA translocation is induced by a

DNA packaging enzyme or terminase, which pushes a certain length of DNA into the procapsid segment by segment. This model strongly agrees with many recent findings.

Most recently, it was reported that under an external electrical force, the channel of the phi29 nanomotor favored DNA entry but blocked DNA departure(149). The one-way traffic property implies that during the packaging of dsDNA via the active motor, dsDNA travels in only one direction from the narrow external end (N-terminal) toward the wider internal end (C-terminal) of the channel. Essentially, the channel functions as a one-way valve. Thus, these results suggest that dsDNA packaging occurs through the combination of two separate tasks. The first one is the active pushing mechanism(6,

149, 206, 207) provided by the ATPase, gp16, bound to either pRNA (the fulcrum) of phi29, or from other dsDNA phage systems, the large subunit of the two terminases grips on the small subunit (the fulcrum) and pushes the dsDNA coupled with ATP hydrolysis. The second task is the control of DNA migration direction with a one-way valve mechanism of the channel (149). The mechanism providing force through the one-way valve connector also agrees with the finding in T4 phage system by Black and co-workers(206, 207). They found that dsDNA was crunched and the compressed if the

DNA entry was blocked at the front end (206, 207). Although the authors interpreted that the force for the crunching and compression is due to the torsion force from the coiled

DNA at the external end, but it is not contradictory to but support on our finding that the

ATPase gp16 functions similar to the DNA-tracking AAA+ family that twisting or rotating the dsDNA (208). Our new model is also supported by the finding that in the T4 DNA packaging system, with both ends of DNA being able to stay outside of the procapsid

64

(206, 207). If the motor would have implemented a pulling instead of pushing function, it is difficult to interpret how the initiation of DNA translocation begins when both ends are located outside the channel (206, 207).

The new model raises the question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel in one direction. It has been proposed that the motor connector adopts a conformational change after DNA packaging is complete

(165). Such a conformational change renders the channel capable to eject the dsDNA from the viral procapsid. It has been reported that the conformation of the phi29 connector is substantially changed after DNA packaging (16, 165, 209). Significant rearrangement of the connector after DNA packaging, a similar feature reported in other phage systems (210-212), may also change the channel configuration to favor the reverse exit of DNA during infection. In bacteriophage p22, a conformational switch of the portal protein primes genome injection (169). From these results, we confirmed that the connector channel indeed exercises conformational changes. Such conformational changes were induced by molecule binding to the C-terminal wider-end that was located within the capsid, and a high electrical voltage shift. The conformational change exhibited three discrete steps, with each step reducing the channel size by 31%.

Our results support the proposal that these three steps of conformational change are regulated by the interaction of DNA with the connector. We demonstrated that the binding or contact of components to the C-terminal would result in the similar discrete steps of conformational change. Conductance assay using mutant connector conjugated with a His-tag to its C-terminal and incubated with anti-his tag antibody or nanogold coated with Ni-NTA revealed a three discrete steps of channel change with

65 blockade of about 32% (Fig 4). It is reasonable to believe that the translocation of DNA into the procapsid or the internal pressure of the fully packaged DNA within the procapsid might lead to the contact of DNA with the C-terminal flexible domain, inducing a conformation changes with two subsequent functions: 1) to prevent the dsDNA from exit; and 2) to prepare a new channel configuration to facilitate the injection of DNA during the host cell infection process. The proposed second step of action has also been evidence in the P22 system (169). To further support this, a connector mutant with the removal of a 25 amino acid segment at each C-terminal of the 12 subunits were constructed with a strep-tag conjugated to the C-terminal. With this mutant, the discrete steps of 32% conductance change were not observed in the presence of streptavidin.

However, conductance assay of the mutant connector by electrical ramping revealed that three-discrete steps of conformational changes were also observed (Fig 6G). The finding suggests that conformational change was a result of the transition of the entire connector structure, and the C –terminal only served as a trigger. This conclusion agrees with the finding in the connector structure of bacteriophage P22 (159). A unique topology of the C-terminal domain was reported to be a ~200-Å-long α-helical barrel that inserts deeply into the virion and is highly conserved in the Podoviridae family. They proposed that the barrel domain would facilitate genome spooling into the interior of the procapsid during DNA packaging, and in analogy to a rifle barrel to increases the accuracy of DNA ejection during infection. During the course of our investigation, we also found that the batch of the polyclonal antibody made a difference, suggesting that not all binding to any location of the C-terminal would induce conformational changes and certain specific epitope at the C-terminal was important for triggering the

66 conformation change.

The mechanism of ion channel gating has been extensively studied (174). The ligand-gated ion channels are regulated by a ligand and are usually very selective to one or more ions such as Na+, K+, Ca2+, or Cl-. Such receptors located at synapses convert the chemical signal of a presynaptically released neurotransmitter directly and very quickly into a postsynaptic electrical signal. Conformational changes of α helices in voltage-gated sodium channels and calcium channels have been proposed to explain the gating mechanism. From crystallographic structural studies of the phi29 connector protein, it is possible to surmise that when a potential difference is introduced over the membrane, the associated electromagnetic field induces a conformational change in the protein channel. From the structural viewpoint of a single chain in the connector protein

(Fig 5A), certain areas of the chain are flexible enough to induce a conformational change in response to an environmental stimuli. The interaction of dsDNA or phi29 terminal protein gp3 induces a conformational change that distorts the shape of the channel proteins sufficiently such that the cavity, or channel, opens to admit ion influx or efflux across the membrane, down its electrochemical gradient. This conformational change leads to the opening or closing of the channel, which will help to control the packaging or release of the viral genome.

The controllable opening and closing of the connector protein has been achieved with different polarity, which also resembles the voltage-gated ion channels. However, they have different functions in the biological environment. The ion channels may have to open and close multiple times in their life cycle, whereas the phi29 DNA connector protein might require less. The significantly different gating behavior of the internal

67 flexible loop-cleaved connector suggests that the flexible loops may play a key role in the voltage gating. Results described above show that after the removal of these loops, both the occurrence and extent of gating reduced tremendously. These loops can induce conformational changes to adjust the channel size in response to an applied potential. Further studies are necessary to investigate if other domains of the connector protein also contribute to its gating and conformational changes.

5. CONCLUSIONS

The real-time direct observation of the viral connector protein opens a new door to study the mechanism of DNA packaging motors. This "modularity" allows us to use these simple and inexpensive model systems to study the regulation of DNA trafficking, the role of the viral portal protein in infection, and pharmaceutical control of the infection.

The gating is also reversible and controllable by voltage or ligand binding, making the modified connector protein an ideal nano-valve for constructing a nanomachine with potential applications in nanotechnology and nanomedicine, such as for drug loading and controlled release, and high-throughput single pore DNA sequencing.

ACKNOWLEDGEMENTS

We thank Feng Xiao and Ying Cai for constructing the recombinant connectors, Peng

Jing for testing the antibody binding, Randall Reif, Chad Schwartz, Daniel Binzel and

Garrett Osswald for assistance in manuscript preparation, and Chris Stites for the preparation of the illustration graphics. The research was supported by GM059944,

EB012135 to PG. PG is a co-founder of Kylin Therapeutics, Inc., Viapore, Inc., and

Biomotor and RNA Nanotech. Co., Ltd.

68

CHAPTER 4 REENGINEERED CONNECTOR FOR SINGLE-STRANDED NUCLEIC

ACIDS SENSING

ABSTRACT

Bacteriophage phi29 viral channel has a channel large enough for the translocation of double-stranded DNA, and has the unique stability and uniform channel size compared with synthetic nanopores. However, the wild type relative large channel designed for dsDNA translocation could not distinguish the single-stranded nucleic acids at the comment experiment conditions. In this report, we designed the phi29 phage portal protein by removing the internal loop segment, resulting two interesting groups of channel size. A group of smaller channel has the size of ~60% was constructed. It is also found that this mutant smaller channel is able to translocate of single-stranded

DNA, and does not allow the transport of double-stranded DNA. This reengineered portal channel expande the future application of phi29 DNA channels in nanomedicine and nanobiotechnology, and provides us on the transport property of biological channels.

Introduction

Nanopores have been used to detect the morphology of polymers, including DNA, RNA and peptides. Biological channels have the advantage of uniform channel size and availability for channel modifications and conjugations. Bacteriophage phi29 viral channel has a viral portal channel large designed for the transport of viral genome, and is one of the most well studied viral channel up to date. However, the wild type relative large channel designed for dsDNA translocation could not distinguish the single- stranded nucleic acids at the comment experiment conditions.

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The channel of phi29 DNA packaging motor connector protein has been successfully reconstituted into lipid membrane (114, 115, 149, 150, 213). With a central channel of

3.6 nm at the narrowest point, the connector protein channel is an idea nanostructure for detection of the translocation of small molecules. The translocation of double- stranded DNA has been studied extensively (114). Both single channel recording and quantitative PCR confirmed that the capability of transporting dsDNA driven by an applied electrical field. The conductance of the channel at various salt concentration and pH were also test (115). It is found that the channel is stable at the pH range from 2 to

12, and the conductance has a linear relationship with the salt concentration. The opening and close of the channel are also controllable via a channel conformational change at higher applied trans-membrane potential.

Some other biological nanopore, such as α-hemolysin (214), has been studied extensively for the analysis of the translocation of both double-stranded and single- stranded DNA. However, due the limit of the channel size, the space for further modification of the internal channel is limited. Solid nanopores were also developed during the past decade to detect the translocation double stranded DNA.

In this report, we designed the phi29 phage portal protein by removing the internal loop segment, resulting two interesting groups of channel size. A group of smaller channel has the size of ~60% was constructed. It is also found that this mutant smaller channel is able to translocate of single-stranded DNA, and does not allow the transport of double-stranded DNA. This reengineered portal channel expands the future application of phi29 DNA channels in nanomedicine and nanobiotechnology, and provides us on the transport property of biological channels.

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MATERIALS AND METHODS

Materials

Phospholipid 1,2-diphytanoyl-sn-glycerol-3-phosphocholine (abbreviated as DPhPC) were obtaiend from Avanti Polar Lipids, Alabaster, AL. n-decane and chloroform were purchase from Fisher and TEDIA, respectively. The oligo of DNA and RNA used in this study were ordered from Integrated DNA Technologies (IDT). All other reagents were purchase from Sigma-Aldrich, if not specified.

Cloning and purification of the phi29 connector protein

Two mutant version of the connector protein were used in this research: C-terminus 14 histidine (His) tagged connector (C-His connector), and C-terminus 14 histidine tagged connector with internal flexible loop cleaved (C-His/Δloop connector). The cloning, expression and purification of these C-His connector has been reported in the recent publication in Guo lab (112, 114). The construction of C-His/Δloop connector is also reported in publication (150), as well as the chapter 3 of this dissertation.

Incorporation of the connector protein into giant liposomes

The giant liposome containing the connector protein was prepared following the recent reports (114, 115, 149, 150). A de-hydration and re-hydration procedure was used to form giant unilaminar vesicles (GUV), with an average diameter of 50 µm. The connector was incorporated into the liposome during the rehydration stage, with the co- incubation of the connector protein with 200 mM sucrose buffer. The formation of the vesicles can be observed by microscopy imaging in bright field. The ratio of the connector to lipid was set to be is the range of 4000:1 to 16000:1 for planar lipid bilayer

71 insertaion experiments.

Insertion of the connector protein into preformed lipid bilayer

Procedures were developed recently (114, 115, 149, 150) for the incorporation of connector protein into lipid bilayer. A Chamber for Lipid Bilayers (BLM Cell, Eastern

Scientific LLC) were used to contain the BLM system. A Teflon Partition was attached between the top and bottom chambers of the BLM cell, creating a compartment separation for two chambers. The telflon partition has a 0.2 mm hole in the middle, and a lipid bilayer was formed covering this hole by painting technique. Conducting buffer were added to both the top and bottom compartments of the BLM cell, and two electrodes were dipped into the compartments respectively. The electrode in the bottom compartment was connected to the headstage of the amplifier (Axopatch 200B), and the electrode in the top compartment was connected to the ground.

With buffer filled in two compartments, formation of lipid bilayer covering the partition’s hole will form a insulation, resulting a zero current between two electrodes across by the lipid membrane. Membrane fusion between the liposome and the lipid bilayer can happen immediately after applying proteoliposome containing the connector protein, and the current through the connector channel can be recorded.

Electrophysiological measurements

A classical patch clamp instrument consisted of amplifier (Axopatch 200B), analog- digital converter (model DigiData 1440 from Axon Instruments) and headstage was used for electrophysiological measurement. The current recording was low-pass filtered at a 1 kHz frequency. The sampling frequency is 2 kHz if not specified. The data recording was into computer PClamp 9.1 software (Axon Instruments), and analyzed by the

72 software of Clampfit and Origin Pro 8.1.

Two mode of applied potential were used in the study: holding potential and ramping potential. The holding potential was a constant potential applied across membrane. +75 mV and -75 mV were used in the experiments. Another voltage mode used in the study was the ramping potential, which is a voltage with constant increase over a period of time. Ramping potential ranged between -100 mV and +100 mV, -200 mV and +200 mV were used in the experiments for the channel stability test and

DNA/RNA translocation orientation test.

Purification of the DNA/RNA used in the experiment

A 20-nt oligonucleotide was purchase from Integrated DNA Technologies (IDT). 100

µL stock of oligonucleotide (100 µM) was mixed with 100 µL 2XTBE buffer. The 200 µL mixture was loaded into 2 columns of 8% urea-TBE gel. After electrophoresis, the single-stranded DNA band was cut under UV shadow. The gel containing the ssDNA was eluted in 37 °C for 4 hours, following by spinning down. The precipitation was then removed and 1/10 volume 3M NaOAC and 2.5X volume of 100% EtOH were added to the elution buffer and stored overnight in refrigerator. The elution was then then centrifuged, and additional slat was removed by rinsing with 70% EtOH. The purified ssDNA was stored in TMS buffer finally.

20-nt ssRNA was purified in a similar method.

Translocation experiments of DNA and RNA

For the translocation experiments, purified ssDNA or ssRNA was premixed with the buffer, and added to the compartment in the BLM cell. After the addition and insertion of connector protein into lipid bilayer, a highly conducted channel would form. The ssDNA

73 or ssRNA will be driven through the aperture in the Teflon partition under the applied electrical field.

RESULTS

Narrowing channel size by reengineering the phi29 DNA packaging motor

The conductance of native (213) and terminus modified connector channels (114, 149) have been measured, and has a uniform channel size with narrow distribution. Figure 2 shows the overlap of the current traces through the individual channels of C-his tagged connector (red) and C-His tagged connector with loop segment deletion (blue). Each jump in the current increase corresponds to a single protein channel reconstitution into the planar lipid bilayer (BLM). The buffer condition is 1 M KCl with 5 mM HEPES, pH 7.4.

The current jump of one C-his tagged connector is 365 pA, equals to 4.86 nS; The current jump of one C-his/Δloop connector channel is 218 pA, equals to 2.91 nS. A continuous current traces recording of multiple C-his/Δloop connector channel insertion is shown in Figure 2B (right). The current traces includes stepwise jumps indicating mutilpe protein channel reconstitution. It is clear from the statistics in the histogram of the channel conductance (Fig 2B right). There are two peaks of the conductance. One major peak with conductance of 2.2 nS represents the channel smaller modified channels, while the minor peak of 3.4 nS represents the channel of regular channels.

The mutilmodal size distribution of C-His connector and C-His/Δloop connector are shown in Figure 2 D and 2E by from dynamic light scattering data. A major peak of around 15 nM are shown in Figure 2D, while there are two peak observed in figure E.

74

The two peak value are 15 nM and 50 nM, respectively.

Sensing of single-stranded DNA by the modified channels

20-nt single-stranded 2’F modified RNA is used for the test of translocation through the channel. Two sets of experiments are designed: Control experiments without adding. Three channels of size-modified connector are incorporated into the membrane consequently, and it is clear that there are no spikes during the recording

DNA (Fig 3A). In the same experiments with 10 nM ssRNA added, spikes appear after each insertion with a similar blockade percentage (Fig 3B). A magnified view shows the individual event of the signal of one ssRNA translocation, which can be descripted by the two parameters: dwell time and blockade percentage. The former represent the time of the ssRNA takes to translocate through the channel, while the latter represents the ratio of cross-sectional area between the ssRNA and the channel. Histograms of these two parameters from 50 events were shown in Fig. 3C and Fig. 3D, respectively. It is seen that the dwell time in the given experiment condition ranges from 1 mS to 70 mS, with a peak value around 15 mS. The histogram of amplitudes has a peak value of 40 pA, equivalent to 21% the modified channel size.

Sensing of single-stranded RNA by the modified channels

Similar translocation experiments were performed using 20-nt single-stranded DNA. A burst of spikes was observed after the incorporation of the modified channel (Fig 4A).

The dwell time various from 0.1 mS to 60 mS (Fig 4B). The peak of the amplitude histogram shows up around 40 mS.(Fig 4C)

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Two-way translocation of single-stranded nucleotides by the modified channels

It has been reported that the dsDNA has a one-way translocation property through C-

His tagged and wild-type connector. Electrophysiological measurement with polarity switch and ramping potentials were used to verify the direction of 2’F ssRNA translocation through the channel. 2’F ssRNA added to both chambers; the ssRNA is driven through the channel powered by the electric field. Under holding potential of +45 mV, blockade events of ssRNA translocation were observed. After switching to -45 mV, events with similar blockade percentage but different also appeared (Figure 5A). This results represents the ssRNA can be enter both both terminus, and pass through the channel. In another experiment with ramping potential from -100 mV to +100 mV, spikes with blockade appeared at both polarities.(Fig 5B) This also suggested that the ssRNA was translocated through both direction of the connector protein.

Stability of the narrowed channel in various salt, pH and voltage condition

The gating property the connector channel at higher potential (> 100 mV) has been observed in several mutants. To improve the stability of channel for broader application needs a higher gating potential. The stability of the connector channel under high voltage was examined under high voltages (-200 m to +200 mV) both under holding potential and ramping potential. At holding potential of higher than +200 mV and -200 mV, the channel was stable while the C-His connector will be gated immediately. Similar results were observed in holding potential, without gating in the range between -200 mV to +200 mV. The conductance of the channel was also test in different buffer, Lithium

76

Chloride, Sodium Chloride and Potassium Chloride. Curves showing the relationshio between current and voltage at low and high pH values: pH 2 (5 mM phosphoric acid buffer) and pH 12 (5 mM Na2HPO4.7H2O buffer) was plot according to the conductivity measured. A is obtained demonstrating that the stability of membrane/connector complex under extreme acidic or alkaline buffer is confirmed by a linear current-voltage relationship curve.

DISCUSSION

Modification of biological nanopore has been studied intensively. Various modifications have been made to both biological nanopores and solid-state nanopore for sensing nucleic acid analysis (215). The modification of biological nanopores can be achieved by introduction of various amino acids (216), Covalent linking of molecular recognition agents (217) formation of enzyme-DNA complex. etc. The solid –states nanopores have been studied mainly by chemical modification. Different assemblies of phage portal protein monomers have been reported on SPP1, phi29, T7 and P2 (218). The 12-mer and 13-mer assembly of SPP1 connector can be both formed at different condition.

For the mutant of internal flexible loop deletion, it is interesting to find that the connector with a channel size similar to native type, and with a size of ~ 60% to native type are both observed (Fib 1B). It is possible that the former has a similar assembly with 12-mer wild-type connector, and the connector with smaller channel size has a different assembly after the loop deletion. It also noted that both type of assembly were expressed and purified from the same batch at the same time. To avoid the possible contamination, we have also conducted a three generation single-colony cell culture.

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The purified C-His/Δloop connector channel from different batches showed similar results, though the ratio of regular-sized to size-reduced channel varied. (Fig

1C).Transmission electron microscopy (TEM) images of the procapsid with loop cleaved connector (data not shown) also showed a smaller procapsid appeared compared with the wild-type one.

The speed of DNA transport through the nanopore is a major bottle-neck for nanopore- based nucleic acid analysis (215). Due to the relative large diameter of the connector channel, the native channel is an idea tool for the study of double-stranded DNA transport (114), but detection of single-stranded nucleic acid remains difficult. With this size-reduced channel of loop–cleaved connector, the detection is possible. With 10 nM of s2’F ssRNA and ssDNA, a burst of events occurred immediately after the insertion of the channel to the lipid membrane. These events have a uniform blockade around 19% of a size reduced channel. This blockade ratio is proportional to the ratio of half of the double stranded DNA blockade through a 60% of native channel. This detection was mainly contributed by the size reduction and speed reduction of the translocation thereafter.

The one-way traffic of dsDNA translocation through has been observed and confirmed

(149, 213) by single channel recording. The connector channel can only translocate the dsDNA from the narrow end to wide end. There have been various models about the mechanism of phi29 DNA packaging, including Push through a one-way valve mechanism (213), ratchet model (188, 219), etc. It is confirmed that the portal protein connector channel can exhibit a three-step conformational change under high voltage

(150). In this study, single-stranded DNA and RNA are able to be identified using a size-

78 reduced channel of connector mutant, and the single channel recording data proves that the ssDNA and ssRNA can traverse through the channel from both side of the channel

(Fig 5). With only one connector channel reconstituted in the channel, current traces under both holding potential and ramping potential shows spikes of dsDNA translocation at both electrical polarity. These results suggest that due to the diameter difference, the segments controlling one-way traffic won’t block the reverse translocation of ssDNA

/ssRNA. This also broadens the application of connector channel in DNA loading/delivery. Deletion of the internal flexible loops may also contribute to the two- way traffic of ssDNA/ssRNA translocation.

CONCLUSION

In this study, the channel size modification of phi29 DNA packaging has been achieved by a mutant of removing internal flexible segment, resulting in a size-reduction protein channel with a size of ~60% of the wild-type. This size modified channel is idea for the sensing and detection of single-nucleic acids, including ssDNA and ssRNA, and does not allow the transport of double-stranded nucleic acids. The two-way translocation of single-nucleic acids has also been observed through the loop cleaved channel. With this size-reduced channel along with the native connector protein channel reconstituted into a lipid bilayer, a hybrid system could be by developed for sensing both dsDNA and ssDNA at the same time.

79

Fig. 4.1 Conductance of the gp10C-His/Δloop connector channel. a, Overlap of current traces of a typical C-His connector channel (red line) and C-His/Δloop connector channels. b, A continuous current traces showing the multiple insertions of C-His/Δloop connectors. C. Histogram of the conductance of C-His/Δloop connectors.

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Fig. 4.2 Sensing of ssRNA by the modified channel. A, Control experiments of current through C-His/Δloop connectors only, without addition of ssRNA. B, Same experiments with 10 nM 2’F ssRNA in both chambers. A burst of spikes occurred after the insertion of first channel. C. and D. The histograms of dwell time and amplitude of ssRNA

81 translocation events.

Fig. 4.3. Sensing of ssDNA by the modified channel. A. Current trace through C-

His/Δloop connectors experiments with 10 nM ssDNA in both chambers. B. and C. The histograms of dwell time and amplitude of ssRNA translocation events.

82

2 3 4

200

0

(pA) Im_scaled

-200

20 40 60 Time (s)

500

0 (pA)

Im_scaled

-500

0 Vm (mV) 40 80 Time (s) Sw eep:1 Visible:1 of 1

Fig. 4.4 Two way translocation of ssRNA and ssDNA through the narrowed channel.

(Top) Two-way traffic verified by switching the holding potential. (Bottom). Two-way traffic verified by the ramping potential (-100 mV - 100 mV)

83

CHAPTER 5 SINGLE-MOLECULE ASSAY OF PHI29 PRNA AND CONNECTOR

PROTEIN INTERACTION

Introduction

Besides the connector protein, Bacteriophage phi29 DNA-packaging motor has another important component, that is a packaging RNA (pRNA). (Fig 5.1 a)The six pRNA form a hexameric and gear the motor during packaging along with gp16 and ATP (63). The interaction of phi29 pRNA with the prohead have been studied extensively, however it is difficult to count the RNA molecules interaction with connector protein directly. Using the highly sensitive imaging system developed during the past few years (220), (221), (222),

(223), (61), (89), (90), we report here the observation of pRNA–connector protein binding at single molecule level. The photobleaching steps of single-fluorophore labeled pRNA are detected by single fluorophore imaging, and the stoichiometry with the connector can be calculated in vitro. The interactions of the pRNA with connector protein under different buffer condition were also studied.

The spatial resolution of traditional optical microscopy ranges around hundreds of nanometers, which is about half of the wavelength. It is often limited by the diffraction of light when measuring molecules close to each other. However, DNA, RNA and protein interaction often occurs in the range of scores of nanometer, which is beyond the capability of traditional microcopy imaging, thus it is desirable to develop a highly sensitive imaging method for the observation of molecular interaction in phi29 DNA packaging motor. Single molecule fluorescence imaging is one powerful tool for the

84 study of biomolecules developed recently (224, 225). Single molecule imaging method, such as Fluorescence resonance energy transfer (FRET), photobleaching, has been used to study various bio-complex, including protein synthesis (226), dynamics of RNA in vivo (227), membrane fusion (228), and molecular motors (229).

An imaging system of single-molecule dual-channel Total Internal Reflection Microscopy

(TIFR) was constructed to study the phi29 DNA packaging motor in Guo lab (89, 90,

221, 221, 223, 230) with high single/noise ratio (Fig 5.1 d). The system has been used to study the single RNA counting (89, 90), ATPase activity of gp16 (58), RNA assembly

(61), distance measurement within motor (230), etc.

There are two functional domains in the phi29 pRNA (231), one central domain is the pRNA hand-in-hand interlocking domain (47, 232), and another one is the gp16 binding domain (53) (Fig 5.1 b). The interaction of pRNA with procapsid may be non-specific, and under certain circumstances, although individual pRNA subunits may bind to the connector or procapsid nonspecifically, but only pRNA forming a static ring that will bind to connector / procapsid. (61). Thus formation of hexametric pRNA ring is essential for the binding and servers as a bridge between gp16 and connector, which constitutes the key components of the motor.

Previous study of the pRNA interaction with other components focused on the interaction with the procapsid, but direction interaction of pRNA and connector in vitro has not been studied. It is of interest to study the pRNA-connector interaction in a procapsid free environment. In this study, we immobilized the purified connector on to substrate via lipid monolayer, and then incubate the single fluorophore labeled pRNA with the immobilized connector protein. After rinsing the unbounded pRNA away by

85 flushing a buffer through the chamber, the samples were observed using single- molecule TIRF microscopy imaging system. The photobleaching step of the pRNA was recorded and analyzed. This photobleaching steps revealed the numbers of pRNA bounded together (Fig 5.1 c).

After comparing the control experiments of pRNA binding to substrate directly without connector, it is found that the majority photobleaching steps for pRNA non-specific binding to the substrate are 1 or 2 steps, while the pRNA binding to connector shows a majority step up to six.

MATERIALS AND METHODS

Preparation of lipid monolayer on quartz slide

The glass slides (25 mm × 75 mm, VWR Scientific) were carefully cleaned in a 3:7 (v/v) mixture of 20% H2O2 and 80% H2SO4 for about 2 h. Then, the slides were rinsed with

Milli-Q purified water completely, and stored under water for next step use. Before using to for lipid monolayer deposition, the glass slides were sonicated in Milli-Q purified water again right before the transfer. A lipid mixture of EggPC:DOGS-NTA-Ni with 1:1 molar ratio in 1 mG / L chloroform were used for the formation of lipid monolayer by KSV LB trough.

Preparation of Cy5 labeled RNA

Single fluorophore labeling of pRNA has been developed in Guo group previously (61,

89, 90). Briefly, the labeling of each pRNA by a Cy5 to each pRNA molecule was achieved byRNA transcription in the presence of fluorescent dye. ADOTMF550/570 and

86

ADOTMF650/670 (AdeGenix, Inc.) were used for the labeling. The labeling efficiency could be calculated as the ratio of concentration of dye to the concentration of the pRNA.

The concentration of labeled pRNA was determined by By UV/vis Spectrophotometry at optical density of 260 nm (with the equation of 1OD260 = 1 µM). The molar concentration of fluorescent dye Cy5 was determined from UV Spectrophotometry with absorbance at

-1 -1 650nm. The concentration is calculated as ε650 equals to 250,000 mol cm .

C-His Connector Conjugation to Ni-NTA Lipid bilayer

FITC labeled connector protein (2.5 x 10-3 mg/ml) were added to monolayer samples and then incubated for 1hr at 37ºC bilayer within a custom made chamber on the surface of slide. The samples were rinsed several times in PBS solution for removing unbound protein. The sample can be observed by microscope in FITC channel with lamp to determine the immobilization of the protein.

The connector protein can also be immobilized on the quartz substrate during LB trough deposition. To immobilize connector on the surface of quartz, 0.4 mg FITC-labeled connector were solve tin the sub-phase (10 mM HEPES, 100 mM NaCl, pH 7.8) of the

LB trough during the LB formation of monolayer.

Binding Cy5 RNA to the connector bound to Ni lipid monolayer

After formation of monolayer immobilizing connector protein, a customized chamber made of glass slide and tape was attached to the quartz substrate and Cy5 SphIi’ RNA

(8.04x10-5 μg/μl to 2.6x10-3 μg/μL) was added to the chamber. After incubation for 1hr at 37ºC, the excess unbound pRNA was rinsed off with 1X TMS. The pRNA/connector complex can be observed by single molecular imaging method described above (89),

(90).

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RESULTS

Immobilization of connector via monolayer

Three sets of experiment’s conditions for immobilizing connector on lipid monolayer were tested (table 1). Mica subtracte were used in experiments #1 (Fig2 A,B) and 2#

(Fig2 C,D), and quartz was used in experiment #3 (Fig2 E, F). 50 mM ATP was added in the buffer in experiment #2 (Fig 2 C, D). The results suggested that Incubation of gp10 connector in 50 mM ATP made the gp10 in lipid monolayer less aggregated; however the improvement is not obvious. Increasing the ATP concentration and sonication treatment of the gp10/ATP mixture could be used to decrease the gp10 aggregation.

Due to the optical property of quartz crystal, the lipid monolayer and gp10 on quartz is clear, and suitable for single molecular observation when binding with pRNA.

Interaction with pRNA

Different concentrations of connector protein were used for the immobilization test (Fig 2 G-J). It is shown that single spots of FITC labeling were clearly observed before photo bleaching, and the florescence disappeared after photo bleaching.

However, the 450 pM concentration of connector protein is relative higher, which resulted aggregation and overlap of single molecule-imaging studies, thus 90 pM connector protein concentration was selected for pRNA binding experiment in the next step.

Single molecule imaging observation of the immobilized florescent labeled pRNA

Binding pRNA onto lipid bilayer with connector was counted using single-molecule imaging with photobleaching. Under the photobleaching microscopy measurement of

88 the fluorescent labeled pRNA/connector complex, the immobilized samples on the surface of the perfusion chamber showed as bright spots due the fluorescence (Fig. 3

B). The reduction of fluorescence intensity of the spots was caused by the continuous excitation with the laser beam over time. The optical single was recorded by the Andor

IQ software and stored in computer system. The average optical intensity within a recorded region was subsequent analyzed by the same software versus time. For each individual shining spot, an area was circled around the fluorescent spot, and the total optical density was accumulated and was sued for data analysis. After calculation of the mean fluorescence intensity, a plot was made with the time as X-axis and time as Y-axis.

The background mean intensity has been subtracted from the signal averaged mean intensity to avoid the interference of background noise.

Each such trace represented a single spot of the pRNA biocomplex immobilized via connector on the substrate (Fig 5.3, c-g). Non-specific binding of pRNA to the substrate, or aggregated pRNA can exhibit a similar traces, but with different photobleaching steps.

The number of photobleaching steps in each spot represents the number of fluorescent molecules within that spot. This highly sensitively detection is not able to be realized in the conventional optical microscope. Photobleaching with steps of 2, 3, 4, 5 and 6 are observed (Fig 5.3 -G), and histograms were shown in Fig 5.3 h.

Single molecule counting of pRNA binding to the connector

Control experiments were conducted to distinguish from the the non-specific binding to the subtracte (Fig 4 A). The pRNA photobleaching most appears in 1-step and 2-step in the sample without connector (Fig. 5.4A blue), and 4-step, 5-step and 6-step have the highest occurrence in the sample with connector (Fig. 5.4A red). This result coincides

89 with the previous research on stoichiometry of pRNA of and its interaction with connector, and the pRNA hexameric ring with the motor (61, 231, 233).

Different concentration of pRNA were also conducted to gain the optimized condition for pRNA connector binding, since abundant pRNA may form nanoparticles and inhibit the binding to connector protein. Experiments with 90 nM pRNA shows a peak in the 6-step photo bleaching. (Fig 5.4)

DISCUSSION

Single molecule Imaging has been used to study the various aspect of phi29 DNA packaging system (220), (221), (222), (223), (61), (89), (90), for example the distance parameters for RNA nanoparticles (220), mechanism of hexamer ring assembly (61),

Counting of pRNAs (89) etc. But the interaction of the connector protein and pRNA has not been. In this study, we investigated the interaction between pRNA and connector protein by single-molecule imaging technique.

Without procapsid, it is difficult to find a suitable substrate for pRNA binding. In this study, the lipid monolayer acts as a substrate immobilizing connector, and acts as a bridge for single molecule study from its native biological environment. Proper concentration of connector protein on quartz substrate and proper amount of pRNA for binding are essential for the single molecule studies, since lower concentration may result lower signal density, which could be difficult for observation as well as to reach a statistical significance. Too-high concentration of samples will form overlapped complex, and bring in additional error for the analysis.

It is observed that the concentration plays a key role not only for the protein

90 immobilization, but also in defining the stoichiometry of pRNA / connector complex, thus it is essential to find a proper reaction concentration. The factor of connector protein concentration was selected as shown in Figure 5.2g and 5.2i, of which 90 nM was selected to show a uniform distribution of immobilized spot with proper density.

In this study, we have shown that pRNA can forms hexmeric complex together with connector protein. Statistics in Figure 5.4A clearly showed the difference of pRNA complex formation with and without connector protein. This interesting result not supports our previous finding of the role of procapsid in the ring formation, but also proves that the pRNA can be assembled into hexametric complex without procapsid.

Formation of pRNA hexamer nanoparticles are extremely interesting, especially in the emerging field of RNA nanotechnology (234). It will be interesting to study the pRNA hexameric nanoparticle even without binding to connector protein. Other instrumentation might also be used for the study of formation and characterization of the nanostructure, including EM, AFM and single channel studies. The development of RNA/protein complex immobilized on lipid monolayer can be served as a platform for other studies of the dynamics of various biological complex in the future.

CONCLUSION

A lipid monolayer based platform has been developed for the study of interaction of pRNA with connector protein. By binding the pRNA to the connector protein immobilized onto the lipid monolayer on glass substrate, the stoichiometry of the pRNA was studied by the single molecule FITR imaging. It is found that the pRNA can forms a hexametric complex with the aid of connector protein. Without the existence of connector protein,

91 non-specific binding was observed with 1 or 2 photobleaching steps. This connector- pRNA-lipid monolayer system provides the possibility of the study of other molecular motors.

92

Table 5.1: Lipid in chloroform / C-His tagged gp10 connector in Hepes Lipid Sample (mol ratio) Component 1# 2# 3# EggPC 49% 49% 49% Substanc DOGS-NTA-Ni 50% 50% 50% e Texas-Red-DHPE 1% 1% 1% FITC labeled C-His 100 µL 100 µL 100 µL Subphase gp10 ATP 50mM Substrate Mica Mica Quartz

The total concentration of the substance is 1 mg/mL in chloroform The C-His gp10 are in 10 mM Hepes, 100 mM NaCl solution, pH 7.8 The C-His gp10 was mixed ATP at 50mM and incubated for 10 minutes at room temperature.

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A C

procapsid

connector

hexameric pRNA

A G U G B U C A A U C A U U G C D 5' U U GCAA GGUA-CG-GUACUU UUGUCAUG GUAUG UGGG CUGA G A CGUU UCAU GC CGUGAA AACGGUAC CAUAC ACCC GACU 3' U G C A U U U U G A U A U C G U A G U C U G C G

Fig 5.1. Structure of phage phi29 DNA packaging motor and design of single molecule experiment. A. Motor including procapsid, connector and hexameric pRNA ring. B. Structure of pRNA monomer. C. Procedure of pRNA binding experiment. D.

Scheme of single-molecule imaging.

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Fig 5.2. Immobilization of connector via monolayer and interaction with pRNA by microscopy imaging. A-F. Immobilization of FITC-labeled connector on different surface at different condition. G-J. Effect of concentration of gp10 connector protein for immobilization and binding.

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Fig 5.3. Single molecule imaging of immobilized Cy Aa’ pRNA. A. Control experiment with connector only and without pRNA. B. Image after pRNA binding in quartz chamber. C-G. Representative traces of photobleaching steps. H. Statistics of the photobleaching steps.

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Fig 5.4. Statistics of control experiments. A. A set of experiments of pRNA photobleaching setp with (red) and without (blue) connector. B. Experiments with different pRNA concentration.

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CHAPTER 6 UNFINISHED WORK, AND PROSPECTS

Previous successful research on the incorporation of connector into artificial lipid membrane leads itself to further investigation of the possibility of incorporating connector protein into cell membrane. Pilot experiments have been designed and performed, and the results provide promising results and can be pursued further.

Binding of connector protein to KB cell and induction of cell apoptosis

It has been a long-term objective to find suitable vehicles for deliver drug into cells to cure certain disease, such as small molecular chemical drugs, or silencing RNA. Since the cell membrane is such a good barrier to penetrate, improved methods are still under investigation. In the pilot experiments, C-His connector and C-strep connector are labeled with FITC fluorescence, and incubated with KB cell at 100 nM in 100 µL RPMI

1640. Different concentration of lipofectamine is added to the media (0, 0.5 µL, 2 µL and

8 µL). The FITC labeled C-Strep connectors bind to the KB cells specifically with the aid of lipofectamine. The merged view of FITC channel and bright view confirm the binding.

The lipofectamine helps the connector protein to interact with the KB cell, while the connector itself without lipofectamine doesn’t bind to KB cell nor induce cell apoptosis.

(Fig. 6.1). Bind to cell and induction of cell apoptosis increase as the amount of lipofectamine increase. These results pace the next step of connector interact with cell via incorporation in liposome vesicles.

Specific interaction of connector/sapc-dops complex to cells

98

It has been reported that SaPC-DOPS has a preferred delivery to cancer cells (235), thus it could be considered to be used as a delivery vehicle along with the connector protein. FITC-connector /SapC-DOPS complex was prepared and characterized by DLS.

The interaction of the complex brain tumor cell line U87 was also tested (Fig 6.2). The fraction numbers indicated that the SapC-DOPS complex forms with FITC-connector incorporated. With four hours incubation, more connectors went into or near the cell.

The flowcytometry experiment was also done in Dr. Qi’s lab.

From the above results, incorporation of the connector into SapC-DOPS vesicles increases the permeability into the cell efficiently. Since brain cancers are difficulty to treat due to the impermeability, this complex could be a good candidate to facilitate the treatment of brain cancer cells.

Cell membrane embodied connector channel by electrophysiological

measurement

The connector protein has the potential due to its three-layer structure. With two independent experiments with experts from Flyion and Nanion, it is find that the connector protein can be inserted into live cell membrane (Fig 6.4, 6.5), and produce a similar current through the nanopore like it is in artificial lipid bilayer. Without connector added, the membrane potential was stable and no events were observed.

There are several step with same current size observed after 5-10 minutes of connector addition to external solution. This could be the event of single connector protein insertion into the cell membrane.

Combined with previous experiment on Flyion’s instrument (we sent blind samples (a)

99 liposome only. (b) liposome with connector, and they only observed insertion events with sample (b)), it can be concluded that the current jump on the cell membrane is due to the addition of connector.

Quantitative comparison of the current jumps between the cell membrane and lipid bilayer will be analyzed. More repeated experiment and investigation are needed to get a statistic of the insertion efficiency to cell membrane.

5h 14 h Bright view FITC Channel Bright view FITC channel

µL

0

µL

0.5

µL

2

µL µL

8

Fig 6.1. Microscopy image of FITC labeled Cstrep connector incubated with KB Cell in t he existence of lipofectamine. The volumes in the y axis indicate the amount of lipofecta mine added. Connector concentration was 100 nM.

100

Bright field DAPI channel FITC channel

1 hou

2

hour

s

4

hour

s

Control: PBS only

Fig 6.2. FITC-connector /SapC-DOPS complex. 10X magnification microscopy images of FITC-connector / SapC-DOPS complex incubated with U87 brain cancer cells. The n ucleis were stained with DAPI (blue)

101

2

hour

s

Vesicle complex observed

4 hours

FITC-connector near nucleus

PBS only Control:

Fig 6.3 FITC-connector /SapC-DOPS complex merged view. Merged view of microscop

y images of FITC-connector / SapC-DOPS complex incubated with U87 brain cancer cel

ls. The nucleis were stained with DAPI (blue), and connector was stained with FITC (gre

en, longer exposure time).

102

a b c - -

V V

+ +

Fig.6.4 Scheme of reconstituting connector into a single cell membrane, and detection o f trans-cell-membrane potential. (a) Capture of a cell on the chip with micro-pore. (b). Ad dtion of connector/DPhPC proteoliposome onto external solution. (c). Confirmation of a connector insertion onto cell membrane by observing a steady current increase.

Similar current steps happened several times, only after connector was added.

Fig 6.5 Current steps across the cell membrane after proteoliposome added.

103

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