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Molecular Engineering of Nucleic Acid

Permanent Link: http://ufdc.ufl.edu/UFE0041192/00001

Material Information

Title: Molecular Engineering of Nucleic Acid Towards Functional and Smart Materials
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Kang, Huaizhi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, aptamer, azobenzene, delivery, dna, drug, hydrogel, liposome, nanomotor, nucleic, photoregulation
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Since the 1950s, traditional scientific research in physics, chemistry and biology has turned towards artificial manipulation of biological activities and construction of molecular level objects. Among all the molecules scientists investigate, nucleic acids and their natural hybridization behavior have aroused tremendous attention. Scientists have explored the chemistry and composition of isolated nucleic acids and have extended the research to the critical and versatile functions of these molecules in biological systems. The first goal of this research was development of a drug delivery system utilizing aptamers for targeted chemotherapy. The aptamers that have used were discovered recently through cell SELEX with high binding selectivity and specificity to target cancer cells. These aptamers were grafted to a liposome particle surface to form a targeting carrier, which selectively bound to targeted cancer cells and released loaded drug molecules. Another area of investigation was the development of advanced biomaterials for tissue engineering and drug delivery. A DNA hybridization mechanism was used to build a DNA-polymer hybrid hydrogel for potential biomedical and bioengineering applications by combining the advantages of both the polymer and DNA. By introducing an extra photoresponsive element, this hybrid hydrogel is photocontrollable and able to encapsulate and release payloads via sol-gel conversion. This is a promising biomaterial for tissue engineering and drug carriers. The third individual research project focused on developing DNA-based nanomotors with the goal of engineering a reversible photo-driven molecular motor. A photoresponsive single DNA hairpin structure was engineered by incorporating photoresponsive moieties, azobenzenes, in the DNA backbone. The azobenzene incorporated DNA nanostructures can absorb at two different wavelengths, and displays reversible motor movement with higher energy conversion efficiencies than linear DNAs. The future goal is to develop high efficiency photo-driven molecular nanomotors using artificial light sources or even solar energy. Overall this research has applied the basic chemistry and physics of nucleic acids, as well as their functionalities in special situations. The ultimate goal of these projects was to use nucleic acids and nanotechnology to design, develop and investigate functional and smart materials, which we envision will be useful in biomedical and pharmaceutical applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huaizhi Kang.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041192:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041192/00001

Material Information

Title: Molecular Engineering of Nucleic Acid Towards Functional and Smart Materials
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Kang, Huaizhi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, aptamer, azobenzene, delivery, dna, drug, hydrogel, liposome, nanomotor, nucleic, photoregulation
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Since the 1950s, traditional scientific research in physics, chemistry and biology has turned towards artificial manipulation of biological activities and construction of molecular level objects. Among all the molecules scientists investigate, nucleic acids and their natural hybridization behavior have aroused tremendous attention. Scientists have explored the chemistry and composition of isolated nucleic acids and have extended the research to the critical and versatile functions of these molecules in biological systems. The first goal of this research was development of a drug delivery system utilizing aptamers for targeted chemotherapy. The aptamers that have used were discovered recently through cell SELEX with high binding selectivity and specificity to target cancer cells. These aptamers were grafted to a liposome particle surface to form a targeting carrier, which selectively bound to targeted cancer cells and released loaded drug molecules. Another area of investigation was the development of advanced biomaterials for tissue engineering and drug delivery. A DNA hybridization mechanism was used to build a DNA-polymer hybrid hydrogel for potential biomedical and bioengineering applications by combining the advantages of both the polymer and DNA. By introducing an extra photoresponsive element, this hybrid hydrogel is photocontrollable and able to encapsulate and release payloads via sol-gel conversion. This is a promising biomaterial for tissue engineering and drug carriers. The third individual research project focused on developing DNA-based nanomotors with the goal of engineering a reversible photo-driven molecular motor. A photoresponsive single DNA hairpin structure was engineered by incorporating photoresponsive moieties, azobenzenes, in the DNA backbone. The azobenzene incorporated DNA nanostructures can absorb at two different wavelengths, and displays reversible motor movement with higher energy conversion efficiencies than linear DNAs. The future goal is to develop high efficiency photo-driven molecular nanomotors using artificial light sources or even solar energy. Overall this research has applied the basic chemistry and physics of nucleic acids, as well as their functionalities in special situations. The ultimate goal of these projects was to use nucleic acids and nanotechnology to design, develop and investigate functional and smart materials, which we envision will be useful in biomedical and pharmaceutical applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huaizhi Kang.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041192:00001


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PAGE 11

In vitro

PAGE 15

Nucleic Acid s and Molecular Engineering in vivo in vitro

PAGE 16

Compositio n and Structure of Nucl e ic Acids

PAGE 17

Synthesis of Chemically Modified Nucleic Acids

PAGE 18

in vitro

PAGE 19

Fluorescence and Applications in vitro in vivo

PAGE 20

Fluorescence and FRET Jablonski diagram

PAGE 22

Fluorescence Spectrometer Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

PAGE 24

K

PAGE 25

Targeted Drug Delivery Systems

PAGE 26

Ligands for Targeting Funct ion Liposomes

PAGE 27

in vivo

PAGE 28

Hydrogels and Their Applications Hydrogels

PAGE 29

Hydrogels for Drug Delivery

PAGE 31

Nucleic Acid s Base d Nanomachines

PAGE 32

Azobenzene trans cis transcis trans cis trans

PAGE 33

in vivo Azobenzene Incorporated DNAs trans cis

PAGE 35

35 F igure 1-1. DNA bases and their corresponding nucleotide structures. DNA nucleotides are shown as A, G, C, and T (deoxy-D-ribose sugar), while U is shown in an RNA nucleotide (D-ribose sugar).

PAGE 36

36 F igure 1-2. Structure of a DNA oligonucleotide and base pairing.

PAGE 37

37 b tnfr n btnfrnr fr F i gure 1-3. Structure of phosphoramidite and four nucleic acid phosphoramidite monomers

PAGE 38

38 b b b b btnfrn trrfnr trrn nbr rrn bbr Fi gure 1-4. Nucleic acid synthesis through DNA synthesizer.

PAGE 39

39 F igure 1-5. Fluorescence mechanism and fluorescence resonance energy transfer (FRET). (A) Jablonski diagram of fluorescence, (B) Fluorescence resonance energy transfer (FRET), (C) The relationship between the absorbance and emission spectra of the FRET pair.

PAGE 40

40 F igure 1-6. The scheme of a typical fluorescence spectrometer with main components. F igure 1-7. Multifunctional liposome nanostructure for targeted delivery.

PAGE 41

41 F igure 1-8. Scheme of a regular crosslinking hydrophilic hydrogel network. F igure 1-9. Photo-induced isomerization of an azobenzene molecule.

PAGE 42

42 bt nbf b bt rrr r Figure 1-10. Scheme of the reversible hybridization/dehybridization of an azobenzene incorporated DNA duplex. The azobenzenes are in trans -form after irradiation with visible light and can stabilize the duplex structure. The azobenzenes are in cis -form after irradiation with UV light and can dissociate the hybridized duplex structure.

PAGE 43

Introduction in vitro in vivo

PAGE 44

Materials and Instrument ation

PAGE 45

Design and Experiments

PAGE 46

Synthesis and Purific ation of Lip o somes

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x Synthesis of Dye Labeled Aptamer

PAGE 48

Aptamer Conjugation to Liposomes and Purification Flow Cytometry and Confocal Microscopy Imaging

PAGE 49

Results and Discussion Design of Multifunc t ion al Liposome Nanostructures for Drug Delivery

PAGE 50

Characterization of the Liposome

PAGE 51

Delivery to Target Cells

PAGE 54

Conclusion in vivo

PAGE 60

Introduction

PAGE 61

transcis Materials and Instrument ation

PAGE 62

Design and Experiments cistranstranscis

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tran cis cis tran Synthesis of Azobenzene Phosphoramidite (Azo -) 1. 21

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2 32 3 Synthesis of Acrydite Phosphoramidite (4) 4

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4 Synthesis of Azobenzene DNA Linker (ADL) and DNA linker (DL)

PAGE 66

Synthesis of DNA Polymer Conjugates (DPCs) Hydrogel Preparation

PAGE 67

Encapsulation and Release of Hydrogels

PAGE 68

B iocompatibility of ADL and ADL Hydrogels Cell Viability Study Results and Discussion Preparation of Photocontrollable Hydroge l

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trans cis cis-trans Reversible Sol-gel Conversion

PAGE 71

trans transTheoretical C alculation s and Modeling of the Hydrogel Microstructure

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Photocontrollable Releasing

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x x x

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Thermodynamic Profiles

PAGE 77

Biocompatibility

PAGE 78

Photocontrollable Cancer Drug Delivery

PAGE 80

Conc lusions in vitro

PAGE 83

83 F igure 3-3. Scheme of synthesis of (A). azobenzene-tethered phosphoramidite monomer (Azo-) and (B). acrydite phosphoramidite monomer.

PAGE 84

84 F igure 3-4. Scheme of incorporation of Azoor acrydite units to DNA sequences by DNA synthesizer.

PAGE 93

In vitro

PAGE 94

Introduction trans cis

PAGE 96

Materials and Instrument ation

PAGE 97

Design and Experiments

PAGE 99

Synthesis and Purification of Photoswitchable Molecular Motors (PSMMs) Characterization of PSMMs

PAGE 100

Photoregulation of PSMMs and Optimization of Operation Con ditions

PAGE 101

Buffer O ptimization

PAGE 102

Light S ources O ptimization cistranstran s cis

PAGE 103

Design Synthesis and Photoregulation of Linear DNAs Results and Discussions Engineering of AzoMoi eties on PSMMs

PAGE 104

Characterizations of AzoDNA

PAGE 105

Photoregulation of the PSMMs and Their Energy C onversion Efficiency Comparison

PAGE 106

trans

PAGE 107

trans cis cistrans transcis Einput = P s x

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x woutput = Fs Woutput Woutput x x woutput x x x NA x x x Woutput Einputx x x Reversibility

PAGE 109

Energy C onversion Efficiency : Comparison with Linear DNAs

PAGE 111

x x x x x

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trans cis trans ciscis trans cisEnergy Conversion Efficiency Comparison with PolyT Loop DNAs

PAGE 113

Conclusion

PAGE 116

trans cis

PAGE 117

117 F igure 4-2. Scheme of available positions for Azoincorporation in the stem region. F igure 4-3. The sequences of the six types of PSMM (PSMMs 1-6). PSMM1, PSMM2, and PSMM3 (or named PSMM1-3 for all three types) are hairpin structures with one to three Azoon the 3 end, and PSMM4, PSMM5, and PSMM6 (or named PSMM4-6 for all three types) are hairpin structures with one to three Azoon the 5 end. The blue bases are stem moieties; red are Azounits, and black bases are on loop moieties.

PAGE 121

Vis UV With cDNA

PAGE 122

A

PAGE 123

B

PAGE 125

Summary of the Molecular Engineering of Nucleic Acid s A Liposomebased Nanostructure for Aptamer Directed Delivery

PAGE 126

DNA Crosslinked Hydrogels for Photocontrollable Release

PAGE 127

A Single Component DNA Nanomotor Regulated by Photons Nano Letters

PAGE 128

in vivo Future Work : NIR Light Controlled DNA Crosslinked Hydrogels for Drug Release

PAGE 129

129 Cells TargetCells A u -AgNanorod(NR) DNA-polymernanogel withNRcore Targeteddilevery NIRlight Figure 5-1 NIR photocontrollable nanogels.

PAGE 130

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