臺灣博碩士論文加值系統

In 1953, James Watson and Francis Crick deduced the double helical structure of DNA and immediately inferred its mechanism of replication. This brilliant accomplishment is ranked as one of the most significant stepping stone in the history of biology because it leads to the understanding of gene function at molecular level. Thus, it has been well known that Watson-Crick base pairing is very stable. However, we have found that it is not always the case; Watson-Crick pairing can sometimes form alternate conformations, i.e. the canonical Watson-Crick G/C or A/T hydrogen-bonded base pairs can be either induced to become perpendicular with the base plane or be transformed into stacked pair. Such facile manipulation of Watson-Crick pair in either trans or cis way greatly increases the repertoire for unusual nucleic acid structural motifs and for sequence-specific DNA recognitions.

In 1953, James Watson and Francis Crick deduced the double helical structure of DNA and immediately inferred its mechanism of replication. This brilliant accomplishment is ranked as one of the most significant stepping stone in the history of biology because it leads to the understanding of gene function at molecular level. Thus, it has been well known that Watson-Crick base pairing is very stable. However, we have found that it is not always the case; Watson-Crick pairing can sometimes form alternate conformations, i.e. the canonical Watson-Crick G/C or A/T hydrogen-bonded base pairs can be either induced to become perpendicular with the base plane or be transformed into stacked pair. Such facile manipulation of Watson-Crick pair in either trans or cis way greatly increases the repertoire for unusual nucleic acid structural motifs and for sequence-specific DNA recognitions.

Abstract 1
1. Introduction 2
2. Experiments Theory
2.1DNA Synthesis 8
2.1.1 Solid phase approach 8
2.1.2 Protection Group Strategy 9
2.1.3 Chemical Synthesis 9
2.2 Sample Purification 13
2.2.1 Reverse phase HPLC 13
2.2.2 Anion exchange HPLC 14
2.2.3 Gel-filtration 14
2.3 NMR Theory 15
2.3.1 NMR phenomenon 15
2.3.2 Vector Model 17
2.3.3 Product Operator Approach 19
2.3.4 Nuclear Overhauser Effect (NOE) 21
2.3.5 One-Dimension Fourier Transform NMR Experiment 21
2.3.6 Two-Dimension Fourier Transform NMR Experiment 22
2.3.7 NMR Experiments in H2O 26
(A) Water suppression method 26
(B) 1D Imino Proton Spectrum 28
(C) 2D NOESY/ H2O 28
2.3.8 NMR Experiments in D2O 31
(a) 2D NOESY 31
(b) DQF-COSY 33
(c) 1H-31P Heteronuclear Correlation Spectrum 33
3. Materials and Methods
3.1 DNA Sample Preparation 34
3.2 Sample Purification 36
3.3 Thermodynamics Studies 38
3.4 NMR experiments 39
3.5 Structure Determination 40
(a) Distance geometry calculation 40
(b) Docking and molecular dynamics 41
4. Results and Discussion
4.1 Novel ActD/5''-GXC/CYG-5'' Complex Structure
4.1.1 Thermodynamics Studies 43
4.1.2 NMR Studies in H2O 45
4.1.3 NMR Studies in D2O 48
4.1.4 Structural Features of the Novel ActD/TA Complex 49
4.1.5 Structural Comparison Between the Novel ActD/5-(GGC)/(CCG)-5''Complex and the Classic ActD/5-(GC)/ (CG)-5’Complex 52
4.1.6 Discussion 53
4.2 Zipper-like Watson-Crick Base Pairs
4.2.1 Thermodynamics Studies 55
4.2.2 NMR Studies in H2O 57
4.2.3 NMR Studies in D2O 58
4.2.4 Structural Determination 63
4.2.5 Structural Features 63
4.2.5 Discussion 65
5. Conclusions 69
References

Addess, K. J., Sinsheimer, J. S. & Feigon, J. (1993) Biochemistry 32, 2498-508.
Altona, C. (1982). Conformational Analysis of Nucleic Acids. Determination of Backbone Geometry of Single-helical RNA and DNA in Aqueous Solution. Recl. Trav. Chim. Pays-Bas.101 (413-433).
Bailey, S. A., Graves, D. E. & Rill, R. (1994) Biochemistry 33, 11493-11500.
Batey, R. T., Rambo, R. P. & Doudna, J. A. (1999). Tertiary motifs in RNA structure and folding. Angew. Chem. Int. Ed. 38, 2326-2343.
Blackburn, E. H. (1991). Structure and Function of Telomeres. Nature, 350, 569-573.
Butcher, S. E., Dieckmann, T. & Feigon, J. (1997). Solution structure of a GAAA tetraloop receptor RNA. EMBO, J. 18, 7490-7499.
Butcher, S. E., Allain, F. H.-T. & Feigon, J. (1999). Solution structure of the loop B domain from the hairpin ribozyme. Nature Struct. Biol. 6, 212-216.
Cavanagh, J. (1996) Protein NMR Spectroscopy Principles and Practice (Fairbrother, W.
J., Palmer III, A. G., Skelton, N. J., San Duego)
Catasti, P., Gupta, G., Garcia, A. E., Ratliff, R., Hong, L., Yau, P., Moyzis, R. K. & Bradbury, E. M. (1994). Unusual structures of the tandem repetitive DNA sequences located at human centromeres. Biochemistry, 33, 3819-3830.
Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R. & Doudna, J. A. (1996a). Crystal Structure of a Group I Ribozyme Domain: Principles of RNA Packing. Science, 273, 1678-1685.
Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Szewczak, A. A., Kundrot, C. E., Cech, T. R. & Doudna, J. A. (1996b). RNA Tertiary Structure Mediation by Adenosine Platforms. Science, 273, 1696-1699.
Chen, F.-M. & Sha, F. (2001) Biochemistry 40, 5218-5225.
Chen, F.-M. (1988) Biochemistry 27, 6393-6397.
Chen, H., Liu, X. & Patel, D. J. (1996) J. Mol. Biol. 258, 457-479.
Cheng, J.-W., Chou, S.-H. & Reid, B. R. (1992). Base Pairing Geometry in GA Mismatches Depends Entirely on the Neighboring Sequence. J. Mol. Biol. 228, 1037-1041.
Chou, S.-H. & Chin, K.-H. (2001) J. Mol. Biol. 312, 753-768.
Chou, S.-H. & Chin, K.-H. (2001) J. Mol. Biol. 314, 139-152.
Chou, S.-H. (2000). NMR studies of DNA structures containing sheared purine:purine and purine:pyrimidine base pairs. J. Biomol. Struct. Dyn. Conversation 11, 2, 303-316.
Chou, S.-H., Cheng, J.-W., Fedoroff, O. & Reid, B. R. (1994a). DNA sequence GCGAATGAGC containing the human centromere core sequence GAAT forms a self-complementary duplex with sheared G:A pairs in solution. J. Mol. Biol. 241, 467-479.
Chou, S.-H., Cheng, J.-W., Fedoroff, O. Y., Chuprina, V. P. & Reid, B. R. (1992a). Adjacent G:A Mismatch Base Pairs Contain BII Phosphodiesters in Solution. J. Am. Chem. Soc. 114, 3114-3115.
Chou, S.-H., Cheng, J.-W. & Reid, B. (1992b). Solution Structure of [d(ATGAGCGAATA)]2: Adjacent GA Mismatches Stablized by Cross-Strand Base-Stacking and BII phosphate Groups. J. Mol. Biol. 228, 138-155.
Chou, S.-H. & Tseng, Y.-Y. (1999). Cross-strand pruine-pyrimidine stack and sheared purine:pyrimidine pairing in the human HIV-1 reverse transcriptase inhibitors. J. Mol. Biol. 285, 41-48.
Chou, S.-H., Tseng, Y.-Y., Chen, Y.-R. & Cheng, J.-W. (1999a). Structural studies of symmetric DNA undecamers containing non-symmetrical sheared (PuGAPu):(PyGAPy) motifs. J. Biomol. NMR, 14, 157-167.
Chou, S.-H., Tseng, Y.-Y. & Wang, S.-W. (1999b). Stable sheared A:C pair in DNA hairpins. J. Mol. Biol. 287, 301-313.
Chou, S.-H., Zhu, L., Gao, Z., Cheng, J.-W. & Reid, B. R. (1996a). Hairpin loops consisting of single adenine residues closed by sheared A:A and G:G pairs formed by the DNA triplets AAA and GAG: Solution structure of the d(GTACAAAGTAC) hairpin. J. Mol. Biol. 264, 981-1001.
Chou, S.-H., Zhu, L. & Reid, B. R. (1994b). The unusual structure of the human centromere (GGA)2 motif: Unpaired guanosines stacked between sheared GA pairs. J. Mol. Biol. 244, 259-268.
Chou, S.-H., Zhu, L. & Reid, B. R. (1997). Sheared purine:purine pairing in biology. J. Mol. Biol. 267, 1055-1067.
Chou, S.-H., Zhu, L. & Reid, R. R. (1996b). On the Relative Ability of Centromeric GNA Triplets to Form Hairpins versus Self-paired Duplexes. J. Mol. Biol. 259, 445-457.
Correll, C. C., Freeborn, B., Moore, P. B. & Steitz, T. A. (1997). Metals, Motifs, and Recognition in the Crystal Structure of a 5S rRNA Domain. Cell, 91, 705-712.
Correll, C. C., Munishkin, A., Chan, Y.-L., Ren, Z., Wool, I. G. & Steitz, T. A. (1998). Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proc. Natl. Acad. Sci. USA, 95, 13436-13441.
Dallas, A. & Moore, P. B. (1997). The Loop E - Loop D region of Escherichia coli 5S rRNA: the solution structure reveals an unusual loop that may be important for binding ribosomal proteins. Structure, 5, 1639-1653.
Evans, J. N. S. (1995) Biomolecular NMR Spectroscopy (New York).
Ferrer, N., Azorin, F., Villasante, A., Gutierrez, C. & Abad, J. P. (1995). Centromeric dedeca-satellite DNA sequences form fold-back structures. J. Mol. Biol. 245, 8-21.
Gao, Y.-G., Robinson, H., Sanishvili, R., Joachimiak, A. & Wang, A. H.-J. (2000). Structure and recognition of sheared tandem GA base pairs associated with human centromere DNA sequence at atomic resolution. Biochemistry, 38, 16452-16460.
Gorenstein, D. G., Schroeder, S. A., Fu, J. M., Metz, J. T., Roongta, V. & Jones, C. R. (1988). Assignments of 31P NMR Resonances in Oligodeoxyribonucleotides: Origin of Sequence-Specific Variations in the Deoxyribose Phosphate Backbone Conformation and the 31P Chemical Shifts of Double-Helical Nucleic Acids. Biochemistry, 27, 7223-7237.
Grady, D. L., Ratliff, R. L., Robinson, D. L., McCanlies, E. C., Meyne, J. & Moyzis, R. K. (1992). Highly conserved repetitive DNA sequences are present at human centromeres. Proc. Natl. Acad. Sci., U.S.A. 89, 1695-1699.
Gutell, R. R. (1996). Comparative sequence analysis and the structure of 16S and 23S rRNA.In, Ribosomal RNA: Structure, Evolution, Processing, and Function in Protein Biosynthesis, (Zimmermann, R. A. & Dahlberg, A. E.eds.), pp.111-128. CRC Press.
Gutell, R. R., Cannone, J. J., Shang, Z., Du, Y. & Serra, M. J. (2000). A story: Unpaired adenosine bases in ribosomal RNAs. J. Mol. Biol. 304, 335-354.
Hare, D. R., Wemmer, D. E., Chou, S.-H., Drobny, G. & Reid, B. R. (1983). Assignment of the Non-exchangeable Proton Resonances of d(CGCGAATTCGCG) Using Two-Dimensional Nuclear Magnetic Resonance Methods. J. Mol. Biol. 171, 319-336.
Henderson, E., Hardin, C. C., Walk, S. K., Tinoco, I. J. & Blackburn, E. H. (1987). Telomeric DNA Oligonucleotides Form Novel Intramolecular Structures Containing Guanine-Guanine Base Pairs. Cell, 51, 899-908.
Hermann, T. & Patel, D. J. (1999). Stitching together RNA tertiary architectures. J. Mol. Biol. 294, 829-849.
Huang, C.-H., Lin, Y.-S., Yang, Y.-L., Huang, S.-w. & Chen, C. W. (1998). The telomeres of streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures. Mol. Microbiology, 28, 905-916.
Huertas, D. & Azorin, F. (1996). Structural polymophism of homopurine DNA sequences. d(GGA)n and d(GGGA)n repeats form intramolecular hairpins stabilized by different base-pairing interactions. Biochemistry, 35, 13125-13135.
Huertas, D., Bellsolell, L., Casasnovas, J. M., Coll, M. & Azorin, F. (1993). Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes stabilized by the formation of G:A base pairs. EMBO, J. 12, 4029-4038.
Kamitori, S. & Takusagawa, F. (1992) J. Mol. Biol. 225, 445-456.
Kamitori, S. & Takusagawa, F. (1994) J. Am. Chem. Soc. 116, 4154-4165.
Kettani, A., Basu, G., Gorin, A., Majumdar, A., Skripkin, E. & Patel, D. J. (2000a). A two-stranded template-based approach to G(CA) triad formation: Designing novel structural elements into an existing DNA framework. J. Mol. Biol. 301, 129-146.
Kettani, A., Gorin, A., Majumdar, A., Hermann, T., Skripkin, E., Zhao, H., Jones, R. & Patel, D. J. (2000b). A dimeric DNA interface stabilized by stacked A(GGGG)A hexads and coordinated monovalent cations. J. Mol. Biol. 297, 627-644.
Kettani, A., Kumar, R. A. & Patel, D. J. (1995). Solution Structure of a DNA Quadruplex containing the Fragile X Syndrome Triplet Repeat. J. Mol. Biol. 254, 638-656.
Klimasauskas, S., Kumar, S., Roberts, R. J. & Cheng, X. (1994) Cell 76, 357-369.
Kuryavyi, V., Kettani, A., Wang, W., Jones, R. & Patel, D. J. (2000). A diamond-shaped zipper-like DNA architecture containing triads sandwiched between mismatches and tetrads. J. Mol. Biol. 295, 455-469.
Leontis, N. B., Ghosh, P. & Moore, P. B. (1986). Effect of magnesium ion on the structure of the 5S RNA from E. coli. An imino proton NMR study of the helix I, IV, V regions of the molecule. Biochemistry, 25, 7386-7392.
Leontis, N. B. & Westhof, E. (1998a). The 5S rRNA loop E: Chemical probing and phylogenetic data versus cyrstal structure. RNA, 4, 1134-1153.
Leontis, N. B. & Westhof, E. (1998b). A common motif organizes the structures of multi-helix loops in 16S and 23S rRNAs. J. Mol. Biol. 283, 571-583.
Liu, X., Chen, H. & Patel, D. J. (1991) J. Biomol. NMR 1, 323-347.
Liu, C. & Chen, F.-M. (1996) Biochemistry 35, 16346-16353.
Lian, C., Robinson, H. & Wang, A. H.-J. (1996) J. Am. Chem. Soc. 118, 8791-8801.
Marvin, D. A., Nave, C., Bansal, M., Hale, R. D. & Salje, E. K. (1992). Two Forms of PF1 Inovirus: X-ray Diffraction Studies on a Structural Phase Transition and a Calculated Liberation Normal Mode of the Asymmetric Unit. Phase Transitions, 39, 45-80.
Marvin, D. A., Nave, C., Ladner, J. E., Fowler, A. G., Brown, R. S. & Wachtel, E. J. (1981). Macromolecular structural transitions in Pf1 filamentous bacterial virus.In, Structural Aspects of Recognition and Assembly in Biological Macromolecules, (Balaban, M., Sussman, J. L., Traub, W. & Yonath, A.eds.).Balaban ISS, Rehovot.
Marvin, D. A. & Wachtel, E. J. P. (1976). Structure and assembly of filamentous bacterial viruses. Phil. Trans. R. Soc. Lon. (Biol. Sci.) B. 276, 81-98.
Mauffret, O., Amir-Aslani, A., Maroun, R. G., Monnot, M., Lescot , E. & Fermandjian, S. (1998). Comparative structural analysis by [1H,31P]-NMR and restrained molecular dynamics of two DNA hairpins from a strong DNA topoisomerase II cleavage site. J. Mol. Biol. 283, 643-655.
Moore, P. B. (1999). Structural motifs in RNA. Annu. Rev. Biochem. 67, 287-300.
Morosyuk, S. V., SantaLucia Jr, J. & Cunningham, P. R. (2001). Structure and function analysis of the converved 690 hairpin in E. Coli 16S rRNA. III. Functional analysis of the 690 loop. J. Mol. Biol. 307, 213-228.
Nowak, R. (1994). Mining treasures from "Junk DNA". Science, 263, 608-610.
Ortiz-Lombardia, M., Cortes, A., Huertas, D., Eritia, R. & Azorin, F. (1998). Tandem 5''-GA:GA-3'' mismatches account for the high stability of the fold-back structures formed by the centromeric Drosophila dodeca-satellite. J. Mol. Biol. 277, 757-762.
Plateau, P. & Gueron, M. (1982). Exchangeable Proton NMR without Base-Line distortion, Using New Strong-Pulse Sequences. J. Am. Chem. Soc. 104, 7310-7311.
Quigley, G. J., Ughetto, G., van der Marel, G. A., van Boom, J. H., Wang, A. H.-J. & Rich, A. (1986) Science 279, 1255-1258.
Reinisch, K. M., Chen, L., Verdine, G. L. & Lipscomb, W. N. (1995) Cell 82, 143-153.
Reinisch, K. M., Chen, L., Verdine, G. L. & Lipscomb, W. N. (1995) Cell 82, 143-153.
Robinson, H., Gao, Y.-G., Yang, X.-L., Sanishvili, R., Joachimiak, A. & Wang, A. H.-J. (2001) Biochemistry 40, 5587-5592.
Sarma, R. H., Mynott, R. J., Wood, D. J. & Hruska, F. E. (1973). Determination of the Preferred Conformations Constrained along the C4''-C5'' and C5''-O5'' Bonds of b-5''-Nucleotide in Solution. Four-Bond 31P-1H Coupling. J. Am. Chem. Soc. 95, 6457-6459.
Sanders, J. K. M. (1988) Modern NMR Spectroscopy (Hunter, New York).
Schmitz, U., James, T. L., Lukavsky, P. & Walter, P. (1999). Structure of the most conserved internal loop in SRP RNA. Nature Struct. Biol. 6, 634-638.
Shepard, W., Cruse, W. B. T., Fourme, R., Fortelle, E. d. l. & Prange, T. (1998). A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution. Structure, 6, 849-861.
Sklenar, V., Miyashiro, H., Zon, G., Miles, H. T. & Bax, A. (1986). Assignment of the 31P and 1H Resonances in Oligonucleotides by Two-dimensional NMR Spectroscopy. FEBS Letters, 208, 94-98.
Snyder, J. G., Hartman, N. G., D''Estantott, B. L., Kennard, O., Remeta, D. P. & Breslauer, K. J. (1989) Proc. Natl. Acad. Sci. USA 86, 3968-3972.
Spackova, N., Berger, I. & Sponer, J. (2000). Nanosceond mlecular dynamics of zipper-like DNA duplex structures containing sheared GA mismatch pairs. J. Am. Chem. Soc. 122, 7564-7572.
Sponer, J., Gabb, H. A., Leszczynski, J. & Hobza, P. (1997) Biophys. J. 73, 76-87.
Tseng, Y.-Y. & Chou, S.-H. (1999). Systematic NMR assignment pathways for DNA exchangeable protons. J. Chin. Chem. Soc. 46, 699-706.
Varani, G., Wimberly, B. & Tinoco, I. J. (1989). Conformation and Dynamics of an RNA Internal Loop. Biochemistry, 28, 7760-7772.
Umezawa, Y. & Nishio, M. (2000) Bioorg. & Med. Chem. 8, 2643-2650.
Viswamitra, M. A. & Pandit, J. (1983). A Proposal For a Specific Double-Helical Structure in Which the Polynucleotide Strands Intercalate Instead of Forming Base-pairs. J. Biomol. Struct.Dyn. 1, 743-753.
Wadkins, R. M., Vladu, B. & Tung, C.-S. (1998) Biochemistry 37, 11915-11923.
Westhof, E. & Fritsch, V. (2000). RNA folding: beyond Watson-Crick pairs. Structure, 8, R55-R65.
Wild, K., Weichenrieder, O., Leonard, G. A. & cusack, S. (1999). The 2 A struture of helix 6 of the human signal recognition particle RNA. Structure, 7, 1345-1352.
Wimberly, B., Varani, G. & Tinoco, I. J. (1993). The conformation of loop E of eukaryotic 5S ribosomal RNA. Biochemistry, 32, 1078-1087.
Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids (John Wiley & Sons, New York).
Yang, X.-L. & Wang, A. H.-J. (1999) Pharmacology & Therapeutics 83, 181-215.
Zhu, L., Chou, S.-H. & Reid, B. R. (1995a). Structure of a Single-cytidine Hairpin Loop formed by the DNA Triplet GCA. Nature Struct. Biol. 2, 1012-1017.
Zhu, L., Chou, S.-H. & Reid, B. R. (1996). A single G-to-C change causes human centromere TGGAA repeats to fold back into hairpins. Proc. Natl. Acad. Sci. USA, 93, 12159-12164.
Zhu, L., Chou, S.-H. & Reid, R. B. (1995b). The structure of a novel DNA duplex formed by human centromere d(TGGAA) repeats with possible implications for chromosome attachment during mitosis. J. Mol. Biol. 254, 623-637.
Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, J.-P. & Pardi,
A. (1997). Interlocking structural motifs mediate molecular
discrimination by a theophylline-binding RNA. Nature Struct. Biol.
4, 644-649.