Deoxyribozymes to hydrolyze DNA phosphodiester bonds

Xiao, Ying

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Description

Title

Deoxyribozymes to hydrolyze DNA phosphodiester bonds

Author(s)

Xiao, Ying

Issue Date

2014-05-30T16:44:15Z

Director of Research (if dissertation) or Advisor (if thesis)

Silverman, Scott K.

Doctoral Committee Chair(s)

Silverman, Scott K.

Committee Member(s)

• Bailey, Ryan C.
• Hergenrother, Paul J.
• Nair, Satish K.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Deoxyribozymes
• DNA hydrolysis

Abstract

In nature, the main role of double-stranded DNA is storage of genetic information. However, once a DNA oligonucleotide is freed from its confining complementary strand, it can fold into complex three-dimensional structures that support catalytic activities. These catalytic single-stranded DNA oligonucleotides are called deoxyribozymes. Up to now, there are no known deoxyribozymes in nature, and the only method to identify them is in vitro selection. Since the identification of the first deoxyribozyme in 1994, many deoxyribozymes have been found to catalyze various chemical reactions, of which the substrates can be either oligonucleotides or small molecules. Our lab has recently reported the first DNA-hydrolyzing deoxyribozyme, 10MD5, which catalyzes the sequence-specific hydrolysis of a DNA phosphodiester bond. The DNA hydrolysis by 10MD5 proceeds with kobs = 2.7 h-1 and rate enhancement of 1012 over the uncatalyzed P–O bond hydrolysis. However, 10MD5 has a sharp pH optimum near 7.5, with greatly reduced yield and rate when the pH is changed only by 0.1 units in either direction. Therefore, 10MD5 was optimized via reselection, leading to variants with broader pH tolerance. The reselection experiments and the follow-up characterization were described in Chapter 2. An artificial phylogeny constructed with sequences of the reselected variants suggested three mutations, T16R, G19Y and C30T, are strongly correlated with the broader pH tolerance. The reselected variants with broader pH tolerance were also found to suffer from relaxed site specificity, which could only be restored by expanding the “recognition site” beyond ATG^T (as in the parent 10MD5) to TATG^TT. These findings reflected functional compromises of the initial family of DNA-hydrolyzing deoxyribozymes. Additionally, a reselected variant, 9NL27, showed unique Zn2+-only hydrolysis activity, even though it differed from 10MD5 by only five out of 40 nucleotides. Evaluation of the five mutations in 9NL27 showed that only two nucleotide mutations in the 10MD5 sequence, T16A and G19C, were sufficient to convert the heterobimetallic 10MD5 deoxyribozyme into a monometallic deoxyribozyme that required Zn2+ alone. Systematic selection experiments were performed to establish broad generality of ssDNA-hydrolyzing deoxyribozymes and to identify a complete set of deoxyribozymes that can collectively cleave any arbitrarily chosen single-stranded DNA substrate at any predetermined site. These selection experiments were described in Chapter 3. Comprehensive selection experiments were performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotides at the cleavage site (e.g. X^G, X= A, T, C, or G) while retaining WatsonCrick sequence generality beyond those nucleotides along with useful cleavage rates. These results established experimentally that broadly sequence-tolerant and site-specific deoxyribozymes were readily identified for hydrolysis of single-stranded DNA. However, further selection experiments did not lead to identification of many new sequence-general deoxyribozymes due to various problems. The efforts toward the complete set of ssDNA-hydrolyzing deoxyribozymes were stopped because of the limited application of ssDNA-hydrolyzing deoxyribozymes alone. Finally, current efforts to achieve double-stranded DNA hydrolysis were described in Chapter 4. A targeted selection approach was designed to identify dsDNA-hydrolyzing deoxyribozymes, in which the deoxyribozyme pool would be conjugated to a dsDNA-binding protein, dHax3-TALE. The protein would recognize and bind to a specific region in the double-stranded DNA substrate, while the deoxyribozyme would catalyze the site-specific hydrolysis of the substrate. The ligation and capture steps were validated, and the selection conditions were determined. However, the key challenge of the selection approach was the requirement of a site-specific and efficient bio-conjugation method to link the deoxyribozyme pool with the TALE protein. Four conjugation strategies were designed, based on disulfide exchange reaction, FBDP-tyrosine reaction, PTAD-tyrosine reaction, or N-terminal threonine oxidation. After comprehensive assays, the strategies based on PTAD-tyrosine reaction and N-terminal threonine oxidation showed high site-specificity and promising reaction yields.

Graduation Semester

2014-05

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Copyright 2014 Ying Xiao

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