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1. Nonenzymatic Cleavage of Single-Stranded DNA to Nucleotide Resolution. 2. Novel Base Specific DNA Cleavage Reactions


Iverson, Brent Lee (1988) 1. Nonenzymatic Cleavage of Single-Stranded DNA to Nucleotide Resolution. 2. Novel Base Specific DNA Cleavage Reactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/pd8h-vx39.


Part 1

A new strategy for the complementary-addressed modification of nucleic acids was investigated involving the enzymatic incorporation of a modified 2'-deoxynucleotide 5'-triphosphate molecule into oligonucleotide strands. These modified 2'-deoxynucleotide 5'-triphosphate compounds (and hence the strands into which they were incorporated) carried a latent reactive group in the form of a methylthioether function. The methylthioether function was activated by treatment with cyanogen bromide to enable alkylation of a complementary nucleic acid strand. The alkylation was shown to involve methyl group transfer and upon piperidine treatment resulted in the cleavage of the DNA at essentially a single residue on the target strand. The system was found to be capable of cleaving oligonucleotides as well as long pieces (5386 bases) of single-stranded DNA to nucleotide resolution.

Part 2

Two novel base specific DNA cleavage reactions were discovered and investigated. The first is an A specific reaction caused by K₂PdCl₄ at low pH. The second is a photochemical reaction with "GG" specificity caused by some nitroaromatic and Co(III) compounds. Reaction of DNA with K₂PdCl₄ at low pH followed by a piperidine workup produces specific cleavage at adenine residues. Product analysis revealed the K₂PdCl₄ reaction involves selective depurination at adenine, affording a gapping reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Photolysis of DNA in the presence of 4-nitroveratrole, 3-nitroanisole or Co(III) compounds such as Co(III)(NH₃)₆ followed by a piperidine workup produces cleavage of the DNA with "GG" specificity, that is the 5'-G of 5'-GG-3' sequences is preferentially attacked. Product analysis revealed that the aromatic guanine base is decomposed into numerous fragments, and the nitroaromatic compound is apparently not consumed in the reaction. An electron transfer mechanism is proposed to account for the photochemical reaction at 5'-GG-3' sequences.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Dervan, Peter B.
  • Dougherty, Dennis A.
  • Richards, John H.
Defense Date:24 August 1987
Funding AgencyGrant Number
Record Number:CaltechTHESIS:01242013-083723752
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7430
Deposited By: Benjamin Perez
Deposited On:24 Jan 2013 16:52
Last Modified:16 Apr 2021 23:01

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