Event Title

Directed Evolution Strategy for Generating of Novel Zinc Finger Peptides with Affinity for Gapped DNA

Location

Parker-Reed, SSWAC

Start Date

30-4-2015 11:00 AM

End Date

30-4-2015 1:55 PM

Project Type

Poster

Description

A significant fraction of oncologists arsenal consists of DNA damage causing agents. Repair proteins that recognize aberrant DNA structural features mitigate this damage and thus the efficacy of such anticancer drugs. DNA damage recognition is crucial for genomic stability, and thus avoidance of carcinogenesis, in healthy cells. The DNA repair enzymes poly(ADP-ribose) polymerase (PARP) and DNA ligase III use zinc finger motifs to facilitate this sequence-independent DNA recognition. Reported here is a strategy to model these protein-DNA interactions using small proteins. Proteins with high affinity for gapped DNA are selected from highly diverse libraries of proteins, based on PARP-like zinc finger domains, using phage display. These populations were engineered at the DNA level using two parallel approaches with built-in randomization then cloned into viral chromosomes. The de novo approach involved synthetic oligonucleotides with specific random codons. In addition, the gene encoding the second PARP zinc finger was cloned from human cells and subjected to 30 cycles of error-prone PCR with a mutation rate of 0.02 per nucleotide. From each approach, a library of approximately 10 billion distinct phage-displayed variants is exposed to immobilized DNA molecules featuring a gapped DNA construct. Selected molecules are either identified by DNA sequencing or subjected to further selection after amplification in bacterial culture. In subsequent experiments, Illumina sequencing will identify consensus sequences that will be cloned into plasmids appropriate for expression in bacteria. Molecules discovered by this strategy will help illuminate the structure-function relationships of proteins that recognize distinct DNA structures and may lead to new anticancer approaches.

Sponsoring Department

Colby College. Chemistry Dept.

CLAS Field of Study

Natural Sciences

Event Website

http://www.colby.edu/clas

ID

1359

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Apr 30th, 11:00 AM Apr 30th, 1:55 PM

Directed Evolution Strategy for Generating of Novel Zinc Finger Peptides with Affinity for Gapped DNA

Parker-Reed, SSWAC

A significant fraction of oncologists arsenal consists of DNA damage causing agents. Repair proteins that recognize aberrant DNA structural features mitigate this damage and thus the efficacy of such anticancer drugs. DNA damage recognition is crucial for genomic stability, and thus avoidance of carcinogenesis, in healthy cells. The DNA repair enzymes poly(ADP-ribose) polymerase (PARP) and DNA ligase III use zinc finger motifs to facilitate this sequence-independent DNA recognition. Reported here is a strategy to model these protein-DNA interactions using small proteins. Proteins with high affinity for gapped DNA are selected from highly diverse libraries of proteins, based on PARP-like zinc finger domains, using phage display. These populations were engineered at the DNA level using two parallel approaches with built-in randomization then cloned into viral chromosomes. The de novo approach involved synthetic oligonucleotides with specific random codons. In addition, the gene encoding the second PARP zinc finger was cloned from human cells and subjected to 30 cycles of error-prone PCR with a mutation rate of 0.02 per nucleotide. From each approach, a library of approximately 10 billion distinct phage-displayed variants is exposed to immobilized DNA molecules featuring a gapped DNA construct. Selected molecules are either identified by DNA sequencing or subjected to further selection after amplification in bacterial culture. In subsequent experiments, Illumina sequencing will identify consensus sequences that will be cloned into plasmids appropriate for expression in bacteria. Molecules discovered by this strategy will help illuminate the structure-function relationships of proteins that recognize distinct DNA structures and may lead to new anticancer approaches.

http://digitalcommons.colby.edu/clas/2015/program/5