Sequence-Specific DNA Damage Using Iodine-125-Labeled Antisense Oligonucleotides

Abstract

A procedure is described that cleaves single-stranded DNA with sequence specificity. This process involves attaching a DNA damaging agent to an oligonucleotide. This oligonucleotide delivers the DNA damaging agent, iodine-125, to a specific DNA sequence by complementary hybridization. 5-[¹²⁵I]Iodo-2-deoxycytidine 5′-triphosphate was enzymatically incorporated into an oligonucleotide that was designed to hybridize to a single-stranded DNA target. ¹²⁵I decays by electron capture and causes breaks in the target DNA. These breaks were observed on a DNA sequencing gel. After 22 days of exposure to the ¹²⁵I-labeled oligonucleotide, significant damage was observed within 1 to 2 bases of the expected site of hybridization. Densitometry showed that after 48 days the amount of damage had approximately doubled. This method facilitates easy design and testing of oligonucleotides that could potentially be used to inactivate gene expression in a wide variety of organisms.