The proposed studies develop a novel, site-specific, and sequence-specfic DNA cross-linking reaction as a signal output for the detection of nucleic acid sequence and structure in bioanalytical applications. A nanopore will be used for single-molecule detection of cross-linked DNA. We have selected the detection of single-nucleotide polymorphisms (SNPs) as a test-bed to demonstrate the general utility of this approach. Many assays use noncovalent, reversible hybridization of a probe to target for detection of nucleic acid sequence. Our approach employing sequence-specific covalent cross-linking to the target strand is novel. In this application, we propose to develop sequence-specific DNA cross-linking as a new single-molecule signal-output for use with nucleic acid-based sensors and probes. The proposed work builds upon our extensive preliminary data including: (i) sequence-selective cross-link formation in DNA duplexes containing a reactive Ap site (Gates), (ii) use of nanopore technology for the bioanalytical characterization of nucleic acid sequence and structure (Gu), and (iii) use of nanopore technology for the single-molecule detection of DNA duplexes containing interstrand cross-links (Gates and Gu). We hypothesize that sequence-selective cross-linking reactions can be used for the sensitive detection of disease-relevant nucleic acid sequences. Experiments to test this hypothesis are divided into the following Three Specific Aims: (1) Use biochemical methods to characterize and optimize the ability of Ap- containing probes to detect disease-relevant DNA sequences via sequence-specific cross-linking reactions. (2) Develop and optimize methods for the quantitative detection of cross-linked DNA duplexes in the ?-hemolysin (HL) protein nanopore, including the use of Ap-containing probe strands that optimize capture and unzipping in the nanopore, engineered protein pores, and barcoded probe strands. (3) Apply the methods developed in Aims 1 and 2 to the detection of disease-relevant DNA sequences in human cell lines. The outcomes of our studies include the characterization of sequence-selective cross-linking reactions for the accurate detection of polymorphisms in disease-relevant human DNA sequences and characterization of the resulting cross-linked duplexes using nanopore technology. The long-term goal of our research is to exploit new sequence-specific cross-linking reactions and the unmistakable current signature of cross-linked duplexes in the ?- HL nanopore to develop a single-molecule detection strategy that can provide a robust signal output to any hybridization-based analytical strategy.
Methods for the selective detection of DNA sequence and structure are important in biology and medicine, for example in the detection of disease-relevant single base changes in the genome (single nucleotide differences in the genetic code) that can underlie human variation in susceptibility to disease, responses to drugs and environmental exposure, drug efficacy, and cancer outcomes. In this work, we will develop a fundamentally novel approach to sequence detection involving covalent chemical cross-linking of a probe strand to the target nucleic acid.
