Creation of RNA molecules that recognize the oxidative lesion 7,8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA

Abstract

We used in vitro evolution to obtain RNA molecules that specifically recognize and bind with high affinity to the oxidative lesion 7, 8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA. A pool of approximately 10(15) RNA molecules containing a random insert of 45 nucleotides in length was subject to 10 successive rounds of chromatographic enrichment using an 8-oxodG affinity matrix, reverse transcription, PCR amplification, and RNA synthesis. Selected RNA molecules bind to 8-oxodG located at the 3' terminus (Kd </= 270 nM) or in the center (Kd </= 2.8 microM) of a 19-nt strand of DNA, with no detectable affinity for the corresponding dG-containing DNA sequences. These 8-oxodG-binding RNAs will be used to monitor levels of 8-oxodG in DNA from biological sources and should provide a unique method for evaluating oxygen-mediated DNA damage. This approach should be applicable for the creation of RNA molecules that can bind to and identify the different modifications of DNA produced by a variety of environmental agents.

Figure 1

Structures of the 8-oxodG-CH-Sepharose affinity matrix, dG, and 8-oxodG.

Scheme 1

In vitro evolution of RNA molecules using an 8-oxodG nucleoside affinity matrix.

Figure 2

Percent RNA recovery for each cycle of in vitro evolution.

Figure 3

Binding affinity in cycle 10 pool RNA to 8-oxodG and dG using an electrophoretic mobility-shift assay. Unlabeled RNA was denatured at 100°C for 1 min, mixed with binding buffer to a final concentration of 20 mM NH₄CH₃CO₂ (pH 6), 300 mM NaCl, and 5 mM MgCl₂, and renatured for 1 h. This RNA was allowed to bind with the indicated 5′-³²P-radiolabeled (∗) DNA substrate (1 pmol) for 30 min at rt before the addition of nondenaturing loading buffer (Promega). Samples were loaded onto a running (25 mA), 10% native polyacrylamide gel (29:1 acryl/bis), electrophoresed for 3–4 h at 10–12°C, and subsequently dried on DE-81 DEAE cellulose paper (Whatman). Quantitation of the single-stranded (ss) DNA and the RNA/DNA complex was obtained by using PhosphorImage analysis with ImageQuant software (Molecular Dynamics). (A) Native gel analysis of binding reactions between cycle 10 pool RNA (30–1,000 pmol) and the terminal-8-oxodG-DNA substrate (1 pmol). (B) Native gel analysis of binding reactions between cycle 10 pool RNA (30–1,000 pmol) and the terminal-dG-DNA substrate (1 pmol).

Figure 4

Evaluation of 8-oxodG binding by cloned RNA molecules. Individual RNA molecules (100 pmol) were denatured, renatured, and then incubated with the 5′-³²P-radiolabeled (∗) terminal-8-oxodG-DNA substrate (1 pmol) as described in Fig. 3. The samples were analyzed on a 10% native polyacrylamide gel, and the single-stranded (ss) DNA and RNA/DNA complex was quantified as previously described. (A) Binding of individual RNA clones to the terminal-8-oxodG-DNA substrate. (B) Binding of individual RNA clones to the centrally located 8-oxodG-DNA substrate.

Figure 5

Computer-generated secondary structure of RNA R10-B35 predicted by using the program mulfold (29, 30). The highlighted nucleotides correspond to fixed primer sequence.

Figure 6

Affinity and specificity of RNA R10-B35 to 8-oxodG. 5′-³²P-Radiolabeled (∗) terminal-8-oxodG-DNA (1 pmol) was mixed with unlabeled dG-DNA (10–10,000 pmol), and the substrates were allowed to compete for binding to R10-B35 RNA (100 pmol) for 30 min at rt. The samples were analyzed, and the data were quantified as described.