Acceleration of the Deamination of Cytosine through Photo-Crosslinking

Abstract

Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures. Through substitution of the groups at the 5'-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours.

Keywords: DNA manipulation; RNA editing; cytosine deamination; nucleobase editing; photo-crosslinking.

Figure 1

Structure of the cytosine derivatives.

Figure 2

(A) Scheme of the photo-crosslink-assisted cytosine deamination reaction using cytosine derivatives. (B) UPLC analysis of the deamination reaction of the photo-cross-linked duplex consisting of ODN(K) and cODN(C) consisting of cytosine derivatives. [ODN(K<>C)] = 5 μM in 50 mM Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl. incubated at 37 °C; photo splitting was performed with a transilluminator (312 nm) at 37 °C. The peak marked with an asterisk (*) indicates the newly formed product as depicted on each plot.

Figure 3

Dependence of LogP and the reaction rate constant of the deamination reaction at 90 °C.

Figure 4

Scheme of deamination of cytosine with different counter base and photo-crosslinker incorporated in the ODN.

Figure 5

(A) Bar graph representing the LogP of each combination of ODN containing a counter base (I, G, or C) and a photo-crosslinker (^OHVK, ^OMeVK, ^CNVK, or ^NH2VK). The detailed data are provided in Table S5. Lower LogP values represent higher hydrophilicity. (B) Correlation of LogP and the reaction rate constant of the deamination reaction at 37 °C.

Figure 6

Proposed mechanism of the deamination of cytosine cross-linked to the cross-linker in the complementary strand.

Figure 7

Scheme of deamination of the cytosine with disjointed complimentary ODNs. Here, K is ^CNVK and X is the counter base of the target cytosine.

Figure 8

Conversion of cytosine to uracil after the photo-crosslink-assisted deamination reaction with ODN(K) and ODN(X/Xp).

Figure 9

Sequence and structure of the disjointed ODNs with phosphate-modified guanine and ^CNVK.

Figure 10

UPLC analysis of the deamination reaction of the photo-cross-linked duplex consisting of ODN(K/pK), ODN(X/Xp), and ODN(tC). [ODN(G/K<>C)] = 5 μM in 50 mM Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl. incubated at 37 °C; photo splitting was performed with a transilluminator (312 nm) at 37 °C.

Figure 11

Conversion of cytosine to uracil after the photo-crosslink-assisted deamination reaction with ODN(K/pK) and ODN(X/Xp).

Figure 12

(A) Deamination reaction scheme with 5′-terminal modified ODNs. (B) Structure of the various 5′-terminal modifications. (C) Conversion ratio of cytosine to uracil using the different 5′-terminal modifications. (D) LogP values of the 5′-terminal modified ODNs.

Figure 13

Compiled LogP values of the counterbase–crosslinker variation compared with 5′phosphate modification.

Figure 14

Proposed mechanism of the deamination of the cytosine cross-linked to the cross-linker in the complementary strand with a free phosphate group.