Modification of nucleic acids
Necessity of site-selective functionalization of nucleic acids
The ability for site-selective functionalization of oligonucleotides (ON) serves as a powerful tool for investigating fundamental biological processes involving DNA- and RNA-protein interactions such as replication,1 repair,2 transcription,3, 4 translation,5 , 6 and gene silencing.7 In addition, this approach enabling the installation of orthogonal functionalities would promote the acceleration of broad DNA biotechnology and nanotechnology based on DNA-induced protein immobilization,8 DNA-patterned synthesis here, 9, 10 aptamers, 11 , 12 and DNA origami. 13, 14 However, there are few post-synthetic modifications of nucleic acids by chemical methods, due to narrow functionalities, as well as low reactivity and site selectivity.
Previous methods for synthesis of modified oligonucleotide
- Using pre-functionalized phosphoramidites
- Enzymatic methods
- Modification by SELEX (systematic evolution of ligands by exponential enrichment)
- Modifications utilizing base-pairing for site-selectivity
Reaction optimization and substrate scope
Reaction conditions of bioconjugation
Examples of modification of proteins via metal carbene
They demonstrated modification of triosephosphate isomerase (TIM) and CALP PDZ domain containing thiol residues. They showed that RNase A and insulin were modified with moderate conversion, but that myoglobin and ubiquitin, whose N-terminal amino group is not exposed, did not respond.
Reported methods of nucleic acids using metal-carbene chemistry
Reaction opitmization
Structure analysis and proposed mechanism
To confirm the structure of the acetonylation of deoxyguanosine by diazoacetone and [Rh(COD)Cl]2, a scale-up reaction was performed and NMR was performed. From the 1H-13C heteronuclear multiple bond correlation (HMBC) (Figure 2-9a), a 3JC6,H9 correlation was observed, proving O6 acetonylation of dG. To test the N7 chelation effect, N7-methyl-deoxyguanosine was additionally treated under the optimized reaction conditions to give a 65% yield of acetonylated product 11a (Figure 2-9b).
Depurination was performed to cleave the modified nucleobase, and HMBC NMR analysis (Figure 2-9c) indicated acetonylated O6 N7-methylguanine judging from the 3JC6,H9 correlation. Nucleophilic attack of O6 of dG on the Rh(I)-carbene leads to the formation of the zwitterionic intermediate. Then, electron repulsion of the Rh-C bond produces the pi-complex of the enol and the Rh(COD)Cl monomer.
Dissociation of the enol and Rh(COD)Cl regenerates the catalyst, and the enol undergoes tautomerization to O6-acetonylated dG.
Reactivity to deoxyguanosine analogs
Application of Rh(I)-catalysis to oligonucleotides
- Reactivity to oligonucleotides without secondary structure
- Reactivity to oligonucleotides containing secondary structure
- Solutions to site-selective modification
- Reactions under high dilute conditions
- Reaction in the presence of protein
Experimenatal
Applications to ligation, photocaging, and DNA-protein crosslinking
Chemical ligation of oligonucleotides
- Fields utilizing chemical ligation of oligonucleotides
- Chemical ligation of ODNs by using bisalkoxyamine linker
The absence of enzyme use and the availability of large-scale synthesis, which is cost-effective, are considered advantages of chemical ligation. Moreover, gene construction by successive chemical ligation and transformation of the gene in bacteria was accomplished to prove the compatibility of the ligated gene by the same group63. Ligation was also used for stacking DNA nanostructures to increase the stability of the hexagonal nanostructure using CuAAC64.
In addition, Kool et al demonstrated the detection of RNA and DNA point mutations by chemical ligation of phosphorothioate and 5'-iodide65. For this purpose, we prepared a duplex consisting of the template sequence 24a paired with two ODN fragments 24b and 24c, carrying a G-overhang on each fragment (Figure 3-1). Reaction with diazoacetone proceeded selectively at two G-overhanging residues to give 25b and 25c , each of which was functionalized with acetonylated-G (act-G) in excellent yield.
Ligation was successful, as confirmed by LC-MS and denaturing PAGE analysis (Figure 3-2); the low mobility band slightly above the template sequence 24a corresponds to the bound product 27 .
One-step synthesis of photocaged oligonucleotides
- Photocaing group
- Photocaged nucleic acids
- Single-step preparation of photocaged oligonucleotides via Rh(I)-catalysis
PPGs in nucleic acids are exemplified by a variety of applications such as photoregulation of gene expression with antisense oligonucleotides70, 71, DNAzymes72, ribozymes73, small interfering RNAs74 and microRNAs75. For example, Woodson and coworkers77 reported the study of temporally controlled Hfq-catalyzed ripening using photocaged ORNs. Their synthesis of photocaged ONs begins with the synthesis of phosphoramidite monomers functionalized with a photocleavable group, which requires lengthy synthetic steps.
The photocleavable group utilized in their report was the p-hydroxyphenacyl group, which can be protected by UV light at 302 nm78 (Figure 3-5). We focused on the laborious ten-step preparation of the photocaged phosphoramidite monomer 28 (Figures 3–6), which inspired us to explore the potential of our method to increase synthetic efficiency. Decomposition of 30a was completed in 10 min using UV light to provide pure deprotected ODN 17a (Figure 3-8).
The efficiency of the one-step preparation of ORNs and ODNs in the photocage was described by these results.
DNA-protein crosslinking
- Significance of DNA-protein crosslinking
- Utilized chemistry for DNA-protein crosslinking
- Proof of concept using reductive amination
RNAP was observed for 23c in the silver stain, there was only the band for RNAP without cross-linking, using 23d which contained a bulge in the middle of the T7 promoter sequence (lanes 2 and 4, respectively). We consider that cross-linking to 23d may not have resulted from the destruction of the RNAP-DNA complex due to the bulge or the absence of a nearby lysine residue83. SYBR gold staining for examination of the presence of DNA revealed the presence of dsODN 23c in the cross-linked product (lane 2).
The location of the band was similar to the location of the top band in the silver (lane 2), but there was no observation of the corresponding band for 23d (lane 4, SYBR gold). To detect the cross-linked product in the cell lysate, we designed a double G-bulge dsDNA probe in which the second acetonyl group was used for conjugation with a fluorescence probe (Figure 3-11. For selective manipulation of the two acetonyl groups) groups, they were sequentially introduced in an iterative manner.
Thus, formation of a first G-bulge dsDNA at the desired position was achieved using the corresponding reverse strand, which was then reacted with diazoacetone to give 31a . Introduction of a second G-bulge by formation of the duplex with the corresponding reverse strand followed by reaction with diazoacetone provided the dsDNA probe with double-G-bulge act 34a ready for the cross-linking experiment. The stability of duplex 34 may be slightly lower than 31 due to an additional mismatch pair, however, it does not appear to affect the reaction based on the observation that the second labeling also proceeds smoothly.
-coupling was performed by reductive amination, in which 34a was incubated with NaCNBH3 in the presence of E. Control experiments were also performed a) with the unmodified form of 34a (lane 4). Fluorescence visualization confirmed that selective cross-linking was successfully achieved even in the presence of cell lysate (lanes 1 – 3); bands with the same mobility compared to that in the positive control experiment were detected (lane 7). To confirm that the band corresponding to the cross-linking of 34a with RNAP indeed derives from sequence-specific conjugation between proteins in the lysates, we performed a DPC experiment in the presence of different concentrations of RNAP (lanes 1 - 3). .
The observation that the intensity of the fluorescent bands on the SDS-PAGE gel increases with increasing concentration of RNAP clearly supports that the bands are derived from the sequence-specific conjugation of RNAP with 34a. Further staining with silver and SYBR gold also yielded consistent results with the expected staining patterns (lanes 1–3 vs. positive control 7). Moreover, the results from several negative control experiments further support the specific cross-linking (lanes 4–6, 8, and 9).
Experimental
