Enzymatic Conjugation of Modified RNA Fragments by Ancestral RNA Ligase AncT4_2

Abstract

Oligonucleotide therapeutics have great potential as a next-generation approach to treating intractable diseases. Large quantities of modified DNA/RNA containing xenobiotic nucleic acids (XNAs) must be synthesized before clinical application. In this study, the ancestral RNA ligase AncT4_2 was designed by ancestral sequence reconstruction (ASR) to perform the conjugation reaction of modified RNA fragments. AncT4_2 had superior properties to native RNA ligase 2 from T4 phage (T4Rnl2), including high productivity, a >2.5-fold-higher turnover number, and >10°C higher thermostability. One remarkable point is the broad substrate selectivity of AncT4_2; the activity of AncT4_2 toward 17 of the modified RNA fragments was higher than that of T4Rnl2. The activity was estimated by measuring the conjugation reaction of two RNA strands, 3'-OH (12 bp) and 5'-PO₄ (12 bp), in which the terminal and penultimate positions of the 3'-OH fragment and the first and second positions of the 5'-PO₄ fragment were substituted by 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-H, respectively. The enzymatic properties of AncT4_2 allowed the enzyme to conjugate large quantities of double-stranded RNA coding for patisiran (>400 μM level), which was formed by four RNA fragments containing 2'-OMe-substituted nucleic acids. Structural analysis of modeled AncT4_2 suggested that protein dynamics were changed by mutation to Gly or indel during ASR and that this may positively impact the conjugation of modified RNA fragments with the enzyme. AncT4_2 is expected to be a key biocatalyst in synthesizing RNA therapeutics by an enzymatic reaction. IMPORTANCE RNA therapeutics is one of the next-generation medicines for treating various diseases. Our designed ancestral RNA ligase AncT4_2 exhibited excellent enzymatic properties, such as high thermal stability, productivity, specific activity, and broad substrate selectivity compared to native enzymes. These advantages create the potential for AncT4_2 to be applied in conjugating the modified RNA fragments containing various xenobiotic nucleic acids. In addition, patisiran, a known polyneuropathy therapeutic, could be synthesized from four fragmented oligonucleotides at a preparative scale. Taken together, these findings indicate AncT4_2 could open the door to synthesizing RNA therapeutics by enzymatic reaction at large-scale production.

Keywords: RNA ligase; RNA therapeutics; ancestral sequence reconstruction; xenobiotic nucleic acids.

FIG 1

Schematic view indicating how to design FcT4_2 and AncT4_2 from T4Rnl2 homolog sequences (A). Phylogenetic tree analysis of T4Rnl1, T4Rnl2, and AncT4_2 and their homolog sequences (B). The homolog sequences are represented in Table S2. The MSA was performed by MAFFT (51), and phylogenetic tree data were obtained by MEGA7 (52). The tree was depicted by iTOL software (54).

FIG 2

(A) Expression test of T4Rnl2 and AncT4_2 by SDS-PAGE. Lanes M, S, I, and P represent the protein marker, soluble fraction, insoluble fraction, and purified fraction after affinity chromatography. (B) UV spectra of T4Rnl2 and AncT4_2. Abs, absorbance. (C) Enzyme kinetic plots of T4Rnl2 and AncT4_2. The data were fitted to a substrate uncompetitive inhibition model. The fitted line is solid. Error bars represent standard deviation (SD). (D) Thermal stability analysis of T4Rnl2 and AncT4_2. The data for T4Rnl2 and AncT4_2 are plotted as gray and solid black circles, respectively. Error bars denote 1 SD. (E) Conjugation reaction of double-strand RNA (dsRNA) containing nicks in the sense and antisense strands. The dsRNA, represented as a schematic image, was conjugated by T4Rnl2 and AncT4_2. Here, the time-dependent concentration changes of the produced sense (left) and antisense strand (right) are plotted.

FIG 3

(A) Oligonucleotide substrates to estimate substrate selectivity of T4Rnl2 and AncT4_2. Two oligonucleotides (3′-OH [blue] and 5′-PO₄ [orange] fragments) formed by 12 bp and cRNA were utilized as the substrates; with XNAs, the −2 and −1 positions of the 3′-OH fragment and +1 and +2 positions of the 5′-PO4 fragment were substituted with 2′-OH (control), 2′-F, 2′-OMe, 2′-MOE, and 2′-H, respectively. (B) Relative activity of T4Rnl2 and AncT4_2 toward oligonucleotides containing XNAs. The specific activity of AncT4_2 toward RNA (control condition) is normalized as 100%. Error bars denote 1 SD. (C) Overall model structure of the AncT4_2 RNA binding form. The 22 mutated residues are represented as red spheres.