Limits...
Non-canonical 3'-5' extension of RNA with prebiotically plausible ribonucleoside 2',3'-cyclic phosphates.

Mutschler H, Holliger P - J. Am. Chem. Soc. (2014)

Bottom Line: We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C.We also observed 5' addition of 2',3'-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers.Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

ABSTRACT
Ribonucleoside 2',3'-cyclic phosphates (N>p's) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p's can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3'-5' nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C. We also observed 5' addition of 2',3'-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

Show MeSH
Concentration-dependent 5′ transfer of A>p,G>p, C>p, orU>p (−7 °C in ice, 72 h, 10 mM MgCl2).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4333585&req=5

fig2: Concentration-dependent 5′ transfer of A>p,G>p, C>p, orU>p (−7 °C in ice, 72 h, 10 mM MgCl2).

Mentions: Next we investigated whether 5NTz could catalyze nucleotide transferof the three other canonical N>p’s. Indeed, we observedribozyme-dependentaddition of G>p, U>p, and C>p (Figure 2). Strikingly,5′ extension was already visible at an apparent N>p concentrationas low as 10 μM for purine and 100 μM for pyrimidine nucleotides.The preference of 5NTz for purines may be due to their stronger stackinginteractions with G8 (Figure 1b). A furtherincrease in 5′-nucleotidyl transfer efficiency at high millimolarnucleotide concentrations was impeded by substrate inhibition, whichwas observed for all N>p’s except C>p. Inhibition wasmostsevere for G>p (Figure 2), possibly duetocompetition of exogenous G>p with the internal G-1 base for pairingto C25 (Figure 1a), an interaction requiredfor loop–loop docking during active-site assembly.7


Non-canonical 3'-5' extension of RNA with prebiotically plausible ribonucleoside 2',3'-cyclic phosphates.

Mutschler H, Holliger P - J. Am. Chem. Soc. (2014)

Concentration-dependent 5′ transfer of A>p,G>p, C>p, orU>p (−7 °C in ice, 72 h, 10 mM MgCl2).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4333585&req=5

fig2: Concentration-dependent 5′ transfer of A>p,G>p, C>p, orU>p (−7 °C in ice, 72 h, 10 mM MgCl2).
Mentions: Next we investigated whether 5NTz could catalyze nucleotide transferof the three other canonical N>p’s. Indeed, we observedribozyme-dependentaddition of G>p, U>p, and C>p (Figure 2). Strikingly,5′ extension was already visible at an apparent N>p concentrationas low as 10 μM for purine and 100 μM for pyrimidine nucleotides.The preference of 5NTz for purines may be due to their stronger stackinginteractions with G8 (Figure 1b). A furtherincrease in 5′-nucleotidyl transfer efficiency at high millimolarnucleotide concentrations was impeded by substrate inhibition, whichwas observed for all N>p’s except C>p. Inhibition wasmostsevere for G>p (Figure 2), possibly duetocompetition of exogenous G>p with the internal G-1 base for pairingto C25 (Figure 1a), an interaction requiredfor loop–loop docking during active-site assembly.7

Bottom Line: We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C.We also observed 5' addition of 2',3'-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers.Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

ABSTRACT
Ribonucleoside 2',3'-cyclic phosphates (N>p's) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p's can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3'-5' nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C. We also observed 5' addition of 2',3'-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

Show MeSH