TABLE 1.

Advantages and disadvantages of different tRNA production methods.

Approach	Method	Advantages	Disadvantages	References
Purification in vivo	Hydrophobic tagging	Modified nucleotides, low cost, simple operation	Multiple complicated procedures, cannot distinguish isoacceptor tRNAs	Kothe et al. (2006)
	DNA probe-elution	Cost-effective, modified nucleotides, simple operation	Low yield, low purity, low binding efficiency	Kaneko et al. (2003)
	DNA probe-digestion	High purity, high specificity, well-established method	Extensive downstream purification, using toxic reagents	Nilsen (2013)
In vitro biosynthesis	Direct	Easy purification, fast, the mutagenesis of the tRNA is easy	Addition of nucleotides at the 3′end, the first base must be a guanine, without modified nucleotides	Li et al. (1999)
	Hammerhead	Fully active in aminoacylation; Not limited to the first base	No 5′phosphorylation, without modified nucleotides, addition of nucleotides at the 3′ end	Fechter et al. (1998)
	RNase P	Not limited to the first base; no addition of nucleotides at the 3′ end	Without modified nucleotides	Hibi et al. (2020)
Chemical synthesis	Solid-phase chemical synthesis	Modifications possible, easy purification, fast, no sequence-specific optimization	Expensive equipment required, length limited, limited availability of labeled or modified phosphoramidites	Ogilvie et al. (1988)