Rational Design of Aptamer-Tagged tRNAs
Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using <i>Escherichia coli</i> reporter assay systems. As the tRNA chassis for engineering...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/20/7793 |
| _version_ | 1797550211354066944 |
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| author | Takahito Mukai |
| author_facet | Takahito Mukai |
| author_sort | Takahito Mukai |
| collection | DOAJ |
| description | Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using <i>Escherichia coli</i> reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in <i>E. coli</i>. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code. |
| first_indexed | 2024-03-10T15:26:18Z |
| format | Article |
| id | doaj.art-23e5a44fb7274ca29fb027bc5bdaece6 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T15:26:18Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-23e5a44fb7274ca29fb027bc5bdaece62023-11-20T17:58:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-10-012120779310.3390/ijms21207793Rational Design of Aptamer-Tagged tRNAsTakahito Mukai0Department of Life Science, College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, JapanReprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using <i>Escherichia coli</i> reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in <i>E. coli</i>. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.https://www.mdpi.com/1422-0067/21/20/7793genetic codeanticodonpseudo-anticodonamber suppression |
| spellingShingle | Takahito Mukai Rational Design of Aptamer-Tagged tRNAs International Journal of Molecular Sciences genetic code anticodon pseudo-anticodon amber suppression |
| title | Rational Design of Aptamer-Tagged tRNAs |
| title_full | Rational Design of Aptamer-Tagged tRNAs |
| title_fullStr | Rational Design of Aptamer-Tagged tRNAs |
| title_full_unstemmed | Rational Design of Aptamer-Tagged tRNAs |
| title_short | Rational Design of Aptamer-Tagged tRNAs |
| title_sort | rational design of aptamer tagged trnas |
| topic | genetic code anticodon pseudo-anticodon amber suppression |
| url | https://www.mdpi.com/1422-0067/21/20/7793 |
| work_keys_str_mv | AT takahitomukai rationaldesignofaptamertaggedtrnas |