Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Serine-proline (Ser-Pro) dipeptide analogues are crucial for studying cis-trans isomerization in cell cycle and transcription.
• Previous methods were limited to synthetic peptides, posing challenges for larger molecules due to yield loss.
• Incorporating non-native dipeptide mimics into proteins was previously difficult.

Purpose of the Study:

• To develop a method for tRNA aminoacylation with dipeptides and their analogues.
• To enable the incorporation of cis- and trans-locked Ser-Pro analogues into full-length proteins.
• To overcome synthetic limitations and expand the study of Ser-Pro isomerization in vivo.

Main Methods:

• Synthesis of 3,5-dinitrobenzyl (DNB)-activated esters of Ser-Pro dipeptide and its alkene analogues.
• Utilized the DNB flexizyme (dFx) ribozyme for tRNA acylation.
• Generated truncated amber tRNA via in vitro transcription.
• Catalyzed chemical misacylation of tRNA with Ser-Pro-DNB activated dipeptide.

Main Results:

• Successfully synthesized DNB-activated esters of Ser-Pro dipeptide and its cis/trans-locked alkene analogues.
• Demonstrated the ability of dFx flexizyme to acylate truncated amber tRNA with the Ser-Pro-DNB dipeptide.
• Established a method for introducing non-native Ser-Pro dipeptide mimics into full-length proteins via in vitro transcription-translation.

Conclusions:

• The developed method allows for the efficient incorporation of Ser-Pro dipeptide mimics into proteins.
• This technique facilitates the investigation of cis-trans isomerization's role in biological regulation.
• Overcomes previous synthetic challenges, opening new avenues for protein engineering and functional studies.