Small Unnatural Amino Acid Carried Raman Tag For Molecular Imaging Of Genetically Targeted Proteins

The emerging Raman probes are superior to fluorescent probes in various aspects,such as less molecular weight?<1 kDa?,narrower vibrational bandwidth?¹ nm?,more stable Raman signal intensity,and strict linear relationship between the signal and the concentration of bio-molecules for quantitative imaging.However,the Raman probe or Raman spectrum is lack of protein specificity,that is,the inability to genetically label and identify specific proteins.Therefore,for a long time,the development of Raman labeling and Raman microscopy has been only applicable to image metabolites or organelles in the biological system,and unable to distinguish specific proteins.These factors greatly limit the development of Raman spectroscopy and Raman microscopy.Meanwhile,Raman signal are much weaker than fluorescence,which seriously impedes their wide application in biological research.In this thesis,we developed a method for genetically targeted protein imaging,which combined novel Raman probe,genetic codon expansion technology,and hyperspectral stimulated Raman?HSRS?microscopy.We report the smallest?total⁰.55 kDa?and potentially the most innocuous label for genetically targeted protein imaging,which incorporates a phenyl ring enhanced Raman tag with single unnatural amino acid?UAA?to target specific proteins.We further demonstrate hyperspectral stimulated Raman scattering?SRS?imaging of Histone3.3 protein in the nucleus,Sec61?protein in the endoplasmic reticulum of HeLa cells,and Huntingtin protein Htt74Q in mutant huntingtin-induced cells.Genetic encoding of small,stable,sensitive and narrow-band Raman tag took one key step forward to enable SRS or Raman imaging of specific proteins,and could further facilitate quantitative Raman spectra based super-multiplexing microscopy in future.Specifically,we employed the genetic codon expansion technique,the orthogonal Methanosarcina barkeri PylRS?MbPylRS?/tRNA_CUA,to identify a specific amber codon?TAG?on messenger RNA?mRNA?.An unnatural amino acid?⁰.23 kDa?carried an alkyne?C?C?would respond to amber coden and inserted into the target protein.To gain the Raman signal,we then chemically clicked UAA with a phenyl ring enhanced Raman tag.The total molecular weight of UAA carried Raman label is only about 0.55 kDa.Meanwhile the Raman signal is improved more than two orders of magnitude in contrast to the single alkyne,and the Raman spectral width is only 18 cm^-1.In the validation experiment,we expressed the exogenous fusion protein Histone3.3-EGFP,the endoplasmic reticulum?ER?protein EGFP-Sec61?and the pathogenic protein Htt74Q-EGFP of huntingtin in HeLa cells,and performed SRS imaging respectively.To completely remove the influence of EGFP,we directly incorporated the UAA into the target protein Histone3.3 or Htt74Q through the genetic codon expansion technique,then connected the phenyl ring enhanced Raman tag with the UAA by Clicking-iT.We successfully performed the HSRS imaging of those genetically targeted proteins.Finally,we modified the aminoacyl tRNA synthase-BCNRS,that specifically identified BCNK?an UAA used for rapid Diels-Alder reactions in living cells?to achieve SRS imaging of specific proteins in living cells.To image specific protein with advantageous Raman probe,we incorporated enhanced Raman probe into the protein of interest via the genetic codon expansion technology,which pave the way for multicolor Raman imaging.This UAA carried Raman probe has many advantages,such as protein specificity,less molecular weight,strong signal,narrow Raman spectrum and excellent optical stability,and promises to replace the traditional fluorescent proteins in future.