PNA-Induced Assembly of Fluorescent Proteins Using DNA as a Framework

Abstract

Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein-PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)-peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA's assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…For example, a predetermined number of a protein (monomeric teal fluorescent protein; mTFP) was successfully assembled and the energy-transfer processes in the assemblies were investigated in detail. 230 A PNA tag (6 nucleobases) was bound to the mTFP protein as follows. A cysteine residue was first attached to the N-terminus of PNA, and reacted with a thioester which was introduced to the Cterminus of mTFP.…”

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

“…Additionally, expressed protein ligation (EPL) can be performed between the C‐terminal thioesters of a protein and the N‐terminal cysteine of a PNA, resulting in a native peptide bond . Gholami et al.…”

Controlled Assembly of SNAP–PNA–Fluorophore Systems on DNA Templates To Produce Fluorescence Resonance Energy Transfer

“…1 Furthermore, self-assembled DNA-protein conjugates can be used as template controlled systems for spatially arraying other molecules with increased relative accuracy and programmability. [1][2][3][4][5] The programmable hybridization of nucleic acid provides a framework to design nanoscale assemblies. [6][7][8][9][10][11][12][13][14][15] Peptide nucleic acid (PNA) provides excellent control properties over a self-assembled system including better stability than DNA duplexes, even for short sequences, and higher mismatch sensitivity.…”

“…5 Moreover, it allowed fundamental insights into the behaviour of proteins in self-assembled architectures by observing the photophysical behaviour of assembled FPs. 57 Here, the combination of the unique recognition characteristics of PNA with the wide application of SNAP tag has been used to provide versatile, well-defined units to create programmed self-assembled protein systems.…”