A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution

Abstract

We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing

“…Next, various salt mix concentrations were tested during post translation incubation. Previously, a high concentration of KCl has been shown to improve the accessibility of ribosomebound peptidyl-tRNA to puromycin (Van Der Mast & Bloemers, 1973) and has been adopted in the recent mRNA and cDNA display methods (Naimuddin & Kubo, 2016;Seelig, 2011;Yamaguchi et al, 2009). We found that without salt addition, conjugate formation is less than 10%, however the addition of 32.5 mM MgCl 2 and 375 mM KCl greatly increased the formation rate to above 40% ( Figure 2B).…”

“…For the translation time, we tested 0, 5, 15, 30 and 60 minutes at 37 , prior to the addition of salt mix (final conc, 32.5 mM MgCl 2 and 375 mM KCl) followed by 60 minutes of incubation. For the optimization of salt mix concentration, we first conducted 30 minutes translation at 37 , then either added no salt or 1x, 1/2x, 1/4x, 1/8x concentration of 65 mM MgCl 2 and 750 mM KCl concentration originally reported to promote covalent bond between puromycin and polypeptide chain (Naimuddin et al, 2011;Naimuddin & Kubo, 2016). For the incubation time after salt addition, we initially performed 30 minutes translation, added salt to final 32.5 mM MgCl 2 , 375 mM KCl concentration, then tested 0, 10, 30, 60 and 90 minutes of incubation time.…”

“…The formation rate of the mRNApeptide conjugate also depends on the incubation conditions during and after the translation to promote efficient incorporation of puromycin to the stalled ribosome at the mRNA-DNA boundary. Previous studies have applied rare codons at the 3' end of mRNA coding region (Nagumo et al, 2016) or used long linker in the DNA tag (Naimuddin & Kubo, 2016) to improve the conjugate formation. In this study, we have adopted both of these features by placing rare GGA codon at the 3' end and included polyA (18 nt) in the puromycin-FITC DNA tag.…”

“…(A) Difference in length of translation before salt addition. Number indicates different incubation time (in minutes) carried out at 37 degrees C. (B) Difference in salt concentrations with original concentration (1x) as 750 mM KCl, 65 mM MgCl 2 used in the previous studies (Naimuddin et al, 2011;Naimuddin & Kubo, 2016) (C) Difference in lengths of incubation time (in minutes) after salt addition. (D) Trypsin digestion of mRNA-tag and mRNA-peptide conjugate products.…”

“…These advantages of recombinant systems are particularly beneficial when one needs a control over conditions for high-throughput screening and directed evolution of peptide/proteins (Contreras-Llano & Tan, 2018;Dodevski, Markou, & Sarkar, 2015;Fujii et al, 2014). Different display methods (phage display (Ledsgaard, Kilstrup, Karatt-Vellatt, McCafferty, & Laustsen, 2018), yeast display (Boder & Wittrup, 1997;Cherf & Cochran, 2015), ribosome display (Zahnd, Amstutz, & Plückthun, 2007), liposome display (Fujii, Matsuura, Sunami, Kazuta, & Yomo, 2013), DNA display (Bertschinger & Neri, 2004;Doi & Yanagawa, 1999;Yonezawa, Doi, Kawahashi, Higashinakagawa, & Yanagawa, 2003), cDNA display (Naimuddin & Kubo, 2016;Yamaguchi et al, 2009), mRNA display (Nemoto, Miyamoto-Sato, Husimi, & Yanagawa, 1997;Roberts & Szostak, 1997;Seelig, 2011)) use various strategies to couple genotype to phenotype and as such have become indispensable tools for directed evolution. Among the display methods, in vitro approaches such as mRNA, cDNA, and ribosome display can screen the highest number of molecules (up to 10 13 ) to be tested because they are not limited by the efficiency of transformation or transfection.…”