PaaA and TbtF Binding

For binding assays, we had the 6xHis tags attached to PaaA (C-term) and TbtF (N-term with MBP) and not attached to the peptide substrate. Initially, we incubated the displayed peptide substrate with its corresponding RiPPs enzyme. After this “pre-binding” step, we incubated this peptide-bound-enzyme with Ni-NTA resin. We removed the flow-through and washed the resin once with buffer. To elute, we heat denatured at 95 oC in 1x reverse transcription (RT) buffer. This is the positive, selected peptides, meaning the mutations allowed binding to the enzymes. After elution, we used Superscript III to reverse transcribe the mRNA followed by PCR to create more of the selected DNA template (Figure 4.6.b).

130

By testing with radio-labeled, WT, PaaP and TbtA, binding to their respective RiPPs enzymes (PaaA and TbtF) can clearly be seen (Figure 4.5b and c). In these radio-labeled gels, little to no displayed peptide can be seen in the flow-through and wash lanes, but the displayed peptide can clearly be seen coming off in the elution. These gels show that displayed peptide can bind to its corresponding RiPPs enzymes. These results open the door to testing binding affinity and leader peptide requirements for interacting and binding to their corresponding RRE present in these enzymes. It has been shown by Roberts and co-workersthat mRNA display can be used to determine binding kinetics which can be used and adapted for determining binding constants for leader peptides to RRE’s.23

Figure 4.5. Selection for binding to RiPPs enzymes. a) Pictorial representation of selection for binding to RiPPs enzymes. b) Radio-labeled (35S), 8% SDS-PAGE gel showing display-peptide (TbtA) binding and eluting with TbtF. c) b) Radio-labeled (35S), 8% SDS-PAGE gel showing display-peptide (PaaP) binding and eluting with PaaA.

131

4.5 Summary and Discussion

The next step after the reverse transcription and PCR would be to sequence the positive selected sequences that correspond to either modification or binding depending on the selection procedure (Figure 4.6). Traditionally, the DNA sequences would be cloned into a blunt or TA- TOPO vector and the insert sequenced normally by Sanger methods.15,19-22 This approach does not give a full depiction of the library until many round of selection and a great number of colonies sequenced. Also, when we tried this approach, we received inconsistent data, namely partial reads and low quality data. There is another approach using next-generation sequencing (high-throughput sequencing) to sequence the DNA that was amplified after selection, called amplicons. Next-generation sequencing (NGS) has many advantages over traditional cloning and sequencing.57 First, there is no cloning, so poor cloning, ligation, and transformation efficiency are not a problem. Second, the PCR product off the reverse transcription reaction is what is sent for sequencing, so there are no more necessary steps required by the scientist carrying out the selection. Lastly, instead of getting one read of a sequence like in traditional Sanger sequencing, NGS gives millions of reads over the amplicons, allowing full coverage and quantitation, by percentage, of what is present in the amplicon. Exact sequences, single- nucleotide polymorphisms (SNPs), and mutations can all be detected at the same time through NGS. More recently, NGS was used with mRNA display in a high-throughput manner to measure binding kinetics between a library of peptide and Bcl-xL protein.23 This was accomplished by adding on unique identifying barcodes and utilizing a Illumina58 HiSeq 2500 platform to sequence the library. This approach gave both the frequency and factional composition of their library, statistics valuable for knowing the complexity and make-up of your selected library. We have planned on doing a similar NGS approach using the Illumina

132

technology and platform; however at the time of this writing, this has yet to be done. We are actively pursuing NGS through GENEWIZ to perform NGS on the amplicons after selection, both binding and activity (Figure 4.6b and c).

Figure 4.6. Reverse transcription and amplification of selected library members. a) Pictorial representation of workflow. b) Amplicons of selected members from PaaA and TbtF binding assays. c) Amplicon of selection from PaaA activity assay. The sizes of the corresponding amplicons are: PaaP: 225 bp, His-PaaP: 258 bp, and TbtA: 282 bp.

In the presented work, we have described a mRNA system for probing the promiscuity of the biosynthesis of a natural product class, RiPPs. We show that it is possible for displayed RiPPs to bind to their respective enzymes while attached to their genomic component, mRNA. We also show that it is indeed possible to impart RiPPs modifications onto a displayed RiPP precursor peptide and confirm that modification. We also outline the procedures necessary to probe both the biosynthetic activity as well as binding affinity of RiPPs to their enzymes by mRNA display. Sequencing, specifically NGS, is what remains to analyze the data collected after selection. With this system, we believe it is possible to fully probe the biosynthetic promiscuity of a RiPP enzyme as well as to determine the leader peptide requirements necessary for binding to the RRE of the enzyme. This can give valuable information on what residues are necessary and can be used to create chimeric leader peptides to combine separate and distinct RiPPs pathways into a single precursor peptide.

133

4.6 Experimental