Supporting Information

Transcription

1 Supporting Information Supplementary Figure-1 Results of Phage Display Screening Against TNT The sequences seen in Supplementary Figure-1 represent the phages sequenced after the third and fourth rounds of phage display screening against a TNT substrate under a binding condition of ph 7.5. The sequence ID represents Substrate - Binding Conditions - Round/Experiment Phage Sample. The abundance listed on the right reflects the number of times a given receptor sequence appeared from the sequencing results. sequencing. After each round, approximately 20 phage samples were submitted for 1

2 Supplementary Figure-2 Results of Phage Display Screening Against DNT The sequences seen in Supplementary Figure-2 represent the phages sequenced after the third, fourth, and fifth rounds of phage display screening against a DNT substrate under a binding condition of ph 7.5. The sequence ID represents Substrate - Binding Conditions - Round/Experiment Phage Sample. The abundance listed on the right reflects the number of times a given receptor sequence appeared from the sequencing results. After each round, approximately 20 phage samples were submitted for sequencing. 2

3 Supplementary Figure 3 Competitive screening against a TNT substrate The 33 sequences in Supplementary Figure-3 represent the piii receptors expressed on the M13 bacteriophage which were amplified and mixed to equal concentrations of 10 6 pfu/ul of each phage for competitive screening. After a single round of phage screening with 0.2% TBST, the remaining bound phage were eluted, captured, and titrated onto LB Xgal/IPTG agar plates. From the blue plaques which appeared after overnight incubation at 37 C, twenty of these were picked for sequence analysis. The last column of Supplementary Figure-3 represents the number of appearances of a given receptor amino acid sequence from the DNA sequencing results. The most abundant of these receptor sequences indicates the highest level of binding. Importantly, 19 of the 20 phages sequenced possessed a Trp-His-Trp motif at the N terminus. 3

4 Supplementary Figure 4 Selectivity screening of phages identified against TNT The phages bearing receptors identified in Supplementary Figure-4a were amplified to a concentration of 10 6 pfu/ul. This number of phages was identified as the Input and was verified via UV spectrometer as well as titration. 1mL of the phage samples were then individually screened against 5mg of TNT, washed, and the remaining bound phage were eluted. This elution, known as the Output number of phages, was quantified using phage titration. The ratio of Output / Input was calculated and the values can be seen in Supplementary Figure-4b. Similarly, phage samples indicated * were also 4

5 screened against 5mg of DNT and the corresponding ratio of Output / Input can be seen in Supplementary Figure-4b. The results reflect the preferential binding of a given phage for the TNT substrate and hence the selectivity of the receptor sequence. 5

6 Supplementary Figure 5 Selectivity screening of phages identified against DNT The phages bearing receptors identified in Supplementary Figure-5a were amplified to a concentration of 10 6 pfu/ul. This number of phages was identified as the Input and was verified via a UV spectrometer and also titration of phage. 1mL of the phage samples were then individually screened against 5mg of DNT, washed, and the remaining bound phage were eluted. This elution, known as the Output number of phages, was quantified using phage titration. The ratio of Output / Input was calculated and the values can be seen in Supplementary Figure-5b. Similarly, phage samples indicated * were also screened against 5mg of TNT and the corresponding ratio of Output / Input can be seen in Supplementary Figure-5b. The results reflect the preferential binding of DNT_ph7.5-Va-11 for the DNT substrate and hence the selectivity of the receptor sequence His-Pro-Asn-Phe-Ser-Lys-Tyr-Ile-Leu-His- Gln-Arg. 6

7 Supplementary Figure-6 Comparison to TNB binding peptide The sequence identified as the best TNT binder was designated TNT-BP and possessed the sequence Trp-His-Trp-Gln-Arg-Pro-Leu-Met-Pro-Val-Ser-Ile. Scram-Ctrl represents the same amino acids as TNT-BP but in a different configuration. TNB-Ctrl represents the positive control as it is known to bind TNT in liquid. These sequences were synthesized with C terminal biotinylated lysine followed by a tri- Glycine linker to allow functionalization with a fluorescent probe Atto-425 for the comparative analysis of the loan peptide receptors (no longer attached to the phage body). Here, the sequences were exposed to TNT coated polystyrene wells, fluorescently labeled, washed, captured, and measured of their fluorescence relative to the BSA background signal. Supplementary Figure-6 represents the level of binding exhibited by the given sequence for the TNT substrate. The sequence TNT-BP exhibited much greater binding to the sequence Scram-Ctrl which possessed the same amino acids in a different configuration. This result demonstrates the importance of the configuration of the amino acids in the binding event, thereby identifying that it is not solely the apparent charge or the functional groups present on the receptor but instead their particular arrangement. TNT-BP exhibit higher level of TNT 7

8 binding as compared to the positive control, TNB-BP. (P < , n = 4). All data presented as mean ± standard deviation. 8