We next investigated the tolerance of the bTG enzyme towards amino substrates by screening various types of amine reagents. We first confirmed that the bTG was not able to accept secondary amines as substrates as no product formation could be observed with a panel of secondary amines (data not shown). We therefore studied the effect of the length of the linker and performed conjugation reactions between CS_BSA2 sequence and dansyl-based molecules containing a 4-carbon linker (molecule A, Figure and Figure ), a 3-carbon linker (molecule B, Figure and Figure ) or a 2-carbon linker (molecule C, Figure and Figure ). A decline in conversion was detected as the length of the linker was decreasing (100% for A, 81% for B and 31% for C, Tables and). As the natural substrate of bTG is the amino group of a lysine, we also tested a lysine molecule (D, Figure and Figure ) and a lysine analogue (E, Figure and Figure ) for conjugation and 100% conversion was observed for both molecules (Tables and). We then investigated smaller water-soluble molecules with a 1carbon linker (F and G, Figures S66, S67 and Figure ) which led to 77% and 100% conversion respectively. Lastly, we also wondered if the bTG would be able to tolerate more bulky and complex molecules. Molecule H (Figure and Figure ) which has been reported as a BRD inhibitor, gave full conversion (Tables and). 91% conversion was obtained for molecule I (Figures and, Tables and) which acts as a kinase inhibitor. Another important class of substrate are PEG chains, as they are commonly employed as linkers to attach a biologically relevant drug to an antibody. The presence of the PEG linker enables a decrease in immunogenicity, a better solubility, and a slower clearance of the antibody drug conjugate from the body. In this regard, a PEG amine (molecule J, Figure and Figure ) was tested and was fully tolerated by the bTG enzyme as 100% conversion was observed (Tables and). Lastly, siRNA-based therapeutics have emerged as a promising drug modality as these synthetic, double stranded RNA molecules are able to degrade a target mRNA in a sequence specific manner. Despite their unlimited potential, their application in clinical settings is still restricted, due to the absence of effective delivery systems. Even though various conjugations of siRNA have already been reported, the current methods either lack specificity, with drug to protein ratios difficult to control, or are based on a specific type of chemistry, which requires careful design and preparation of the siRNA and protein moieties. To the best of our knowledge, no direct enzymatic conjugation of siRNA to protein scaffolds has been reported to date. In our experiments, we tested molecule K, a siRNA that regulates the expression of AHSA1, a chaperone of heat shock 90 kDa (HSP90), which enhances the ATPase activity of the latter. AHSA1 plays a crucial role in cellular processes related to protein folding and stability and has been found to be overexpressed in numerous cancer cells. This siRNA is composed of an antisense of 21 nucleotides and a sense strand (19-mer) containing a hexylamino tri acetamide amine at its 3'-end (Figure ). We decided to test the bioconjugation with the two most promising sequences CS_4 and CS_5. We first performed conjugations tests (data not shown) and the best performing conditions were found with 3 equivalents of siRNA. The conversion was difficult to follow by LCMS as the peaks corresponding to the bTG, siRNA and parent monobody coeluted. We decided to proceed to the purification of the two reaction mixtures and CS_4_K conjugate was isolated with 56% yield and 93% purity and conjugated product CS_5_K was obtained with 76% yield and 99% purity (Figures and, Tables and). To confirm that the covalent coupling between the monobodies and the siRNA will not alter their gene silencing potency, lipofection of the siRNA conjugate was performed in HEPA1-6 cells (Figure ). The results indicate a silencing efficiency of 87% for both CS_4_K and CS_5_K compounds at 100 nM, which relates similarly to the positive control (siRNA only), showing that the siRNA properties were not affected by the bTG bioconjugation. This primary amine screening therefore confirms the broad scope of the bTG enzyme towards primary amino substrates. These results suggest that a variety of payloads can be attached by the bTG, including dyes, drugs, siRNA, and cell penetrating peptides that could further be used as carriers to deliver the monobody into cells. 35