TY - JOUR
T1 - Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes
AU - Katz, Noa
AU - Tripto, Eitamar
AU - Granik, Naor
AU - Goldberg, Sarah
AU - Atar, Orna
AU - Yakhini, Zohar
AU - Orenstein, Yaron
AU - Amit, Roee
N1 - Publisher Copyright: © 2021, The Author(s).
PY - 2021/3/11
Y1 - 2021/3/11
N2 - We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.
AB - We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.
UR - http://www.scopus.com/inward/record.url?scp=85102422584&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s41467-021-21578-6
DO - https://doi.org/10.1038/s41467-021-21578-6
M3 - مقالة
C2 - 33707432
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 1576
ER -