TY - JOUR
T1 - Combinatorial design of a nanobody that specifically targets structured RNAs
AU - Cawez, F.
AU - Duray, E.
AU - Hu, Yaozhong
AU - Vandenameele, J.
AU - Estevens Romão, Ema
AU - Vincke, Cecile
AU - Dumoulin, M
AU - Galleni, Moreno
AU - Muyldermans, Serge
AU - Vandevenne, Marylene
PY - 2018/5/25
Y1 - 2018/5/25
N2 - Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.
AB - Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.
KW - RNA/protein interactions
KW - antibody fragment engineering
KW - nanobody
KW - non-coding RNAs
KW - structured RNAs
UR - http://www.scopus.com/inward/record.url?scp=85046151520&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2018.03.032
DO - 10.1016/j.jmb.2018.03.032
M3 - Article
VL - 430
SP - 1652
EP - 1670
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 11
ER -