TY - JOUR
T1 - Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions
AU - Katzman, Bella
AU - Vyazmensky, Maria
AU - Press, Olga
AU - Volokita, Micha
AU - Engel, Stanislav
N1 - Publisher Copyright:
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2015/3/1
Y1 - 2015/3/1
N2 - In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches. In vitro selection methodologies for target-interacting compounds generally rely on affinity separation, which limits the overall efficiency of the selection process. To overcome common drawbacks associated with the conventional selection methods, the authors develop a method for detecting molecular interaction events - tethered ribozyme ligation (TRL), with which the entire selection process takes place in a homogeneous solution. TRL can be used for both selection of target-interacting compounds from random libraries and high-throughput screening for inhibitors of specific intermolecular interactions.
AB - In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches. In vitro selection methodologies for target-interacting compounds generally rely on affinity separation, which limits the overall efficiency of the selection process. To overcome common drawbacks associated with the conventional selection methods, the authors develop a method for detecting molecular interaction events - tethered ribozyme ligation (TRL), with which the entire selection process takes place in a homogeneous solution. TRL can be used for both selection of target-interacting compounds from random libraries and high-throughput screening for inhibitors of specific intermolecular interactions.
KW - Class I RNA ligase
KW - DNA-encoded compounds
KW - Display selection
KW - MDM2
KW - Streptavidin
UR - http://www.scopus.com/inward/record.url?scp=84924602787&partnerID=8YFLogxK
U2 - 10.1002/biot.201400551
DO - 10.1002/biot.201400551
M3 - Article
AN - SCOPUS:84924602787
SN - 1860-6768
VL - 10
SP - 379
EP - 385
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 3
ER -