Targeting domain and related signal activated molecular delivery

Abstract

Provided herein are signal activatable molecular constructs for enzyme-assisted delivery of molecules and related components, such as a sensor domain, compositions, methods and systems.

1 . A targeting domain comprising
 a targeting domain duplex RNA of about 19 to about 30 bp length, the targeting domain duplex RNA comprising a guide strand complementary bound to a passenger strand,   wherein
 the passenger strand is nicked in two passenger strand segments each about 2 to about 17 bp long and allowing the targeting domain duplex RNA to adopt a folded conformation and an unfolded conformation, 
 in the folded conformation, opposite ends of the targeting domain duplex RNA are in a configuration that minimizes processing of the guide strand by dicer and/or an argonaute enzyme, and 
 in the unfolded conformation, the opposite ends of the targeting domain duplex RNA are in a configuration allowing processing of the guide strand by dicer and/or an argonaute enzyme. 
   
     
     
         2 . The targeting domain of  claim 1 , wherein the targeting domain duplex RNA is a small interfering RNA (siRNA), a dicer substrate small interfering RNA (DsiRNA), or a synthetic miRNA analogues (miRNA). 
     
     
         3 . The targeting domain of  claim 1 , wherein the targeting domain has a length of about 19 to about 22 bp or of about 25 to about 30 bp. 
     
     
         4 . The targeting domain of  claim 1 , wherein the targeting domain 1s locked m the folded conformation by a suitable linkage. 
     
     
         5 . The targeting domain of  claim 1 , wherein the targeting domain duplex RNA comprise at least about 5% 2′-O-methyl modifications or one or two mismatches. 
     
     
         6 . The targeting domain of  claim 1 , wherein the guide strand and/or the passenger strand comprise one or more modified ribonucleotides and/or a phosphorothioate segment. 
     
     
         7 . The targeting domain of  claim 6 , wherein the one or more modified ribonucleotides comprise 2′-O-methyl ribonucleotide, 2′-fluoro ribonucleotide, 2′-amino ribonucleotide and/or LNA residues. 
     
     
         8 . The targeting domain of  claim 6 , wherein the one or more modified ribonucleotides are located at a 5′ terminus of the passenger strand and the modified ribonucleotides are configured to minimize processing by nucleases. 
     
     
         9 . A method to provide a molecular complex for enzyme-assisted molecular delivery, the method comprising
 contacting the targeting domain of  claim 1  with a locking sensor,   the locking sensor comprising   a locking sensor RNA duplex having a toehold segment, a displacement segment and an activation segment,   the displacement segment presenting a first strand; the activation segment presenting a second strand; the displacement segment complementarily binding the activation segment; and the toehold segment being presented for binding to signal molecule;   wherein:   the locking sensor RNA duplex is configured to attach the opposite ends of the targeting domain of  claim 1  in a folded conformation, through covalent linkage of the first strand with a first end of the opposite ends of the targeting domain and through covalent linkage of the second strand with a second end of the opposite ends of the targeting domain; and the displacement segment, activation segment and toehold segment are configured to allow release of the targeting domain from the folded conformation upon binding of a signal molecule to the toehold segment and consequent displacement of the displacement segment from the activation segment,   the contacting performed for a time and under condition to allow covalent attachment of the opposite ends of the targeting domain to the first strand and the second strand of the target binding portion of the locking sensor in a molecular complex comprising the targeting domain in a folded conformation, the molecular complex configured to release the targeting domain in an unfolded conformation upon binding of a signal molecule to the toehold segment and consequent displacement of the displacement segment from the activation segment.   
     
     
         10 . A system for providing a molecular complex for enzyme-assisted molecular delivery, the system comprising
 at least one targeting domain of  claim 1  and at least one locking sensor to provide a molecular complex
 the locking sensor comprising
 a locking sensor RNA duplex having a toehold segment, a displacement segment and an activation segment, 
 the displacement segment presenting a first strand; the activation segment presenting a second strand; the displacement segment complementarily binding the activation segment; and the toehold segment being presented for binding to signal molecule; 
 
 wherein:
 the locking sensor RNA duplex is configured to attach the opposite ends of the targeting domain of  claim 1  in a folded conformation, through covalent linkage of the first strand with a first end of the opposite ends of the targeting domain and through covalent linkage of the second strand with a second end of the opposite ends of the targeting domain; and the displacement segment, activation segment and toehold segment are configured to allow release of the targeting domain from the folded conformation upon binding of a signal molecule to the toehold segment and consequent displacement of the displacement segment from the activation segment, 
 
   wherein the targeting domain is bound to the locking sensor in the folded conformation through covalent attachment of the opposite ends of the targeting domain to the first strand and second strand of the targeting domain binding portion of the protection segment of the sensor of  claim 1 , the molecular complex configured to release the signal molecule to the toehold segment and consequent displacement of the displacement segment from the activation segment.   
     
     
         11 . An activatable molecular complex comprising
 the targeting domain of  claim 1 ; and   a locking sensor, the locking sensor comprising
 an activation segment; 
 a displacement segment complementary to the activation segment; and 
 a toehold segment capable of binding to a signal molecule, 
   
       the targeting domain bound to the locking sensor in the folded conformation through covalent attachment of the opposite ends of the targeting domain to a first strand presented on the displacement segment and a second strand presented on the activation segment, 
       wherein
 the activatable molecular complex is configured to exhibit a first conformation and a second, activated, conformation in which, 
 
       in the first conformation the displacement segment complementarily binds the RNA portion of the activation segment to form a locking sensor RNA duplex, the toehold segment is presented for binding to a signal molecule; and the targeting domain is in a folded conformation; and 
       in the second activated conformation, the toehold segment and the displacement segment of the locking sensor either complementary bind a third polynucleotide or are absent, the activation segment of the locking sensor is either presented in a single stranded configuration cleavable by ribonuclease enzymes, or folded to provide an RNAase H binding site presented for binding, or is absent, and the targeting domain is released from the folded conformation 
     
     
         12 . The activatable molecular complex of  claim 11 , wherein in presence of a signal molecule, the second activated conformation has a free energy of at least about 5 kcal/mol lower than that the free energy of the first inactive conformation. 
     
     
         13 . The activatable molecular complex of  claim 11 , wherein in the first conformation the displacement segment and the activation segment form a double stranded duplex, the duplex being up to 30 bp in length. 
     
     
         14 . The activatable molecular complex of  claim 13 , wherein the duplex comprise at least about 5% 2′-O-methyl modifications or one or two mismatches. 
     
     
         15 . A method for enzyme-assisted molecular delivery, the method comprising
 contacting the molecular complex of  claim 11  in the first conformation, with a signal molecule capable of binding to the toehold segment of the molecular complex of  claim 16  for a time and under condition to allow switching of the molecular complex from the first conformation to the second active conformation.   
     
     
         16 . A system for controlled release of a targeting domain from an activatable molecular complex, the system comprising
 at least two of one or more activatable molecular complexes of  claim 11 , and a signal molecule able to bind to the toehold segment of the one or more activatable molecular complexes of  claim 11 , for simultaneous combined or sequential use to control release of the targeting domain from the folded conformation to the unfolded conformation within the second activated conformation of the activatable molecular complex of  claim 11 .   
     
     
         17 . A method for controlled activation of a molecular complex, the method comprising
 contacting the activatable molecular complex of  claim 11  in the first condition, with a signal polynucleotide complementary to the toehold segment to allow switching of the molecular complex from the first condition to the second activated condition of the activatable molecular complex.   
     
     
         18 . The method of  claim 17 , wherein the contacting is performed by providing the activatable molecular complex in a cell a expressing the signal molecule. 
     
     
         19 . The method of  claim 18 , wherein the providing is performed by administering the activatable molecular complex to an individual in vivo. 
     
     
         20 . An activated molecular complex, the activated molecular complex comprising
 the targeting domain of  claim 1 ; and   a locking sensor, the locking sensor comprising
 an activation segment 
 a displacement segment complementary to the activation segment; and 
 a toehold segment capable to bind to a signal molecule, 
   wherein,   
       the targeting domain is bound in the unfolded conformation to the displacement segment and the activation segment through covalent attachment of one of the opposite ends of the targeting domain to a first strand presented in the displacement segment and a second strand presented on the activation segment, and
 wherein 
 in the activated molecular complex, the displacement segment and the toehold segment either complementary bind a third polynucleotide or are absent, and the targeting domain is in a conformation configured to allow processing by dicer and/or an argonaute enzyme following cleavage of the activation segment from the targeting domain by a suitable ribonuclease. 
 
     
     
         21 . A method for enzyme-assisted molecular delivery, the method comprising
 contacting the activated molecular complex of  claim 16  with the suitable ribonuclease and with dicer and/or an argonaute enzyme for a time and under condition to allow release of the guide strand from the activated molecular complex.   
     
     
         22 . A composition comprising one or more of the targeting domain of  claim 1  together with a suitable vehicle. 
     
     
         23 . A composition, comprising one or more of the activatable molecular complex of  claim 11  together with a suitable vehicle. 
     
     
         24 . A method for treating a disease in an individual through enzyme-assisted signal activated molecular delivery in cells, the method comprising:
 administering to the individual an effective amount of one or more of the activatable molecular complex of  claim 11 .