U.S. patent application number 14/882346 was filed with the patent office on 2016-05-12 for targeting domain and related signal activated molecular delivery.
The applicant listed for this patent is CALIFORNIA INSTITUTE OF TECHNOLOGY, CITY OF HOPE. Invention is credited to William A. GODDARD, III, Si-ping HAN, John J. ROSSI, Lisa SCHERER.
Application Number | 20160130581 14/882346 |
Document ID | / |
Family ID | 49223357 |
Filed Date | 2016-05-12 |
United States Patent
Application |
20160130581 |
Kind Code |
A1 |
HAN; Si-ping ; et
al. |
May 12, 2016 |
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.
Inventors: |
HAN; Si-ping; (YORBA LINDA,
CA) ; GODDARD, III; William A.; (PASADENA, CA)
; SCHERER; Lisa; (MONROVIA, CA) ; ROSSI; John
J.; (AZUSA, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CALIFORNIA INSTITUTE OF TECHNOLOGY
CITY OF HOPE |
PASADENA
DUARTE |
CA
CA |
US
US |
|
|
Family ID: |
49223357 |
Appl. No.: |
14/882346 |
Filed: |
October 13, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13848687 |
Mar 21, 2013 |
9206419 |
|
|
14882346 |
|
|
|
|
61613617 |
Mar 21, 2012 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/199; 435/375; 536/24.5 |
Current CPC
Class: |
C12N 2310/14 20130101;
C12N 2310/3519 20130101; C12N 2310/321 20130101; C12N 15/111
20130101; C12N 15/113 20130101; C12N 2320/32 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/848,687, filed on Mar. 21, 2013, which, in
turn, claims priority to U.S. Provisional Application No.
61/613,617, filed on Mar. 21, 2012 and entitled "Pseudoknot
construct for signal activated RNA interference" (Docket No. CIT
6141-P), the disclosure of each of which is incorporated herein by
reference in its entirety. The present application might also be
related to U.S. patent application Ser. No. 13/167,672, filed on
Jun. 23, 2011 and entitled "Signal Activated Molecular Delivery"
(Docket No. P823-US), and International Application No.
PCT/US11/41703, filed on Jun. 23, 2011 and entitled "Signal
Activated Molecular Delivery" (Docket No. P823-PCT), the disclosure
of each of which is also incorporated by reference in its entirety.
The present application might also be related to U.S. patent
application Ser. No. 11/978,219 (Docket No. P1607-US), filed on
Oct. 26, 2007, and U.S. patent application Ser. No. 14/641,261
(Docket No. P1632-US), filed on Mar. 6, 2015.
FIELD
[0002] The present disclosure relates to a targeting domain and
related signal activated molecular delivery and in particular to
signal activatable constructs, and related components,
compositions, methods and systems.
BACKGROUND
[0003] Molecular delivery has been a challenge in the field of
biological molecule analysis, in particular when aimed at obtaining
controlled delivery of analytes of interest to specific
environments. Whether for medical applications or for fundamental
biology studies, several methods are commonly used for the delivery
of various classes of biomaterials and biomolecules.
[0004] Controlled delivery of targets to specific environments,
e.g. specific cell types and/or tissues of individuals in vitro
and/or in vivo is currently still challenging, especially when
directed at providing controlled release of the target in a
controllable conformation, typically associated to a biological
activity.
SUMMARY
[0005] Provided herein, are a targeting domain and related signal
activatable constructs for enzyme-assisted molecular delivery, and
related components, compositions, methods and systems. In
particular, in several embodiments, signal activatable constructs
herein described comprise a targeting domain and activatable
molecular complexes and activated complexes suitable for controlled
release of a targeting domain, which can comprise molecules of
various chemical natures.
[0006] According to a first aspect, a targeting domain is
described. The targeting domain comprises a targeting domain duplex
RNA having a length of about 19 to about 30 bp and comprising a
guide strand complementary bound to a passenger strand nicked in
two passenger strand segments having from about to 2 bp to about 17
bp length 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 minimizing processing of the guide strand by
dicer and/or an argonaute enzyme. 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.
[0007] According to a second aspect, a locking sensor is described
for enzyme-assisted molecular delivery of a targeting domain herein
described, and related compositions, methods and systems. The
locking sensor comprises a locking sensor RNA duplex having a
toehold segment a displacement segment, and an activation segment.
The locking sensor RNA duplex comprises a first strand presented on
displacement segment and a second strand presented on the
activation segment and is configured to attach opposite ends of the
targeting domain 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. In
the locking sensor herein described, the displacement segment
complementarily binds the activation segment and the toehold
segment is presented for binding to a signal molecule. In the
locking sensor herein described, the targeting domain and the
locking sensor are further configured to allow release of the
targeting domain from the folded conformation upon binding of the
signal molecule to the toehold segment and consequent displacement
of the displacement segment from the activation segment.
[0008] The composition comprises one or more locking sensors
together with a suitable vehicle. The method comprises: contacting
the locking sensor herein described with a targeting domain herein
described, the contacting performed for a time and under condition
to allow covalent attachment of the targeting domain to the first
strand and the second strand of the locking sensor in a molecular
complex comprising the targeting domain in a folded conformation.
The system comprises: a locking sensor and a targeting domain
herein described for simultaneous combined or sequential use in the
method to provide a molecular complex is described for
enzyme-assisted molecular delivery herein described.
[0009] According to a third aspect a molecular complex is described
for enzyme-assisted molecular delivery, and related compositions
methods and systems. The molecular complex comprises a targeting
domain herein described and a locking sensor comprising a locking
sensor RNA duplex having a toehold segment, a displacement segment
and an activation segment. In the molecular complex, opposite ends
of the targeting domain covalently bind to the locking sensor
through covalent linkage of a first end of the opposite ends of the
targeting domain with a first strand of presented on the
displacement segment and through covalent linkage of a second end
of the opposite ends of the targeting domain with a second strand
presented on the activation segment. In the molecular complex, the
displacement segment is complementarily bound to the activation
segment and the toehold segment is presented for binding to a
signal molecule. In the molecular complex, the targeting domain and
the locking sensor are configured to allow release of the targeting
domain from the folded conformation upon binding of the signal
molecule to the toehold segment and consequent displacement of the
displacement segment from the activation segment. The composition
comprises one or more molecular complexes herein described together
with a suitable vehicle. The method comprises: contacting the
molecular complex with a signal molecule able to bind to the
toehold for a time and under condition to allow release of the
targeting domain from the folded conformation to the unfolded
conformation. The system comprises: at least two of a molecular
complex, and a signal molecule able to bind to the toehold segment
of the molecular complex, for simultaneous combined or sequential
use to control release of the targeting domain from the folded
conformation according to the methods herein described.
[0010] According to a fourth aspect, an activatable molecular
complex is described and related, activated complexes, compositions
methods and systems. The activatable molecular complex comprises: a
targeting domain herein described and a locking sensor comprising a
toehold segment a displacement segment and an activation segment
having at least an RNA portion. In the molecular complex, the
targeting domain covalently binds the locking sensor through
covalent linkage of a first of opposite ends of the targeting
domain with a first strand of presented on the displacement segment
and through covalent linkage of a second end of the opposite ends
of the targeting domain with a second strand presented on the
activation segment. The activatable molecular complex is configured
to exhibit a first conformation and a second, activated
conformation wherein, in the first conformation the displacement
segment complementarily binds the RNA portion of the activation
segment to form a locking sensor RNA duplex, and the toehold
segment is presented for binding to a signal molecule; and the
targeting domain is in a folded conformation. 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.
[0011] The composition comprises one or more activatable complexes
and a suitable vehicle. The method comprises contacting an
activatable molecular complex in a first conformation, with a
signal molecule able to bind to the toehold segment of the
activatable molecular complex for a time and under condition to
allow switching of the molecular complex from the first
conformation to the second active conformation. The system
comprises at least two of one or more activatable molecular
complexes herein described, and a signal molecule capable to bind
the toehold segment of the molecular complexes for simultaneous
combined or sequential use to control release of the targeting
domain from the folded conformation in the molecular complex.
[0012] According to fifth aspect, an activated molecular complex is
described and related compositions methods and systems. The
activated molecular complex comprises a targeting domain herein
described and a 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. In the
activated molecular complex 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, 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.
[0013] The related composition comprises one or more activated
molecular complexes and a suitable vehicle. The related method to
provide the activated molecular complex comprises contacting the
activatable molecular complex herein described in the first
conformation, with a signal molecule binding to the signal binding
portion to allow switching of the molecular complex from the first
conformation to the second activated conformation of the molecular
complex. The related method for controlled release of a targeting
domain from an activated complex comprises: contacting the
activated molecular complex with a 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.
[0014] According to sixth aspect, a method for treating a disease
in an individual through RNAase assisted signal activated molecular
delivery in cells, is described, and related compositions and
systems. The method comprises administering to the individual an
effective amount of one or more of the signal activatable
constructs as described in the second aspect. The related
pharmaceutical composition comprises one or more signal activatable
constructs herein described with a pharmaceutical acceptable
vehicle.
[0015] According to a further aspect, complexes, herein described
can be provided by a method comprising providing a polynucleotide
guide strand, a polynucleotide A strand and a polynucleotide B
strand, wherein the polynucleotide A strand comprises from the 5'
end to 3' end the toehold segment, the displacement segment and a
first passenger strand segment of the two passenger strand segments
of the targeting domain in a 5' to 3' configuration. IN the method
the polynucleotide strand B comprises from the 5' end to 3' end a
second passenger strand segment of the two passenger strand
segments of the targeting domain and the activation segment. The
method further comprises contacting the polynucleotide guide
strand, the polynucleotide A strand and the polynucleotide B strand
for a time and under condition to allow annealing of the strand to
form the molecular complex of claim 5.
[0016] The constructs, systems, compositions and methods herein
described allow in several embodiments to performed cell type
specific molecular delivery.
[0017] The constructs, systems, compositions and methods herein
described also allow in several embodiments integration of signal
detection, signal transduction and targeting in a single compact
molecular construct with easier delivery and/or administration as
well as enhanced efficiency of signal transduction with respect to
some approaches of the art.
[0018] The constructs, systems, compositions and methods herein
described also allow in several embodiments intracellular
information processing and controlling in which the presence of one
set of biomolecules (e.g. protein or nucleic acid) is coupled with
inhibition or activation of another set of biomolecules in the
cells.
[0019] The methods and systems herein described can be used in
connection with applications wherein cell-type specific modulation
of cells is desired, including but not limited to medical
application, biological analysis, research and diagnostics
including but not limited to clinical, therapeutic and
pharmaceutical applications, such as cell type specific drug
delivery, cell type specific modeling or therapy, including but not
limited to gene therapy and RNAi.
[0020] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description and examples sections, serve to explain the
principles and implementations of the disclosure.
[0022] FIG. 1 shows a schematic illustration of a targeting domain,
according to an embodiment herein described showing the general
structure (FIG. 1, panel A), an open active conformation (FIG. 1,
panel B) and a folded inactive conformation (FIG. 1, panel C)
[0023] FIG. 2 shows a schematic illustration of molecular complexes
herein described showing exemplary attachments of the locking
sensor to the targeting domain according to embodiments herein
described.
[0024] FIG. 3 shows a schematic illustration of exemplary
activatable molecule complexes herein described, shown in an
inactive conformation (FIG. 3, panel A and FIG. 3, panel B) and
active conformation (FIG. 3, panel B and FIG. 3, panel D). In the
illustration of FIG. 3, the correspondence between active and
inactive form is indicated by arrows.
[0025] FIG. 4 illustrates the exemplary activatable molecule
complexes of FIG. 3 with arrows indicating unstructured regions
minimizing activation of the PKR pathway.
[0026] FIG. 5 shows exemplary sites of exemplary chemical
modification of molecular complexes herein described illustrated
with reference to the exemplary activatable molecule complexes of
FIG. 3.
[0027] FIG. 6 shows exemplary configuration of activatable molecule
complexes herein described illustrated as variant geometry with
respect to the activatable molecule complexes of FIG. 3.
[0028] FIG. 7 shows exemplary targeting domain herein described in
a folded conformation covalently linked to exemplary locking
sensors herein described (FIG. 7, panel A and FIG. 7, panel C) and
in an folded conformation following binding of an exemplary signal
molecule (FIG. 7, panel B and FIG. 7, panel D). In the illustration
of FIG. 7, the correspondence between active and inactive form is
indicated by arrows.
[0029] FIG. 8 shows exemplary targeting domain herein described in
a folded conformation covalently linked to exemplary locking
sensors herein described (FIG. 8, panel A and FIG. 8, panel C) and
in an folded conformation following binding of an exemplary signal
molecule (FIG. 8, panel B and FIG. 8, panel D). In the illustration
of FIG. 8, the correspondence between active and inactive form is
indicated by arrows.
[0030] FIG. 9 shows a schematic representation of an exemplary
method to provide an exemplary targeting domain in a molecular
complex according to embodiments herein described.
[0031] FIG. 10 shows a schematic representation of an exemplary
molecular complex herein described wherein the locking sensor
comprises a signal binding portion configured to release the
targeting domain from the folded conformation Medusa G A1 B6b (SEQ
ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 9) (FIG. 10,
panel A) to an unfolded conformation (SEQ ID NO: 1, SEQ ID NO:5,
SEQ NO: 8, SEQ NO: 9, and SEQ NO: 16) (FIG. 10, panel B) through
displacement of the signal binding portion following binding of a
polynucleotide signal, to provide a molecular presenting an
activation segment and in an active conformation following
processing of the activation segment by a ribonuclease enzyme.
[0032] FIG. 11 shows a schematic representation of an exemplary
method to release an exemplary targeting domain from the exemplary
molecular complex of FIG. 10.
[0033] FIG. 12 shows a schematic illustration of an exemplary
molecular complex, Medusa G[[A1]]A2B6b (SEQ ID NO: 1, SEQ ID NO: 48
(5'-C C U C A G A C G C A mA mG-3'), SEQ ID NO: 49 (5'-G A U G A G
C U C U U C G U C G C U G U C U C-3'), SEQ ID NO: 8, and SEQ ID 9).
In particular, FIG. 12, panel A shows the inactive conformation and
FIG. 12, panel B_shows the active conformation_(SEQ ID NO: 1, SEQ
ID NO: 48, SEQ ID NO: 49, SEQ NO: 8, SEQ NO: 9, and SEQ NO:
16).
[0034] FIG. 13 shows a schematic illustration of an exemplary
molecular complex, Medusa GA1B6b. In particular, FIG. 13, panel A
shows the inactive conformation (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 8, and SEQ ID NO:9) and FIG. 13, panel B shows the active
conformation (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO:
9 and SEQ ID NO: 16).
[0035] FIG. 14 shows a schematic illustration of an exemplary
molecular complex, Medusa G2A3B7. In particular, FIG. 14, panel A
shows the inactive conformation (SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID 15) and FIG.
14, panel B_shows the active conformation (SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 50 (5'-mA mA mA mA mA G C G G A G A C A G C G A C G
A A G A G C U C A U C G mA mA mA mA mA T-3'))
[0036] FIG. 15 shows a schematic illustration of an exemplary
molecular complex G A1 B4 (SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID
NO: 7) with an activation segment for RNAase H processing. In
particular, FIG. 15, panel A shows the inactive conformation (SEQ
ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 7) and FIG. 15, panel B
shows the active conformation (SEQ ID NO: 1, SEQ ID NO: 51 (5'-mA
mA mG mG mU C C C T G A T C G A C G A A G A G C U C A U C A G G G U
A A C mU A mG A mG A U mC C C U C A G A C G C A A G mC mU mG mA mU
mG mA mG mC mU mC mU mU mC mG mU mC mG mC mU mG mU mU T-3') and SEQ
ID NO: 16).
[0037] FIG. 16 shows a schematic representation of an exemplary
method to release an exemplary targeting domain from the exemplary
molecular complex of FIG. 15.
[0038] FIG. 17 shows schematic illustration of exemplary molecular
complexes. In particular, molecular complex GA1B1 (SEQ ID NO: 1,
SEQ ID NO: 5, and SEQ ID NO: 6) is depicted in FIG. 17, panel A and
complex GA1B4 (SEQ ID NO: 1, SEQ ID NO: 52 (5'mA mA mG mG mU C C C
T G AT C G A C G A A G A G C U C A U C A G G G U A A C U A G A G A
U C C C U C A G A C G C A A G mC mU mG mA mU mG mA mG mC mU mC mU
mU mC mG mU mC mG mC mU mG mU mU T-3') and SEQ ID NO: 16) is
depicted FIG. 17, panel B.
[0039] FIG. 18 shows a schematic illustration of an exemplary
molecular complex complex G A1 B6b (SEQ ID NO: 1, SEQ ID NO: 5, SEQ
ID NO: 8, and SEQ ID NO: 9) in an inactive conformation.
[0040] FIG. 19 shows a schematic representation of potential
interactions that can lower RNAi activity. In particular, FIG. 19,
panel A illustrates exosome interactions and FIG. 19, panel B
illustrates PKR interactions (SEQ ID NO: 1, SEQ ID NO: 15, and SEQ
ID NO:5).
[0041] FIG. 20 shows a schematic illustration of a siRNA (FIG. 20,
panel A), a Dicer substrate siRNA (FIG. 20, panel B and FIG. 20,
panel C), and a miRNA analogue with Dicer cleavage sites (FIG. 20,
panel C).
[0042] FIG. 21 shows schematic illustration of exemplary molecular
complexes. In particular, molecular complex G1A1B6b (SEQ ID NO: 1,
SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 9)_is depicted in FIG.
21, panel A and complex G2A3B7 is depicted FIG. 21, panel B.
[0043] FIG. 22 shows schematic illustration of an exemplary
molecular complex with a fall-away sensor. In particular, FIG. 22,
panel A shows the "OFF" conformation and FIG. 22, panel B_shows the
"ON" conformation
[0044] FIG. 23 shows schematic illustration of exemplary molecular
complexes with a fall-away sensor. In particular, molecular complex
GH1J1 is depicted in FIG. 23, panel A and complex GH2J2 is depicted
FIG. 23, panel B.
[0045] FIG. 24 shows a schematic illustration of an exemplary
molecular complex, Medusa RNAseH: GA1B4(S) (SEQ ID NO: 1, SEQ ID
NO: 5, and SEQ ID NO: 7). In particular, FIG. 24, panel A shows the
inactive conformation (SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO:
7) and FIG. 24, panel B shows the active conformation (SEQ ID NO:
1, SEQ ID NO: 51 and SEQ ID NO: 16).
[0046] FIG. 25 shows a schematic illustration of an exemplary
molecular complex, Medusa Xrna1: GA1B6b(S) (SEQ ID NO: 1, SEQ ID
NO: 5, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 16). In
particular, FIG. 25, panel A shows the inactive conformation (SEQ
ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 9) and FIG.
25, panel B shows the active conformation (SEQ ID NO: 1, SEQ ID NO:
5, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 16).
[0047] FIG. 26 shows a schematic illustration of an exemplary
molecular complex, Medusa Xrna1: G2A3B7 (SEQ ID NO: 10, SEQ ID NO:
11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:
15). In particular, FIG. 26, panel A shows the inactive
conformation (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15) and FIG. 26, panel B
shows the active conformation (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 and SEQ ID
NO: 50). EG indicates Ethylene Glycol.
[0048] FIG. 27 shows a schematic illustration of an exemplary
molecular complex with a fall-away sensor. In particular, FIG. 27,
panel A shows an example (G H1 J1 (SEQ ID NO: 1, SEQ ID NO: 20, SEQ
ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 53 (5' mC U
mG A mU G mA G mC U mC U mU C mG U mC G mC U mG mU mC mU mG mC mG
mC 3') with a J1 sensor (SEQ ID NO: 53) and FIG. 27, panel B shows
an example (G2 H2 J2 (SEQ ID NO: 10, H2 (H2a: SEQ ID NO: 54 (5' C C
C U C A G A C G mC mG 3') SEQ ID NO:55 (5'G C A G A G C G A C G A A
G A G C)) and (H2b: SEQ ID NO: 56 (5'G G A G A C A G C G C G C U C
U G C A 3') SEQ ID NO: 57 (5'mG mG mU A A C mU Am G A mG A mU 3'))
with a J2 sensor (SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28).
EG indicates Ethylene Glycol.
[0049] FIG. 28 shows a schematic illustration of an exemplary
molecular complex with a fall-away sensor. In particular, FIG. 28,
panel A shows the "OFF" conformation and FIG. 28, panel B shows the
"ON" conformation.
[0050] FIG. 29 shows the stability of individual segments of an
exemplary complex the Guide (SEQ ID NO: 1), the Sensor A (SEQ ID
NO: 5), and the Sensor B (SEQ ID NO: 6). In particular, FIG. 29,
panel B shows the schematic of an exemplary complex, and FIG. 29,
panel A shows visualized gels of the individual segments next to
ladders (marked).
[0051] FIG. 30 shows a luciferase assay of exemplary Medusa
complexes G A1 B1 (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6), G A1
B1 plus signal (SEQ ID NO: 1, SEQ ID NO: 51 and SEQ ID NO: 16), G
A1 B4 (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO:7), G A1 B4 plus
signal (SEQ ID NO: 1, SEQ ID NO: 50 and SEQ ID NO: 16), G A1 B6b
(SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 9), G A1
B6b plus signal (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID
NO: 9 and SEQ ID NO: 16), G Ac B6c (SEQ ID NO: 1, SEQ ID NO: 2, and
SEQ ID NO: 18), G Ac B6b (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8
and SEQ ID NO: 9), and G Ac B4 (SEQ ID NO: 1, SEQ ID NO: 57) with
controls where the y-axis represents relative luciferase units and
x-axis represents the exemplary complexes used in the assay. Panels
A, B, C, and D represent different luciferase assays.
[0052] FIG. 31 shows a Northern blot of exemplary Medusa complexes
with and without signal strands and controls. Lane M, RNA size
markers, number of nucleotides is indicated. Lane 1, G Ac B4 SEQ ID
NO: 1, SEQ ID NO: 58 (5' mA mA mG mG mU C C C T G A T C G A C G A A
G A G C U C A U C A G G G U A A C mU A mG A mG A U mC C C U C A G A
C G C A A G T-3'); lane 2, G Ac Bc (SEQ ID NO: 1 and SEQ ID NO: 59
(5'G G U A A C U A G A G A U C C C U C A G A C G C A A G T-3');
lane 3, G A B6b(SEQ ID NO:1, SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID
NO: 9); lane 4, G A B6b S (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO:
8, SEQ ID NO:9 and SEQ ID NO: 16); lane 5, G A B6c (SEQ ID NO: 1,
SEQ ID NO: 5, and SEQ ID NO: 19); lane 6, G A B6c S (SEQ ID NO: 1,
SEQ ID NO: 5, SEQ ID NO: 19, and SEQ ID NO: 16); lane 7, G Ac B6b
(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 9); lane
8, G Ac B6c (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 19); lane 9,
G RP (SEQ ID NO: 1 and SEQ ID NO: 4). Region between Lanes M and 1
contain unrelated constructs.
[0053] FIG. 32 shows the results of a dual luciferase assay with
the exemplary Medusa construct A Gc6, G Ac Bc6, G A B6b S, G2 A3
B7, G2 A3 B7S and G RP at 1.0, 0.25 and 0.6 nMolar
concentrations.
[0054] FIG. 33 shows a Northern blot of exemplary Medusa complexes
with and without signal strands and controls. Lane M, RNA size
markers, number of nucleotides is indicated. Probe (oligo 544)
hybridizes to intact guide strand G (29 nucleotides) seen in all
lanes and the approximately 21 nucleotide Dicer product, indicated
by the arrow, seen with G Ac Bc6, G2 A3 B7 and G2 A3 B7 S (lanes 2,
5 and 6, respectively).
[0055] FIG. 34 shows an unlocked (G Ac Bc (SEQ ID NO: 1, SEQ ID NO:
2, and SEQ ID NO: 3)) and a locked (G RP (SEQ ID NO: 1, and SEQ ID
NO: 4)) RNAi targeting domain. G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3) and G RP (SEQ ID NO: 1, and SEQ ID NO: 4) have
identical sequences, but in G RP the 3' of Passenger A is directly
linked to the 5' of Passenger B, comprising a single "reversed
topology" passenger strand. This linkage locks the RNAi targeting
domain into a folded conformation that minimizes proper Dicer
processing.
[0056] FIG. 35 shows the assembled G RP (SEQ ID NO: 1, and SEQ ID
NO: 4) product. The individual strands composing G RP SEQ ID NO: 1,
and SEQ ID NO: 4) or G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ
ID NO: 3) were ordered from a commercial company, Thermo
Scientific. For assembly the strands composing G Ac Bc (SEQ ID NO:
1, SEQ ID NO: 2, and SEQ ID NO: 3) or G RP (SEQ ID NO: 1, and SEQ
ID NO: 4) were combined at 1 micromolar concentration in
1.times.PBS buffer (approximately 150 mM KCl with other
components), heated to .about.90 degrees Celsius, and allowed to
cool to room temperature. During this process the strands
self-assemble into either G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, and
SEQ ID NO: 3) or G RP (SEQ ID NO: 5). The resulting G RP (SEQ ID
NO: 1, and SEQ ID NO: 4) products were assessed by running through
8% non- denaturing polyacrylamide gel in 1.times.TBE buffer
following standard practices in the art. The first lane on the left
shows the band corresponding to the Guide strand alone. In the G RP
(SEQ ID NO: 1, and SEQ ID NO: 4) lane, there is a clear band
showing a construct corresponding to the G RP construct in the
correct conformation. In this conformation, Dicer processing is
minimized. In addition, there are a number of higher molecular
weight lanes, corresponding to incorrect, multimeric assemblies of
G (SEQ ID NO: 1) and RP (SEQ ID NO: 4) strands. These higher
molecular weight products can have spurious Dicer processing and
RNAi activity. If desired, these products can be removed by
filtering using HPLC, or filtration membranes with the appropriate
molecular weight cutoff, or by extracting them using native
polyacrylamide gel electrophoresis.
[0057] FIG. 36 shows the results of a dual luciferase assay with G
Ac Bc and G RP exemplary Medusa complexes at 5.0, 1.0 and 0.2
nMolar concentrations.
[0058] FIG. 37 shows the definition of Dicer processing. For a
duplex RNAi targeting domain with a guide strand, such as the one
shown (G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) from
FIG. 34), correct processing occurs when PAZ domain of Dicer binds
to 3' of the Guide strand and the endonuclease domain of Dicer
cleaves the 5' end of the guide strand in the position indicated.
For a perfectly base-paired RNAi targeting domain, this should
produce a 21 to 23 nucleotide long product, highlighted in gray.
For an imperfect duplex, the product can be up to 25 nucleotides
long.
DETAILED DESCRIPTION
[0059] Herein described are signal activatable constructs for
enzyme-assisted molecular delivery and related components,
compositions, methods and systems.
[0060] The term "signal activatable construct" as used herein
indicates a molecular complex that can have more than one
conformation, and at least one of the conformations results from
the binding of a signal molecule to the molecular complex.
Typically, the conformation associated to the binding of a signal
molecule to the molecular complex is also associated to a chemical
and/or biological activity that characterizes the conformation as
active with respect to the identified activity. Accordingly, signal
activatable constructs herein described can have at least one
active conformation and at least one inactive conformation with
respect to the enzymatic activity of the enzyme assisted molecular
delivery. Switching between an inactive conformation to an active
conformation is triggered by binding of the signal molecule to the
construct.
[0061] Signal activatable constructs and related components herein
described comprise one or more polynucleotides. The term
"polynucleotide" as used herein indicates an organic polymer
composed of two or more monomers including nucleotides, nucleosides
or analogs thereof. The term "nucleotide" refers to any of several
compounds that consist of a ribose or deoxyribose sugar joined to a
purine or pyrimidine base and to a phosphate group and that is the
basic structural unit of nucleic acids. The term "nucleoside"
refers to a compound (such as guanosine or adenosine) that consists
of a purine or pyrimidine base combined with deoxyribose or ribose
and is found especially in nucleic acids. The term "nucleotide
analog" or "nucleoside analog" refers respectively to a nucleotide
or nucleoside in which one or more individual atoms have been
replaced with a different atom or a with a different functional
group. Exemplary functional groups that can be comprised in an
analog include methyl groups and hydroxyl groups and additional
groups identifiable by a skilled person.
[0062] Exemplary monomers of a polynucleotide comprise
deoxyribonucleotide, ribonucleotides, LNA nucleotides and PNA
nucleotides. The term "deoxyribonucleotide" refers to the monomer,
or single unit, of DNA, or deoxyribonucleic acid. Each
deoxyribonucleotide comprises three parts: a nitrogenous base, a
deoxyribose sugar, and one or more phosphate groups. The
nitrogenous base is typically bonded to the 1' carbon of the
deoxyribose, which is distinguished from ribose by the presence of
a proton on the 2' carbon rather than an --OH group. The phosphate
group is typically bound to the 5' carbon of the sugar. The term
"ribonucleotide" refers to the monomer, or single unit, of RNA, or
ribonucleic acid. Ribonucleotides have one, two, or three phosphate
groups attached to the ribose sugar. The term "locked nucleic
acids" (LNA) as used herein indicates a modified RNA nucleotide.
The ribose moiety of an LNA nucleotide is modified with an extra
bridge connecting the 2' and 4' carbons. The bridge "locks" the
ribose in the 3'-endo structural conformation, which is often found
in the A-form of DNA or RNA. LNA nucleotides can be mixed with DNA
or RNA bases in the oligonucleotide whenever desired. The locked
ribose conformation enhances base stacking and backbone
pre-organization. This significantly increases the thermal
stability (melting temperature) of oligonucleotides. LNA
oligonucleotides display unprecedented hybridization affinity
toward complementary single-stranded RNA and complementary single-
or double-stranded DNA. Structural studies have shown that LNA
oligonucleotides induce A-type (RNA-like) duplex conformations. The
term "polyamide polynucleotide", "peptide nucleic acid" or "PNA" as
used herein indicates a type of artificially synthesized polymer
composed of monomers linked to form a backbone composed of
repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
The various purine and pyrimidine bases are linked to the backbone
by methylene carbonyl bonds. Since the backbone of PNA contains no
charged phosphate groups, the binding between PNA/DNA strands is
stronger than between DNA/DNA strands due to the lack of
electrostatic repulsion. PNA oligomers also show greater
specificity in binding to complementary DNAs, with a PNA/DNA base
mismatch being more destabilizing than a similar mismatch in a
DNA/DNA duplex. This binding strength and specificity also applies
to PNA/RNA duplexes. PNAs are not easily recognized by either
nucleases or proteases, making them resistant to enzyme
degradation.
[0063] PNAs are also stable over a wide pH range. In some
embodiments, polynucleotides can comprise one or more
non-nucleotidic or non nucleosidic monomers identifiable by a
skilled person.
[0064] Accordingly, the term "polynucleotide" includes nucleic
acids of any length, and in particular DNA, RNA, analogs thereof,
such as LNA and PNA, and fragments thereof, possibly including
non-nucleotidic or non-nucleosidic monomers, a each of which can be
isolated from natural sources, recombinantly produced, or
artificially synthesized. Polynucleotides can typically be provided
in single-stranded form or double-stranded form (herein also duplex
form, or duplex).
[0065] A "single-stranded polynucleotide" refers to an individual
string of monomers linked together through an alternating sugar
phosphate backbone. In particular, the sugar of one nucleotide is
bond to the phosphate of the next adjacent nucleotide by a
phosphodiester bond. Depending on the sequence of the nucleotides,
a single-stranded polynucleotide can have various secondary
structures, such as the stem-loop or hairpin structure, through
intramolecular self-base-paring. A hairpin loop or stem loop
structure occurs when two regions of the same strand, usually
complementary in nucleotide sequence when read in opposite
directions, base-pairs to form a double helix that ends in an
unpaired loop. The resulting lollipop-shaped structure is a key
building block of many RNA secondary structures. The term "small
hairpin RNA" or "short hairpin RNA" or "shRNA" as used herein
indicate a sequence of RNA that makes a tight hairpin turn and can
be used to silence gene expression via RNAi.
[0066] A "double-stranded polynucleotide", "duplex polynucleotide"
refers to two single-stranded polynucleotides bound to each other
through complementarily binding. The duplex typically has a helical
structure, such as double-stranded DNA (dsDNA) molecule or double
stranded RNA, is maintained largely by non-covalent bonding of base
pairs between the strands, and by base stacking interactions.
[0067] The constructs and complements herein described are suitable
in many embodiments for enzyme assisted molecular delivery. The
term "molecular delivery" as used herein indicates any process by
which controlled activation of molecular complexes regulates the
release of a chemical compound for various purposes.
[0068] The term "enzyme-assisted" as used herein is defined to mean
any chemical process where a protein or other chemical entity is
used to catalyze or increase the rate of a chemical reaction. The
protein used for this purpose can include, but is not limited to,
chains of amino acids (natural or unnatural), that may or may not
contain other chemical variations and can have a defined secondary
structure. The chemical reaction can include, but is not limited
to, reactions of RNA or portions of RNA, DNA or portions of DNA,
and/or any nucleotide or derivative thereof. Typically, enzymes
catalyze reactions through binding to specific or aspecific target
molecular portions usually indicated as binding sites.
[0069] In several embodiments, the enzyme-assisted molecular
delivery herein described is an XRN1 assisted molecular delivery.
In several embodiments, the enzyme-assisted molecular delivery
herein described is an XRN1 assisted molecular delivery. The term
"XRN1" as used herein refers to an exoribonuclease enzyme that is
capable of degrading ribopolynucleotides by removing terminal
nucleotides from the 5' terminus of the ribopolynucleotide. As used
herein the term "XRN1" comprises any enzymes whether naturally
occurring or synthetically modified including any enzyme modified
in one or more residues which substantially retain an
exoribonuclease activity such as the one herein described.
Naturally occurring XRN1 enzymes which are members of the XRN1
family can be found in many organisms including yeast, nematode,
fruit fly, and human. XRN1 is also referred as Pacman, KEM1, SEP1,
DST2, RAR5 SKI1 and DST2 to one skilled in the art.
[0070] In several embodiments, the enzyme-assisted molecular
delivery herein described is an RNAase H assisted molecular
delivery. The term "RNAse H" as used herein refers to a nonspecific
endonuclease that is able to catalyze RNA cleavage via a hydrolytic
mechanism. In particular RNase H's ribonuclease activity cleaves a
3'-O-P bond of RNA in a DNA:RNA duplex to produce 3' hydroxyl and
5' phosphate terminated products. RNAase H cleaves the RNA strand
in DNA:RNA duplexes. The minimal substrate for RNAse H cleavage
activity is usually a 5 to 7 base pair long stretch of duplex
DNA:RNA. As used herein the term "RNAase H" comprises any enzymes
whether naturally occurring or synthetically modified including any
enzyme modified in one or more residues which substantially retain
an endonucleasic activity such as the one herein described.
Naturally occurring RNAase H enzyme which are members of the RNAse
H family can be found in nearly all organisms, from archaea to
prokaryote and eukaryote are identifiable by a skilled person. In
human cells, RNAse H commonly cleaves the RNA sequence of a DNA:
RNA duplex at a position that is 5 nucleotides from the 5' end of
the RNA sequence forming the duplex. If the duplex is longer than 7
base pairs, RNAse H can cleave at additional positions to the 3' of
the first cleavage site. The mammalian RNAse H class enzymes cleave
the RNA portion of DNA:RNA duplexes. RNAse H class enzymes
constitute the dominant mechanism of activity for many antisense
oligonucleotide drugs. RNAse H can be typically active both in the
cytoplasm and the nucleus.
[0071] In particular in some embodiments, the enzyme assisted
molecular delivery is directed to release a targeting domain with a
biological environment and in particular within a cell, and the
release of the targeting domain can be catalyzed by XRN1 or RNAaseH
in combination with dicer and/or an argonaute enzyme.
[0072] A "domain" in the sense of the present disclosure indicates
a part of a given polynucleotide having a structure specifically
associated with a function and that exist independently of the rest
of the polynucleotide. The structure/function association in a
domain is typically conserved during the chemical and/or biological
reaction associated with the polynucleotide.
[0073] A "targeting domain" as used herein indicates a domain of a
polynucleotide associated with the function of binding or reacting
with a predetermined target within a biological environment and in
particular within a cell.
[0074] The term "target" as used herein indicates an analyte of
interest. The term "analyte" refers to a substance, compound,
moiety, or component whose presence or absence in a sample is to be
detected. Analytes include but are not limited to biomolecules and
in particular biomarkers. The term "biomolecule" as used herein
indicates a substance, compound or component associated with a
biological environment including but not limited to sugars, amino
acids, peptides, proteins, oligonucleotides, polynucleotides,
polypeptides, organic molecules, haptens, epitopes, biological
cells, parts of biological cells, vitamins, hormones and the like.
The term "biomarker" indicates a biomolecule that is associated
with a specific state of a biological environment including but not
limited to a phase of cellular cycle, health and disease state. The
presence, absence, reduction, upregulation of the biomarker is
associated with and is indicative of a particular state. The
"biological environment" refers to any biological setting,
including, for example, ecosystems, orders, families, genera,
species, subspecies, organisms, tissues, cells, viruses,
organelles, cellular substructures, prions, and samples of
biological origin.
[0075] Exemplary targeting domains in the sense of the present
disclosure comprise siRNA, saRNA, microRNA and additional
polynucleotides identifiable by a skilled person.
[0076] In embodiments herein described, the targeting domain of the
disclosure a duplex RNA duplex RNA of about 19 to about 30 bp
length comprising a guide strand complementary bound to a passenger
strand nicked in two passenger strand segments having from about to
2 bp to about 17 bp length 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 which minimizes processing of the
guide strand by dicer and/or an argonaute enzyme. 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.
[0077] Reference is made to the schematic illustration of FIG. 1
which shows an exemplary targeting domain according to an
embodiment herein described, in a depiction schematically
illustrating the RNA duplex comprised in an exemplary targeting
domain (FIG. 1, panel A) the unfolded conformation of the exemplary
targeting domain of FIG. 1, panel A (FIG. 1, panel B) and the
folded conformation of the exemplary targeting domain of FIG. 1,
panel A (FIG. 1, panel C).
[0078] In the illustration of FIG. 1 the targeting domain comprises
a guide strand (1,2) complementary and complementary binding to
passenger strand (3,4) to form an RNA duplex.
[0079] The term "complementary" as used herein indicates a property
of single stranded polynucleotides in which the sequence of the
constituent monomers on one strand chemically matches the sequence
on another other strand to form a double stranded polynucleotide.
Chemical matching indicates that the base pairs between the
monomers of the single strand can be non-covalently connected via
two or three hydrogen bonds with corresponding monomers in the
another strand. In particular, in this application, when two
polynucleotide strands, sequences or segments are noted to be
complementary, this indicates that they have a sufficient number of
complementary bases to form a thermodynamically stable
double-stranded duplex. Double stranded of complementary single
stranded polynucleotides include dsDNA, dsRNA, DNA: RNA duplexes as
well as intramolecular base paring duplexes formed by complementary
sequences of a single polynucleotide strand (e.g. hairpin
loop).
[0080] The term `complementary bind", "base pair", "complementary
base pair" as used herein with respect to nucleic acids indicates
the two nucleotides on opposite polynucleotide strands or sequences
that are connected via hydrogen bonds. For example, in the
canonical Watson-Crick DNA base pairing, adenine (A) forms a base
pair with thymine (T) and guanine (G) forms a base pair with
cytosine (C). In RNA base paring, adenine (A) forms a base pair
with uracil (U) and guanine (G) forms a base pair with cytosine
(C). Accordingly, the term "base pairing" as used herein indicates
formation of hydrogen bonds between base pairs on opposite
complementary polynucleotide strands or sequences following the
Watson-Crick base pairing rule as will be applied by a skilled
person to provide duplex polynucleotides. Accordingly, when two
polynucleotide strands, sequences or segments are noted to be
binding to each other through complementarily binding or
complementarily bind to each other, this indicate that a sufficient
number of bases pairs forms between the two strands, sequences or
segments to form a thermodynamically stable double-stranded duplex,
although the duplex can contain mismatches, bulges and/or wobble
base pairs as will be understood by a skilled person.
[0081] The term "thermodynamic stability" as used herein indicates
a lowest energy state of a chemical system. Thermodynamic stability
can be used in connection with description of two chemical entities
(e.g. two molecules or portions thereof) to compare the relative
energies of the chemical entities. For example, when a chemical
entity is a polynucleotide, thermodynamic stability can be used in
absolute terms to indicate a conformation that is at a lowest
energy state, or in relative terms to describe conformations of the
polynucleotide or portions thereof to identify the prevailing
conformation as a result of the prevailing conformation being in a
lower energy state. Thermodynamic stability can be detected using
methods and techniques identifiable by a skilled person. For
example, for polynucleotides thermodynamic stability can be
determined based on measurement of melting temperature Tm, among
other methods, wherein a higher Tm can be associated with a more
thermodynamically stable chemical entity as will be understood by a
skilled person. Contributors to thermodynamic stability can
include, but are not limited to, chemical compositions, base
compositions, neighboring chemical compositions, and geometry of
the chemical entity.
[0082] In particular, in the exemplary targeting domain described
in FIG. 1, segment (3) is configured to complementary bind segment
(1) in a (1):(3) duplex, and segment (4) is configured to
complementary bind a portion of segment (2) in a (2):(4) duplex.
Mismatches and bulges in either the (1):(3) or (2):(4) duplexes are
permitted as long as the as long as the melting temperatures Tm of
(1):(3) and (2):(4) duplexes are predicted to be greater than the
operating temperature (e.g. 37.degree. C. in embodiments in which
detection of formation of RNA duplex is desired through methods
known to one skilled in the art such as Native PAGE followed by
visualization or UV-vis spectroscopy). In embodiments herein
described, duplex formation can be verified by Native PAGE or UV
vis spectroscopy or additional techniques identifiable by a skilled
person.
[0083] In particular in the illustration of FIG. 1, a 19 nt region
is indicated on the guide strand (1,2) which complementary binds
the passenger strand (3,4) to provide a thermodynamically stable
double stranded polynucleotide at a desired operating temperature.
Accordingly, in the exemplary illustration of FIG. 1, the 2 base
overhang of segment (2) does not need to be complementary to the
segment (4), and the region of segment (1) outside the 19 nt region
does not need to be complementary to the segment (3) to obtain a
thermodynamically stable double stranded structure.
[0084] In the illustration of FIG. 1, panel A, the guide strand
comprises segment (1) covalently linked at one end to a segment (2)
and the passenger strand comprising a segment (3), covalently
linked at one end to a segment (4). The term "covalent binding" or
"covalently linked" as used herein indicates connection between two
segments through formation of a chemical bonding that is
characterized by sharing of pairs of electrons between atoms, known
as the covalent bond. Examples covalent binding can include, but
are not limited to covalent bonds formed between any two of the
following: RNA or portions RNA, DNA or portions of DNA, any
nucleotide or derivative thereof, and/or enzyme.
[0085] In particular the exemplary illustration of FIG. 1, panel A,
guide strand (1,2) is the guide strand of an RNAi trigger, which in
the illustration of FIG. 1 is a siRNA, but can be other RNAai
triggers such as a Dicer substrate siRNA, a miRNA or other Dicer
substrates (see e.g. FIG. 20).
[0086] In the illustration of FIG. 1, panel B, the passenger strand
(3,4) is nicked into passenger strand segments (3) and (4). The
term "nicked" as used herein with reference to a polynucleotide
strand of a double stranded polynucleotides indicates a gap in the
direct covalent linkage between two nucleotides of the
polynucleotide chain forming the strand that are engaged in
complementary binding within double stranded polynucleotide.
Accordingly, an RNA duplex comprising a nicked passenger strand can
be obtained by cleaving the covalent linkage between suitable
nucleotides e.g. by using suitable endoribonucleases (such as an
RNAase III enzyme) or by synthesis of the a double stranded
polynucleotide with selected dideoxyribonucleotides used to
introduce the nick as will be understood by a skilled person.
Additional approaches will also be identifiable by the skilled
person directed to obtain a passenger strand in which two of the
nucleotides forming the polynucleotide chain engaged in the
complementary binding with the guide strand are not directly
covalently linked to each other. For example, in the illustration
of FIG. 1, panel B segments (3) and (4) can be connected by an
unstructured covalent linker (e.g. PEG or polynucleotide loop) as
long as the linker allows (3) and (4) to adopt a folded
conformation that prevents RNAi activity. In the illustration of
FIG. 1, panel B if an unstructured linker is introduced between 3
and 4, the unstructured linker can have a fully stretched length of
at least 2 nm.
[0087] In the illustration of FIG. 1, panel C, to inactivate the
RNAi substrate, a link is introduced between the two ends of the
duplex to force the fold illustrated. In particular in the
illustration of FIG. 1, panel C, a covalent link is included
between the terminal backbone positions or bases on two ends of the
duplex to lock the two ends of the targeting domain. Alternatively,
the lock can bind together two interior positions on (2):(4) and
(1):(3), in a configuration allowing the resulting structure
migrate at a substantially different rate in 10% Native PAGE gel
compared to the conformation of FIG. 1, panel A and FIG. 1, panel
B
[0088] In the exemplary illustration of FIG. 1, panel C the linkage
between the two opposite ends of the targeting domain provide a
configuration of the opposite ends such that those ends are at an
angle of about 10.degree. between each other. Additional suitable
configurations of the opposite ends suitable for a folded
conformation of the targeting domain comprise angles up to about
90.degree. as will be understood by a skilled person. Calculation
of the angle between the opposite ends of the targeting domain can
be performed by estimating the length of a duplex segment to be
approximately 0.34 nm per base-pair, and the maximum length of an
unstructured polynucleotide of less than or equal to 20 nucleotides
to be approximately 0.5 nm per nucleotide, and then using
trigonometry to calculate the maximum possible angle assuming the
estimated duplex lengths and the maximum unstructured
polynucleotide length. If the different segments are linked via a
non-polynucleotide linker, such as a C3 or a polyethylene glycol
linker, then for a short linker the one can use the maximum
possible length of the linker as calculated from the length of the
constituent molecular bonds and angles to calculate the maximum
angle via trigonometry. For unstructured polynucleotide linkers
longer than 20 nucleotides or polymer linkers longer than 20
polymer units, the average end to end distance can also be
calculated using polymer physics and an approximate range of motion
can be established by considering the energetic penalty of
stretching the unstructured polymer beyond its average end to end
distance. In these cases the bending angle should be estimated to
remain below the maximum angle of 90 degrees with at least 90%
probability. In a solution targeting domains herein described are
expected to change in accordance with temperature, length of
linkage between the opposite ends and additional parameters
identifiable by a skilled person.
[0089] Additionally, Forster Resonance Energy Transfer (FRET)
experiments can be used to experimentally estimate the angle in
solution by attaching a quencher fluorophore pair or two
fluorophores known to have FRET activity on the arms of the
construct adjacent to the angle. The distance between the FRET
pairs, and hence the distance between their attachment points on
the construct, can then be estimated by recording and analyzing
fluorescence signals from the construct in solution.
[0090] In the exemplary illustration of a targeting domain in the
sense of the present disclosure provided in FIG. 1, switching from
the unfolded conformation of FIG. 1, panel B to the folded
conformation of FIG. 1, panel C and vice versa is determined by
various factors such as presence or absence of the covalent linkage
length of the covalent linkage between the opposite ends of the
domain, operating temperature, the salt concentration, the
structure of the linkage between the opposite ends of the domain,
the presence in solution of molecules that can affect the structure
of the linkage, and additional factors identifiable by a skilled
person.
[0091] Reference is also made to the exemplary illustration of FIG.
2 in which different attachment of the targeting domain in a folded
conformation are shown. In some embodiments no extension of the 3'
of the guide strand beyond the two base overhang is included to
increase proper processing of the guide strand. In some embodiments
if the 3' of the guide strand is extended, a non-nucleic acid
linker can be comprised in particular when minimization of the
interference of the 3' overhang with processing of the guide strand
and its proper functioning in RISC is desired. In this connection
reference is made for example to Wang et al (2009) incorporated
herein by reference in its entirety.
[0092] Variations in the targeting domain and related molecular
construct shown in the illustration of FIG. 1 and FIG. 2 are
identifiable by a skilled person in view of the present disclosure.
For example, (1):(3) does not have to be the same length as
(2):(4), ii) separately, (1):(3) and 2:4 can exceed the length of
an RNA duplex of about 18 bp; iii) neither (1):(3) or (2):(4) can
be so short that the melting temperature of the duplex is
calculated to be less than the operating temperature of the
construct considering all modifications according to the present
disclosure; and v) in embodiments wherein the construct is to be
used in a cell the RNAi substrate overall can be no longer than 30
bp to prevent immunogenic toxicity.
[0093] In some embodiments, constructs herein described are signal
activatable construct that comprise a locking sensor configured for
providing different conformations upon binding of a signal molecule
to a suitable segment of the locking sensor through interrelation
of various segments of the locking sensor. In particular, in
several embodiments the locking sensor is configured to provide
different conformations following binding of the locking sensor to
a signal molecule that can be signal polynucleotide or another
molecule able to complementarily bind a suitable portion of the
locking sensor. The term "signal polynucleotide" as used herein
indicates a polynucleotide that is capable of acting as a signal
molecule for the signal activated constructs and related components
herein described. Accordingly, a signal polynucleotide herein
described is capable of triggering a switch between an inactive
conformation and an active conformation of the signal activated
molecular construct upon binding to a segment of the signal
activated construct.
[0094] The term "segment" as used herein indicates a portion of a
signal activated construct having chemical and/or biological
properties that are functional to changes in conformation of the
signal activated construct or components thereof, and/or to a
related ability to perform the enzyme assisted release herein
described.
[0095] In some embodiments the locking sensor comprises a toehold
segment, a displacement segment, and an activation segment. In
particular in those embodiments herein described each of an
activation segment, a toehold segment, and a displacement segment
comprises at least one polynucleotide portion configured so that i)
the toehold segment is complementary to a signal polynucleotide or
other suitable signal molecule; ii) the activation segment is
complementary to the displacement segment; iii) the displacement
segment is complementary to the signal polynucleotide.
[0096] Reference is made to the illustration of FIG. 3 to FIG. 8,
showing molecular complexes of the disclosure in which an exemplary
targeting domain (100) is locked in a folded conformation (FIG. 3,
panel A, 3, panel C, 4, panel A, 4, panel C, 5, panel A, 5, panel
C, 6, panel C, 6, panel C, 7, panel A, 7, panel C, 8, panel A, 8,
panel C) or presented in an unfolded conformation (FIG. 3, panel B,
3, panel D, 4, panel B, 4, panel D, 5, panel B, 5, panel D, 6,
panel B, 6, panel D, 7, panel B, 7, panel D, 8, panel B, 8, panel
D).
[0097] In the molecular complex of FIGS. 3 to 8 the locking sensor
(200) comprises a toehold segment (7), a displacement segment (6),
and an activation segment (5) complementary bound the displacement
segment (6). In the molecular complex of FIGS. 3 to 8 the locking
sensor (200) is covalently linked to the targeting domain (100)
through covalent linkage of first strand (60) and second strand
(50) to an end of the targeting domain (100) as illustrated in each
figure.
[0098] In the exemplary embodiments of FIGS. 2 and 3 the signal
activatable construct adopts thermodynamically stable inactive and
active conformations depending on binding presence of a signal
polynucleotide. In particular, the signal activatable construct
adopts an inactive conformation in absence of the signal molecule
(FIG. 3, panel A, 3, panel C, 4, panel A, 4, panel C, 5, panel A,
5, panel C, 6, panel A, 6, panel C, 7, panel A, 7, panel C, 8,
panel A, 8, panel C), and switch to an active conformation upon
binding of the signal molecule (FIG. 3, panel B, 3, panel D, 4,
panel B, 4, panel D, 5, panel B, 5, panel D, 6, panel B, 6, panel
D, 7, panel B, 7, panel D, 8, panel B, 8, panel D).
[0099] In the illustration of FIG. 3 to FIG. 8 the signal molecule
is provided by a signal polynucleotide (FIGS. 3 to 6 and FIG. 8)
and an aptamer (FIG. 7). Additional signal molecule can be applied
to the exemplary constructs of FIG. 3 to FIG. 8 as will be
understood by a skilled person. Suitable signal molecules and in
particular signal polynucleotides can be artificially synthesized
in or typically are already present in the environment wherein
activation of the construct is desired cytoplasm of cells and
analogous biochemical environments, such as a cell lysate.
Exemplary signal polynucleotides according to the present
disclosure include but are not limited to a synthetic
polynucleotide, RNA sequence present in cytoplasm or nuclei of
cells, such as mRNA, non-coding RNA, microRNA, microRNA precursors,
small interfering RNA, aptamers, tRNA, and by-products of abortive
RNA transcription, RNA splicing or RNA degradation. The signal
polynucleotide can be present in a free form or bound to RNA
binding proteins such as RISC. Additional exemplary signal
molecules comprise protein and small molecules as will be
understood by a skilled person.
[0100] In the illustration of FIG. 3 to FIG. 8, the inactive
conformation of the molecular complexes is converted into the
active conformation following binding of signal polynucleotide to
the toehold segment (7), and the displacement segment (6), to
displace the activation segment (5) which in the active
conformation is presented as a single stranded signal
polynucleotide for processing by ribonucleases such as an XRN1.
[0101] The term "displacement", "strand displacement reaction" or
"branch migration reaction" as used herein generally indicates the
process in which two polynucleotide strands with partially or full
complementarity hybridize, displacing in the process one or more
prehybridized strand or sequence. The strand displacement process
can be experimentally tested or measured according to techniques
herein described (see e.g. Examples 2 to 5) and identifiable by a
skilled person.
[0102] Accordingly, in embodiments exemplified by of FIG. 3 to FIG.
8 the activation segment of the locking sensor complementary binds
the displacement segment in an inactive conformation. In those
embodiments, complementary binding of a signal molecule which can
be a signal polynucleotide to the toehold segment result in an
activated conformation the displacement segment and the toehold
segment are displaced by the signal molecule. In particular when
signal molecule is a signal polynucleotide the signal
polynucleotide complementary binds the toehold segment and the
displacement segment to form a signal duplex displacing the
activation segment from the displacement segment.
[0103] In particular in some embodiments, in the locking sensor
herein described and related constructs, binding of the signal
molecule and in particular complementary binding between the signal
polynucleotide and the displacement segment is more
thermodynamically stable than complementary base paring between the
displacement segment and the activation segment, and complementary
binding between the displacement segment and the activation segment
is more thermodynamically stable than complementary base paring
between different portions of the activation segment.
[0104] In signal activatable constructs herein described, the
relative thermodynamic stability of the various segments of the
locking sensor is configured to trigger a switch from an inactive
conformation to an active conformation upon binding of a signal
molecule. Accordingly, switching from a conformation to another can
be controlled based on a comparison of the free energy of the
related systems. The term "free energy" as used herein is defined
to mean a thermodynamic quantity that can be used to determine the
spontaneity of a chemical reaction of transformation. Where the
chemical transformation is the conversion of one polynucleotide
conformation to another polynucleotide conformation, comparing the
free energies of the polynucleotide conformations can be used to
indicate which conformation will predominate. For example, free
energy can be used to estimate thermodynamic stability of
polynucleotide double-strand duplex and/or polynucleotide secondary
structure that is more thermodynamically stable, but it is not
limited to this use. Free energy can be estimated by computational
methods, among other means.
[0105] In several embodiments, the inactivated conformation of the
locking sensor or related signal activatable constructs, the
melting temperature of double-stranded duplex formed by the
activation segment and the displacement segment is at least about
25.degree. C. so that the double-stranded duplex formed by the
activation segment and the displacement segment is more
thermodynamically stable formed by different portions of the
activation segment, activation segment and toehold segment at room
temperature. This is to ensure that in the absence of the signal
molecule, the construct adopt the inactive conformation, with the
activation segment complementarily binds to the displacement
segment, rather than associating with the activation segment. The
strand melting temperature (Tm) of the double-stranded duplex
formed by the protection segment and the displacement segment can
be experimentally tested or measured (see e.g. Example 6 to 8).
Accordingly, the experiment to characterize the strand displacement
reaction as described in Example 8 can use a construct comprising
both the sensor domain and the targeting domain. In particular, the
fluorophore quencher pair can be placed at multiple positions along
the duplex formed by the displacement segment and the second
segment or the displacement segment and the protection segment to
allow assessment of strand displacement. Thermodynamic stability is
affected by various parameters such as composition of the specific
solution, pressure, temperatures as well as other conditions
identifiable by a skilled person.
[0106] In configurations of the activation segment, toehold segment
and displacement segment in an inactive conformation suitable to
transform to an active conformation in presence of the
complementary signal polynucleotide, are such that the binding of
the of the complementary signal polynucleotide to the toehold
segment and the displacement segment has a melting temperature (Tm)
of at least about 25.degree. C. In some of those embodiments,
sequence length and composition of toehold segment and displacement
segment is such that binding of the signal polynucleotide to the
toehold segment and displacement segment is at least as stable as
the binding between the activation segment and the displacement
segment to minimize partial displacement of the activation segment
from the displacement segment upon binding of the signal
polynucleotide.
[0107] For example. in embodiments exemplified by FIGS. 3-5 and 8,
the toehold segment and the signal polynucleotide can have at least
3 consecutive base pairs to initiate binding to the signal
polynucleotide and the strand displacement process, and the toehold
typically comprise be at least 4 consecutive base pairs to allow
functioning at the human body temperature of 37.degree. C.
Additionally, in some embodiments, sequences of the displacement
segment and activation segment can be configured with respect to
the complementarity of the displacement segment and signal
polynucleotide so that up to every base-pair exchange is at least
equal-energy, to minimize incomplete displacement process. For
example, according to some embodiments, if at certain position of
the duplex, the displacement segment and the activation segment
have a GC base-pair, then the signal polynucleotide can also have a
GC base pair with the displacement segment at the corresponding
position; if the displacement segment and the activation segment
have a 2'-O-methyl G base pairs with a C at certain position, also
the signal polynucleotide can base pair to the displacement segment
with a 2'-O-methyl G base pairs with a C. In some embodiments, the
complementary binding between the displacement segment with the
signal polynucleotide can be at least as stable, and possible more
stable, than the complementarily binding between the displacement
segment and the activation segment. Accordingly, mismatches between
the displacement segment and the activation segment at certain
position, can correspond to mismatches between the signal
polynucleotide and the displacement segment. In some embodiments
stabilizing modifications such as 2'-O-methyls can be localized in
the displacement segment, since that displacement segment of the
construct base pairs with both the signal polynucleotide and the
activation segment. In determining the configuration, length and
sequence the delivery conditions can also be considered (e.g.
temperature and salts concentrations).
[0108] In embodiments exemplified by the illustration of FIG. 7,
displacement is performed upon binding of a signal molecule other
than a signal polynucleotide (e.g. protein or small molecule). The
related stability during the design can be calculated by
determining the related thermodynamic free energy. In particular
the signal molecule can be selected so that the signal molecule
displacement-toehold segment complex has a lower free energy and
greater thermodynamic stability of the locking sensor RNA duplex
and therefore binds and displaces the duplex. In the illustration
of FIG. 7, the construct is designed so that the thermodynamic free
energy of binding for the construct where (6) and (7) are bound to
the ligand is lower than the thermodynamic free energy of binding
of (6) with (5). Determination of the free energy for a complex
where the signal molecule is a non-nucleic acid signal molecule can
be performed for example by measuring transition of the energy of
construct assembled with varying concentrations of the signal
molecule in a calorimeter, or by the techniques exemplified in
Examples 6 to 9 as will be understood by a skilled person.
[0109] The minimum length of the duplex is determined by stability
considerations. If the embodiment is designed for use at room
temperature, then the activation:displacement duplex should have a
minimum melting temperature of 25 degrees Celsius. If the
embodiment is designed for operation in a physiological
environment, then the duplex should have a minimum melting
temperature of 37 degrees Celsius.
[0110] The maximum allowable length for the activation:displacement
duplex is determined to avoid cellular toxicity and spurious
activation. The maximum allowable duplex length is 29 base-pairs to
avoid activation of PKR enzymes in the cell and spurious processing
by endonucleases of the RNA interference maturation pathway.
[0111] A good range of duplex lengths is 14 bp to 19 bps. This
confers sufficient stability with the incorporation of chemically
modified nucleotides without PKR activation and without spurious
processing.
[0112] Starting with an initial duplex length, a person skilled in
the art can experimentally test for thermodynamic stability,
nuclease resistance and PKR activation using live cells or cell
lysates via methods such as Northern blotting, immunoprecipitation,
or FRET assays. If the duplex is thermodynamically unstable in the
cellular environment, the duplex length should be increased. If
endonuclease cleavage of the duplex occurs, the duplex length
should be decreased. If PKR activation occurs, the duplex length
should be decreased.
[0113] In particular, in the inactive conformation of the molecular
construct according to the illustration of FIG. 3, an RNA strand
displacement activated sensor is shown wherein the complementary
binding of displacement segment (6) with activation segment (5)
forms a thermodynamically active duplex sensor (6):(5) that can be
7 to 18 bps in length, the toehold segment (7) can be 5 to 10 bp in
length, and the displacement segment (6) and the activation segment
(5) are sufficiently complementary to RNA activation signal for the
signal to displace 5. Displacement can be verified by those skilled
in the art using suitable techniques such as quencher-fluorophore
experiments.
[0114] In the illustration of FIG. 3, if the toehold segment (7) is
in a terminal loop configuration as shown in FIG. 3, panel C, the
loop should be larger than 4 bases with no upper limit. In the
illustration of FIG. 3, to allow formation of the active structure,
the length of the duplex formed by displacement segment (6) and
toehold segment (7) with the activation signal, should not exceed
the length of the targeting domain plus activation segment (5) and
unbound portions of toehold segment (7).
[0115] In the illustration of FIG. 4 the constructs of FIG. 3 are
shown in an illustration wherein unstructured region in
correspondence with the covalent linking of strand (60) and (50)
with the targeting domain is indicated by arrows. Introduction of
unstructured regions, such as the ones indicated in FIG. 4, is
functional to minimize PKR activation usually triggered by stacking
of duplexes and that in this particular case can be formed by the
targeting and sensor:signal stems can activate the PKR pathway,
leading to cellular toxicity. In particular, in the illustration of
FIG. 4, panel B the arrows indicate an exemplary schematic
representation of an exemplary unstructured regions that help
reduce likelihood of PKR activation, that can be placed between the
sensor and targeting regions. In the illustration of FIG. 4, panel
B bulges and mismatches can also be placed in the sensor:signal
duplex.
[0116] In the illustration of FIG. 4, panel D, the length of
segment (5) and the un-paired portion of segment (7) is controlled
to be short enough to enforce a bent conformation between the
targeting domain (100) and the locking sensor (200). In the
illustration of FIG. 4, panel D, the maximal length of an
unstructured RNA strand can be calculated as approximately 0.5 nm
per base, the length of a duplex RNA segment is approximately 0.3
nm per base-pair, the length of the unstructured region indicated
by the arrow in D should be shorter than the length of the sensor
duplex plus the length of the targeting duplex.
[0117] In several embodiments, modified bases can be used
throughout the constructs herein described to increase
thermodynamic stability, and nuclease resistance, decrease
toxicity, and/or increase specificity. Suitable modifications
comprise, for example, 2'-O-methyls, introduction of a non-nucleic
acid linker and/or an unstructured RNA segment, and terminal
modifications. In particular, 2'-O-methyls can be used in
particular in displacement segment (6) and toehold segment (7) to
increase thermodynamic stability and prevent unwinding by RNA
binding proteins. In addition, non-nucleic acid linkers can be used
confer desirable properties to the construct and/or portions
thereof. Exemplary non nucleic acid linkers suitable to be used
herein comprise C3 linkers and tri and hexa-ethylene glycol linkers
as well as any biocompatible polymeric linker group with
no-nonspecific association with DNA. In particular, molecular
constructs herein described can comprise A linker group with a
lower persistence length than nucleic acids (e.g.: C3, polyethylene
glycol) to increase flexibility at the attachment point. Such a
linker group can reduce interference of long overhangs against
Dicer binding. Molecular constructs herein described can also
comprise a non-nucleic acid linker group to interfere with
degradation by exonucleases and endonucleases, including RNAi
pathway enzymes. Molecular constructs herein described can further
comprise an unstructured RNA segment to have non-canonical
interactions with other RNA segments, leading to unpredictable
tertiary conformations. Molecular constructs herein described can
further comprise a terminal modification can prevent binding of the
PAZ domain of Dicer, as well as other terminal modifications useful
for preventing Dicer binding, such as Inverted dT Fluorescein and
other groups incompatible with the PAZ domain listed from last
patent.
[0118] In the illustration of FIG. 5, position of chemical
modifications is schematically illustrated. In particular, in the
illustration of FIG. 5 exemplary regions are shown where chemical
modifications of the activatable constructs herein described can be
introduced to obtain one or more of the above mentioned effects.
According to the illustration of FIG. 5 suitable regions comprise
the strands (50) and (60) linking the locking sensor to the
targeting domain (modification A and Modification B) and the
terminal portion of the toehold (7) lining the toehold (7) to the
displacement segment (6) (Modification C).
[0119] In the illustration of FIG. 6, an exemplary activatable
construct is shown including toehold having different structures.
In particular, in the illustration of FIG. 6, panel A and FIG. 6,
panel B, the sensor binding portion of the locking sensor is
switched to the 5' side of the passenger strand. in the
illustration of FIG. 6, panel C_and FIG. 6, panel D an extra
hairpin is added to the toehold increase the overall length of the
duplex region of the sensor binding strand, thus increasing the
specificity.
[0120] In the illustration of FIG. 7 an exemplary construct is
shown in which activation between the inactive conformation to the
active conformation of the construct can be performed by
non-nucleic acid RNA activation signal such as aptamers. In
particular, in the illustration of FIG. 7, panel A to FIG. 7, panel
C a schematic illustration is provide of strand displacement
reactions also work for aptamers in which, the activating RNA
strand is replaced by ligand which binds to the sensor. In the
illustration of FIG. 8 an exemplary construct is shown presenting
an alternate sensor geometry. In particular. In the designs
illustrated in FIGS. 3 to 7, the 5' and 3' ends of the passenger
strand are extended to form the strands (60) and (50) and then the
displacement segment (6) toehold segment (7) on one side and the
activation segment (5) on the other side of the locking sensor (see
FIGS. 3 to 7)
[0121] In the illustration of FIG. 8, the 5' end of the passenger
strand and the 5' end of the guide strand are extended to form the
sensor, the former constraints on the length of (1):(3) and (2)(4)
still apply; the geometric constraint on 5:6 is that when the
linkers connecting 5:6 to the targeting domain are fully stretched
(e.g. about 0.5 nm per nucleotide), the angle formed by 1-2 should
still be less than 90 degrees. A skilled person can identify the
constraints of this conformation based on trigonometry
calculations.
[0122] In several embodiments of the signal activatable constructs
herein described exemplified by the construct of FIGS. 3 to 8, in
absence of a signal polynucleotide, the activation segment and the
displacement segment form a first duplex through complementary
binding portions, wherein in the presence of the signal
polynucleotide, the displacement segment complementary binds the
signal polynucleotide and the activation segment is displaced and
presented for processing by XRN1. In particular, the XRN1 enzyme
can degrade the activation segment (5) presented in the active form
of the molecular construct allowing binding and processing of the
targeting domain by Dicer or other enzyme of the RNAai inactivation
pathway.
[0123] Various other configurations of the activatable constructs
herein described can be identified by a skilled person upon reading
of the present disclosure.
[0124] A schematic representation of an overall method to provide a
targeting domain and an activable construct herein described is
illustrated in FIG. 9. In particular the exemplary construct of
FIG. 9 inactivates in human cells as shown in Examples 2 and 5 and
has a folding with small energetic cost which results in good
thermodynamic stability for the folded state as shown in example 5.
In the illustration of FIG. 10, the construct of FIG. 9 is shown
with an indication of possible chemical modifications directed to
increase stability of the construct and activation efficiency
following binding of an RNA activating signal to the toehold
segment. In particular, 2'-O-methyl nucleotides in the indicated
positions increase thermodynamic stability and nuclease resistance
for the INACTIVE state and increase stability of toehold binding to
the signal polynucleotide. The 3' terminus of the signal binding
toehold has and inverted dT modification to increase exonucleases
resistance and prevent spurious binding to the PAZ domain of Dicer.
The C3 linker in the indicated position minimizes interference of
the 5' overhang from interfering with Dicer processing in the
ACTIVE state, as shown in example 4. The C3 linker, in conjunction
with adjacent 2'-O-methyl and phosphorothioate modifications on the
same strand, prevents 5' exonucleases degradation from proceeding
beyond the overhang into the targeting domain.
[0125] In the activation: displacement duplex, it is preferred that
thermodynamically stabilizing modifications are made to the side
that binds the signal polynucleotide. This ensures the
thermodynamic and kinetic favorability of binding to the correct
signal polynucleotide.
[0126] In the illustration of FIG. 3 to FIG. 8, upon processing of
the activation segment (5) in the active conformation of the
molecular construct, the targeting binding domain (100) is
presented in a conformation suitable to be processed by an enzyme
of the RNAai inactivation pathway. In particular, in the targeting
domain illustrated in FIGS. 3 to 8, processing is expected to be
performed by Dicer in combination with an argonaute enzyme of the
RNAi inactivation pathway. In variants where the targeting domain
has a different length (e.g. 19 to 22 bp) processing of a targeting
domain in an unfolded form and in particular within a construct in
active conformation according to the present disclosure can be
performed by one or more argonaute enzymes.
[0127] A schematic representation of an exemplary switching of a
construct herein described from an inactive to active form and
subsequent processing of the targeting domain by an RNAai
inactivation pathway enzyme is illustrated in FIG. 11. In
particular in the illustration of FIG. 11, a molecular construct is
provided in an inactive form together with a suitable signal
polynucleotide in the form of a viral RNA transcript (FIG. 11,
frame 1), upon binding of the viral RNA transcript to the toehold
of the construct the displacement segment is displaced from the
activation segment (FIG. 11, frame 2) to provide an activated
construct in which the toehold segment and the displacement segment
are complementary bound to the viral RNA transcript and the
targeting domain is released in an unfolded conformation (FIG. 11,
frame 3). In the construct in active conformation of FIG. 11, the
activation segment of the locking sensor is presented as a single
strand at the 5' terminus of the targeting domain for binding to a
XRN1 enzyme (FIG. 11, frame 4) which degrades the activation
segment up to the 5' end of the targeting domain (FIG. 11, frame 5)
thus providing a targeting domain in an unfolded conformation
suitable to be processed by Dicer or other suitable enzyme of the
RNAai inactivation pathway (FIG. 11, frame 6). Variations of the
method schematically illustrated in FIG. 11, will be identifiable
by a skilled person upon reading of the present disclosure.
[0128] For example, in addition to having different configuration
of the constructs, modifications can be performed to increase the
stability and/or the efficient processing of the activated
construct through RNAai activity. In particular additional process
steps to increase RNAi activity can comprise reduction of long 5'
and 3' overhangs near the PAZ binding domain of the RNAi substrate
(3' end of the Guide strand) inhibit Dicer processing as will be
understood by a skilled person. Additional suitable approaches to
improve RNAi activity on the targeting domain comprise: i) increase
the flexibility of the linker between the overhang and the RNAi
substrate by using a non-nucleic acid linker; ii) allowing an
exonuclease to degrade the overhang and using chemical
modifications to stop the exonuclease at a specific point; and/or
iv) creating an endonuclease domain (e.g., a RNAse H domain) to
allow clipping of the overhang by an endonuclease.
[0129] The illustration of FIGS. 12 to 15 show possible constructs
modified to increase efficiency of RNAai activity following switch
of an XRN1 based construct from an inactive conformation (OFF
conformation) to an active conformation (ON conformation). In
particular, FIG. 12 shows an XRN1 activated version with reduced
turn OFF. FIG. 13 shows an XRN1 activated version with turn OFF
improved using 2'-O-me modifications to stabilize sensor stem. FIG.
14 shows an XRN1 activated version with ON/OFF activity ratio
improved by adding features to reduce PKR recognition resulting in
a less stable duplex RNA.
[0130] In some embodiments, in the locking sensor herein described
the activation segment can comprise a DNA portion and an RNA
portion, the DNA portion of the activation segment complementary to
the RNA portion of the activation segment. In those embodiments,
when the displacement segment is displaced from the activation
segment the DNA portion of the activation segment complementarily
binds the RNA portion of the activation segment to provide an
RNAase H binding site presented for binding.
[0131] Reference is made to the illustration of FIG. 15 wherein an
exemplary embodiment of the RNAase H activated design is described.
In particular, in the illustration of FIG. 15, panel A the
activation segment is shown to comprise a DNA portion at the 5'
terminus configured in connection with the remaining portion of the
activation segment so that upon displacement of the displacement
segment, the such DNA portion complementary binds to RNA portions
of the activation segment forming a three-way activation junction
(see FIG. 15, panel B). This kind of junction can have a melting
temperature of at least about 15.degree. C. In particular, in some
embodiments, a three-way activation junction such as the one
illustrated in FIG. 15, panel B can comprise a DNA: RNA duplex of
at least 5 consecutive base pairs that is composed of unmodified
nucleotides. In embodiments in which the activatable constructs
herein described comprise a targeting domain that is activated by
RNAaseH based design, selection of the sequences is to be performed
so that in absence of the signal molecule, the complementary
binding between RNA portions of the activation segment and the
displacement segment is thermodynamically more stable of the
complementary binding of the RNA portion of the activation segment
with the DNA portion of the activation segment. Also the
configuration is such that upon binding of the signal molecule to
the toehold segment and consequent displacement of the displacement
segment from the activation segment, the RNA portion of the
activation segment complementary binds the DNA portion of the same
segment in a thermodynamically stable three way junction.
[0132] The melting temperature of the three-way activation junction
of an activated construct such as the one exemplified in FIG. 15,
panel B can be experimentally tested or measured using standard
methods after removing the displacement segment from the construct.
In this particular, embodiment, formation of the three-way
activation junction is associated to the correct placement of the
DNA:RNA duplex, and hence, positioning of the cleavage site of
RNAse H in the construct. Possible variations of this structure can
be envisioned by a skilled person in view of the present
disclosure. For example, phosphorothioate backbone modifications
can be applied to the DNA activation sequence to enhance DNA
stability without affecting RNAse H activity. The strand melting
temperature (Tm) of the activation junction can be experimentally
tested or measured (see e.g. Examples 6 to 8).
[0133] The illustration of FIG. 15 illustrates an exemplary sensor
locked siRNA design that utilizes RNAse H activation domain to
remove the 5' overhang. The guide strand is 29 nucleotides long
with 13 base pairs and 14 base-pairs complementary to the two
pieces of the passenger strand. On the left is the inactive domain.
The 3' and 5' extensions of the passenger strands form an 18 base
pair sensor duplex. The 3' sensor toehold is 5 nucleotides on.
[0134] In the targeting domain, the 5' of the guide strand is
modified with 2'-O-methyl bases to increase thermodynamic stability
and nuclease resistance. The rest of the guide strand is unmodified
to avoid interference with RISC functioning. The passenger side
contains interspersed 2'-O-methyl bases to increase thermodynamic
stability.
[0135] In the sensor, the side of the sensor which binds to the
activation signal is entirely 2'-O-methyl to increase nuclease
resistance, thermodynamic stability, and avoid destabilization by
RNA chaperone proteins. The 3' terminus of the sensor toehold has
an inverted dT modification to inhibit binding of Dicer to the
sensor stem. The sensor stem is also kept below 19 base pairs to
avoid Dicer processing. The 5' extension of the sensor stem is
responsible for formation of the RNAse H processing domain. In
addition to the DNA bases, the RNA bases in the 5' extension are
2'-O-methyl modified to increase nuclease resistance.
[0136] In the configuration of activatable constructs based on the
RNAase H activated design according to factors to considered
comprise: i) specific secondary and tertiary structure of the
construct; ii) thermodynamic and kinetic stability in the presence
of RBPs; iii) spurious processing; iv) PKR sensing; v) nuclear vs.
cytoplasmic trafficking; vi) nuclease degradation; v) signal
binding; vi) signal background; and/or vii) RNAi pathway processing
as will be understood by a skilled person
[0137] In the illustration of FIG. 16 an activation process for
RNAse H activated construct is shown In particular an inactive
construct is provided (Medusa in FIG. 16, panel A) which is then
contacted with a signal polynucleotide (arrow I of FIG. 16) for
signal binding and consequent displacement of the displacement
segment from the activation segment (FIG. 16, panel B) and
resulting switch of the construct from the inactive conformation to
the active form (arrow II of FIG. 16) which is then subjected to
reaction with RNAaseH (FIG. 16, panel B) for release of the
targeting domain of the construct in an active form (arrow III of
FIG. 16) for processing by Dicer or other enzyme of the RNAai
pathway (FIG. 16, panel D).
[0138] In the construct of FIG. 16, panel D as well as in other
constructs herein described (see e.g. FIGS. 3 to 10) in which the
active form presents an activated targeting domain for processing
with Dicer or other enzyme of the RNAai pathway, the activated
domain binds at the end opposite to the one presenting the
activated targeting domain, a signal duplex formed by the toehold
segment and the displacement segment and the signal polynucleotide.
In some of those embodiments, modifications of residues of the
displacement segment and/or activation segment can be performed to
increase efficiency of the RNAai processing of the activated
targeted domain as will be understood by a skilled person.
[0139] For example, the thermodynamic stability of toehold binding
to the activation signal can be increased via incorporation of
2'-O-methyl bases or locked nucleic acid (LNA) bases.
[0140] The exemplary illustrations of FIGS. 17 to 18 show possible
constructs modified to increase efficiency of RNAai activity
following switch of an RNAaseH based construct from an inactive
conformation to an active conformation. In particular, FIG. 17
shows a construct in which the nucleotides of the toehold segment
and displaced segment are 2-O-methyl ribonucleotides to increase
stability of the construct and minimize unwinding of the locking
sensor duplex RNA in absence of a signal complementary molecule.
FIG. 18 shows an additional construct where portions of the
displacement segments and the targeting domain comprise 2-O-methyl
modified ribonucleotides and an inverted dT can be incorporated at
the 3'-end of an oligo, leading to a 3'-3' linkage which inhibits
both degradation by 3' exonucleases and extension by DNA
polymerase. A further modification of the residues of the duplex is
introduction of phosphorothioate linkage to protect the oligo from
nuclease degradation as will be understood by a skilled person. In
the illustration of FIG. 18 a C3 linker is also included in the
target binding portion of the locking sensor to introduce
unstructured linker to minimize activation of PKR degradation.
[0141] In the construct of FIG. 18 the 5' extension of the
passenger strand can be degraded by XRN1 or another 5'
exoribonuclease, or Dicer can interact with the targeting domain to
process the guide strand without degradation of the 5' overhang
(see also construct of FIG. 10). To allow this interaction, a C3
linker is placed at the position joining the 5' overhang to the
passenger strand. In the ACTIVE state, the C3 linker serves two
purposes. First, in case of exoribonucleolytic degradation of the
5' overhang, the C3 linker, in conjunction with adjacent
2'-O-methyl modifications and phosphorothioate backbone
modifications, stops exoribonucleolytic processing. Second, the C3
linker gives extra flexibility prevent the 5' overhang from
interfering with the processing of the targeting domain by Dicer.
In the INACTIVE state, the C3 linker, along with the 2 un-paired
bases on the opposite side of the sensor stem, connect the sensor
stem to the targeting domain with sufficient slack to allow the
structure to form correctly.
[0142] FIG. 19 schematically illustrates exemplary PKR interactions
that can interfere with the stability and functionality of
activatable constructs herein described herein provided for
guidance purpose and are not intended to be limiting the scope of
the present disclosure. A first set of interactions comprises
exosome interactions in which the exosome binds onto the 3' tail
degrades strand A of the illustrated construct or stably binds
strand A of said construct (see FIG. 19, panel A). This type of
interactions are verifiable by a skilled person for example
degradation can be verified by Northern blot, and a strand A with
PEG spacer to block degradation can also be tested by Northern blot
as an additional verification. In particular in FIG. 19 panel A the
3' extension on sensor A extends the duplex just past the 30 bp
limit for PKR activation in FIG. 19, panel B additional hypothesis
for PKR is illustrated in which PKR binding excludes Dicer
processing thus interfering with the RNAai activity.
[0143] Accordingly, a cellular immunity sensor for duplex RNA can:
i) activate when two PKR proteins dimerize on minimal substrates of
28 to 30 bp; ii) be tolerant of mismatches; iii) Substrate needs to
be coaxial; iv) A single mismatch every 8 bp reduces activation; v)
activation can be suppressed by chemical modifications, but 2'-O-Me
has no such function.
[0144] FIG. 20 shows exemplary variations to the basic structure of
the targeting domain illustrated in its inactive conformation, and
in particular small interfering RNA (siRNA), dicer substrate small
interfering RNA (DsiRNA), synthetic miRNA analogues (miRNA). In
particular in the illustration of FIG. 19: the siRNA is 17b to 19
bp with symmetric 2 nt 3' overhangs and is therefore not processed
by Dicer (first panel); DsiRNA is equal to or longer than 19 bp to
allow Dicer processing equal to or lower than 30 bp to avoid
cellular toxicity and is processed by Dicer; the shRNA is similar
to DsiRNA but in a hairpin form and is processed by Dicer; the
miRNA is roughly 17 bp to 30 bp RNA hairpins; mismatches and
bulges; and is processed by Dicer or Dicer free pathways. Exemplary
sensor locked siRNA are shown in FIGS. 10 and 18.
[0145] Although only polynucleotide targeting domains are shown in
the illustration of FIGS. 1 to 20 and in other figures of the
present disclosure, in various embodiments of signal activatable
construct herein described a targeting domain can comprise a
molecule other than RNA or a polynucleotide configured to be
delivered to a target with the cells in the presence of the signal
polynucleotide. Exemplary types of cargo molecule that can be
comprised in all or in part as a targeting domain according to the
current disclosure include but are not limited to peptides, small,
molecules aptamers, antibodies, and other chemical compound
identifiable by a person skilled in the art.
[0146] In those embodiments, the targeting domain formed by the
cargo molecule or attaching the cargo molecule, can be carried and
delivered by constructs herein described wherein the segments of
the sensor domain are arranged in various configurations which
allow switching of the construct from an inactive conformation to
an active conformation with respect to the enzyme assisted release
of the targeting domain as will be understood by a skilled person
upon reading of the present disclosure. For example in embodiments,
wherein the targeting segment is configured for delivery of a cargo
molecule, the cargo molecule can be covalently linked to the 3'
terminus of the passenger strand or to the 5' of the guide strand
for targeting domain of 25 bp or longer. In those embodiments
wherein the cargo molecule comprises a cargo such as a
polynucleotide aptamer, the cargo molecule can be non-covalently
attached to the construct for example through complementarily
binding to the 5' terminus of the guide strand segment of the
targeting domain or other base pairing segment linked to the
displacement segment in a configuration that does not interfere
with the binding of the signal molecule and allows release of the
cargo with the displacement segment following RNAai processing of
the guide strand. In particular, in some of the embodiments wherein
a duplex formed between the cargo molecule and the passenger strand
segment of the targeting domain or other base pairing segment, the
duplex can have a melting temperature of at least 15.degree. C.
[0147] Signal activatable constructs and related components herein
described can be designed and manufactured based on techniques
described herein and/or identifiable by the skilled person upon
reading of the present disclosure. In particular the configuration
of the segments of the constructs can be identified and designed
based on calculation of the thermodynamic stability of the various
conformation of the segments and constructs as a whole. For
example, thermodynamic stability of polynucleotide conformation
dependents on several factors identifiable by a skilled person,
including its i) chemical composition (for example, DNA:RNA duplex
is less than RNA: RNA duplex); ii) base composition (for example,
G/C base paring is more stable than A/T base paring, which is
approximately as stable as G/T, G/U wobble base pairing, and the
formation of a stable RNA hairpin requires at least 3 G/C base
pairs or at least 5 A/U, G/U base pairs); iii) nearest neighbors
such as presence of mismatches, open ends, and junctions near a
base-pair can substantially influence its energy contribution
according to the second-nearest neighbor model (for example, the
stacking of successive base-pairs is primarily responsible for the
stability of DNA helices); iv) non-canonical base pairing (for
example, RNA and DNA can form triple helix and quadraplex
structures via Hoogsteen base-pairing, which is less stable base
pairings than canonical base pairing); v) Geometry (e.g.
polynucleotide sequences can only adopt secondary structures that
are geometrically consistent or similar with the known tertiary
structural characteristics of RNA and DNA helices); vi)
Environmental factors, such as pH value, counter-ion concentration
and temperature and additional factors identifiable by a skilled
person.
[0148] Accordingly, designing the polynucleotide sequences
comprised in the signal activatable construct can be performed
identifying the combination of length, sequence, complementarity
and substitutions that is associated with a desired relative
thermodynamic stability resulting in the configuration herein
described and the environment wherein the enzyme assisted molecular
delivery is desired. For example, in several embodiments, in
absence of a signal polynucleotide, an inactive conformation of the
signal activatable construct typically has approximately 3 extra
G/C base pairs or 5 extra A/U or G/U base pairs as compared to the
activated conformation formed in presence of the signal
polynucleotide. Specific sequences of desired signal
polynucleotides can be identified by a skilled person based on
environment (and in particular, specific cells and tissues) where
delivery is desired. Also, the number of complementary base pairs
between the protection segment and displacement segment is
typically more than that between the protection segment and the
activation segment. For applications where molecular delivery in
cells is desired, polynucleotide sequences can be designed
according to the corresponding physiological conditions, such as
approximately, pH 7.3-7.4, about 150 millimolar potassium or sodium
chloride or equivalent salt, and about 37.degree. C.
[0149] For base pairing between unmodified DNA segments or between
unmodified RNA segments, the base-pairing energies and the most
stable secondary structure conformations can be estimated by
computational methods known to and well established in the art.
Several packages are available and published in documents also
discussing in detail factors affecting the energy and stability of
nucleic acid secondary structures. Exemplary publications
describing the packages and factore comprise for i) NUPACK web
server: J. N. Zadeh, et al., (2011); ii) NUPACK analysis
algorithms: R. M. Dirks et al., (2007); R. M. Dirks et al., (2003);
R. M. Dirks et al., (2004); iii) NUPACK design algorithms: J. N.
Zadeh et al., (2011); iv) mfold web server: M. Zuker, (2003); A.
Waugh et al., (2002); M. Zuker et al., (1998); v) UNAFold &
mfold: N. R. Markham et al., (2008); M. Zuker, et al., (1999); M.
Zuker, (1994); J. A. Jaeger et al., (1990); M. Zuker, (1989); vi)
Free energies for RNA: D. H. Mathews et al., (1999); A. E. Walter
et al., (1994); vii) Methods and theory of RNA secondary structure
prediction: D. H. Mathews et al., (2007); D. H. Mathews et al.,
(2006); D. H. Mathews et al. 3.sup.rd edition, John Wiley &
Sons, New York, Chapter 7, (2005); D. H. Mathews et al., (2004); M.
Zuker, (1984); M. Zuker et al., (1981) D. H Mathews et al (2010);
viii) Exemplary mfold & UNAFold applications: J.-M. Rouillard
et al., (2003); J.-M. Rouillard, et al., (2002). In addition, since
some polynucleotide structures typically fluctuate between an
ensemble of secondary structure conformations, the composition of
the relevant ensemble can be determined using computational methods
known in the art (see for example, see Ye Ding et al., (2005),
herein incorporated by reference in its entirety).
[0150] Accordingly, in several embodiments, design of a
polynucleotide sequence of the sensor domain of the signal
activatable construct herein described, can be performed for
sequences or portions of sequences consisting of unmodified DNA
and/or RNA base pairs, by computational methods and/or software
packages to calculate the free energy of the sequence and the
secondary structure conformation. In embodiments, wherein
polynucleotide sequences comprise derivatives of nucleotides, such
as chemically modified bases and analogues, and/or chimeric
polynucleotide sequences composed of a mixture of
deoxyribonucleotides and ribonucleotides, design can be performed
by computationally designing unmodified RNA structures with the
desired secondary structure conformations and thermodynamic
stability, and then introducing one or more chemical modifications
to achieve the desired thermodynamic stability. Exemplary chemical
modifications comprise replacement of nucleotides that are needed
to be base-paired to form a desired secondary structure with
modified nucleotides that are known to increase thermodynamic
stability (e.g. 2'-O-methyl modified nucleotides, LNA, PNA and
Morpholino). Additional exemplary modifications comprise
replacement of nucleotides that are not desired according to a
certain thermodynamic stability with modified nucleotides to ensure
that the resulting modified structures are likely to retain the
desired secondary structure conformations and thermodynamic
stability (e.g. replace a ribonucleotide base with a
deoxyribonucleic base). A person skilled in the art will be able to
identify other suitable modifications upon reading of the current
disclosure.
[0151] The signal activatable construct designed according the
present disclosure can be synthesized using standard methods for
oligonucleotide synthesis well establish in the art, for example,
see Piet Herdewijn, (2005), herein incorporated by reference in its
entirety.
[0152] The synthesized oligonucleotide can be allowed to form its
secondary structure under a desirable physiological condition,
(e.g. 1.times. phosphate buffered saline at pH 7.5 with 1 mmolar
concentration MgCl.sub.2 at 37.degree. C.). The formed secondary
structure can be tested using standard methods known in the art
such as chemical mapping or NMR. For example, see Stephen Neidle,
(2008), herein incorporate by reference in its entirety. The
designed construct can be further modified, according to the test
result, by introducing or removing chemical modifications,
mismatches, wobble pairings, as necessary, until the desired
structure is obtained.
[0153] In some embodiments, in presence of a signal polynucleotide,
the free energy of the construct in an activated conformation is at
least about 5 kcal/mol lower than that of the construct in an
inactive conformation.
[0154] In some embodiment, the free energy of complementary
base-paring between the protection segment and the displacement
segment is at least about 10 kcal/mol lower that the free energy of
complementary base-paring between the DNA activation sequence and
the RNA activation substrate.
[0155] In some embodiment, the targeting domain comprises a first
segment and a second segment, wherein the first segment and the
second segment form a polynucleotide duplex through complementarily
binding with each other; and the 3' terminus of the second segment
is adjacently connected with the protection segment of the sensor
domain both segments.
[0156] In some embodiments, the guide strand, passenger strand,
activation segment, displacement segment and toehold segment of the
signal activatable construct are mainly composed of RNA and/or RNA
derivatives.
[0157] The term "derivative" as used herein with reference to a
first compound (e.g. RNA or ribonucleotide) indicates a second
compound that is structurally related to the first compound and is
derivable from the first compound by a modification that introduces
a feature that is not present in the first compound while retaining
functional properties of the first compound. Accordingly, a
derivative of a molecule of RNA, usually differs from the original
molecule by modification of the chemical formula that might or
might not be associated with an additional function not present in
the original molecule. A derivative molecule of RNA retains however
one or more functional activities that are herein described in
connection with complementary base paring with other nucleotides.
Typically, ribonucleotides and deoxyribonucleotides can be modified
at the 2', 5', or 3' positions or the phosphate backbone chemistry
is replaced. Exemplary chemical modifications of a ribonucleotide
according to the current disclosure include 2'-o-methyl RNA,
2'-Fluoro RNA, locked nucleic acid (LNA), peptide nucleic acid
(PNA), morpholino, phosphorothioate oligonucleotides, and the like
that are identifiable by a skilled person (see e.g. "Modified
Nucleosides: in Biochemistry, Biotechnology and Medicine. Piet
herdewijn (Editor), Wiley-VCH, 2008, herein incorporated by
reference in its entirety). Also applicable are nucleosides which
are not normally comprised in DNA and RNA polynucleotides, such as
inosine. In some embodiments, a single oligonucleotide can be
composed of more than one type of the above derivatives.
[0158] In particular, according to several embodiments herein
described, the guide strand and passenger strand of the targeting
domain comprise unmodified ribonucleotides. In other embodiments,
the guide strand and passenger strand of the targeting domain can
comprise modified ribonucleotides, such as 2'-O-methyl
modification, 2'-fluoro modification, 2'-amino modification or LNA;
the exposed 5' terminus of the passenger strand can have
modifications configured to minimize processing by the XRN1. For
example, 5' terminus of the passenger strand can have at least 1,
and in particular 2 2-O-methyl ribonucleotide. Similarly the 3'
terminus of the guide strand can have modifications configured to
block processing by the endonucleases enzyme Dicer. For example, 3'
terminus of the first segment can have at least 1, and in
particular 2 deoxyribonucleotides. In some embodiments, the
protection segment can comprises unmodified ribonucleotides and/or
some modified ribonucleotides, such as 2'-O-methyl modification,
2'-fluoro modification, 2'-amino modification or LNA. In
particular, in some embodiments, the two nucleotides immediately
flanking the desired RNAse H cleavage site within the RNA
activation sequence can be formed by unmodified
ribonucleotides.
[0159] In some embodiments, the activation segment comprises a DNA
activation sequence formed by unmodified deoxyribonucleotides. In
particular in some of these embodiments the construct is an RNAaseH
based construct.
[0160] In some embodiments, the displacement segment and the
toehold segment can comprise modified ribonucleotides or
derivatives, such as 2'-O-methyl modification, 2'-fluoro
modification, 2'-Amino modification or LNA; the exposed terminus of
the toehold segment can also have modifications configured to block
processing by endonucleases enzyme Dicer. For example, the exposed
terminus of the toehold segment can comprise at least one, and in
particular two, phosphorothioate deoxyribonucleotides.
[0161] In several embodiments, the toehold segment can comprise a
polynucleotide sequence (herein also toehold sequence) that is at
least 3 nucleotides in length and is fully complementary to at
least a portion of the signal polynucleotide. This configuration of
the toehold segment is expected to allow binding of a signal
polynucleotide to bind to the signal activatable construct and
initiate the branch migration process. A smaller toehold sequence
is expected to result in better sequence specificity for signal
discrimination, while a longer toehold sequence is expected to
result in an increased ability to bind to the signal
polynucleotides to form a desired secondary structure with respect
to the ability of a shorter toehold segment. In some embodiments,
the toehold segment can be arranged in single-stranded form and
free of secondary structure. In particular, in some of those
embodiments, the toehold sequence can be 4 to 12 nucleotides in
length. In some embodiments, the toehold segment is composed of
unmodified ribonucleotide. In particular, in other embodiments, the
toehold segment comprises modified nucleotide configured for
improved nuclease resistance. Exemplary modifications include but
are not limited to 2'-O-methyl modification, 2'-Fluoro
modifications, inclusions of LNA and PNA, and the like that are
identifiable by a skilled person.
[0162] In some embodiments, the signal can be a single signal
polynucleotide of a length shorter than 30 nucleotides, the toehold
segment and the displacement segment is fully complementary to the
signal polynucleotide. In other embodiments, the signal can be
formed by multiple homologous signal polynucleotides. In these
embodiments, the signal polynucleotides can be tested with a sensor
design. Mismatches and wobble pairings or permissive bases such as
inosine can be placed at positions in the 3:5 duplex corresponding
to the variable sequences. In particular, in several embodiments,
the Tm for the duplex formed by the signal polynucleotides with the
toehold segment and the displacement segment is typically at least
25.degree. C. and is typically at least equal to the operating
temperature under which the construct will be used. In some
embodiments, the 3' terminus of the sensor domain can have Dicer
blocking groups which are identifiable by a skilled person.
[0163] In some embodiments, where the toehold segment is arranged
as or within a single-stranded loop (see exemplary embodiments in
FIGS. 4, panel C, FIG. 5, panel C and FIG. 7, panel C), the loop
can be sufficiently large to avoid topological constraints that
present a kinetic barrier to displacement of the activation segment
from binding to the displacement segment by the signal
polynucleotide. To test whether the loop is as large as desired,
the strand displacement process of the construct can be tested
using the methods such as the one described in Example 8. Further,
in some embodiments the signal polynucleotide used in the
experiment, can be selected to approximate the expected state of
the signal in the cell. In particular, in embodiments wherein the
signal polynucleotide is expected to be a short oligonucleotide or
RNA segment, such as a miRNA, a short oligonucleotide of the same
sequence as the signal polynucleotide can be used in experiments to
simulate the topological constraints imposed by having the toehold
segment in a hairpin loop. In embodiments wherein the signal is an
mRNA sequence, a polynucleotide having the same sequence as the
mRNA as the signal nucleotide can be used to simulate the
topological constraints imposed by having the toehold segment in a
hairpin loop. In embodiments wherein the region known to bind to
the toehold segment is in a hairpin loop, the signal nucleotide
used in the displacement experiment can have the toehold sequence
in a hairpin loop to simulate the topological constraints imposed
by having the toehold segment in a hairpin loop.
[0164] In embodiments wherein strand displacement does not occur,
the size of the hairpin loop can be increased to decrease the
topological constraint by increasing the loop size. For example, in
some embodiments, the size can be increased using an unstructured
polynucleotide or polymer linker between the toehold segment and
the other segments (e.g. either between the toehold segment and the
activation segment or, usually less favorably, between the toehold
segment and the displacement segment). In particular in various
embodiments, the loop can have at least about 20 unstructured
nucleotides.
[0165] Single stranded regions in the hairpin loop and in other
areas can be protected by chemical modifications if not conflicting
with other design objectives. 2'-O-methyl, 2'-fluoro, LNA, 2'-amino
and other modified RNA nucleosides can replace RNA.
Phosphorothioate deoxyribonucleotides can replace unmodified
deoxyribonucleotides for RNAseH segment.
[0166] In some embodiment, wherein the locking sensor comprises
more than one polynucleotide the melting temperature of the duplex
formed by the displacement segment and the activation segment is at
least 5.degree. C. above the expected operating temperature under
which the construct is used, (e.g. 37.degree. C. for the use in
human cells) in order to prevent spurious activation.
[0167] In some embodiments, the toehold segment can be connected to
the displacement segment through covalent linkage. In particular,
in some embodiments, the toehold segment can be arranged to the 3'
terminus of the displacement segment (see exemplary embodiments in
FIGS. 3 to 5). In some embodiments, the toehold segment can be
arranged as a single strand terminal sequence of the sensor domain;
in other embodiments, the toehold segment can be provided as a
single strand middle sequence of the sensor domain, which can be
arranged within a loop structure of the sensor domain. In
particular, in some embodiments, where the toehold domain can be
arranged within a loop structure of the sensor domain, the loop can
comprise at least 20 nucleotide unmodified nucleotides, which in
some cases can be ribonucleotides. In some embodiments, the toehold
segment can be at least 3 nucleotides in length. In particular, in
some embodiments, the toehold segment can be at least 4 nucleotides
in length.
[0168] In some embodiments, the activation segment can be kept to
the minimum length necessary for efficient formation of an
activation junction to kinetically minimize spurious activation
usually associated with binding of a large terminal loop in the
sensor domain to a partially deprotected activation site as a
result of partial displacement of (5) by a partially complementary
polynucleotide that is not the intended signal polynucleotide.
Accordingly, the activation segment can be at least 5 nucleotides
in length, and in particular less than 10 nucleotides in length.
Additional lengths of the loop can be identified by a skilled
person taking into account that the possibility of having
complementary binding of a strand to the loop that result in
displacement of the displacement segment from the activation
segment in view of a desired experimental design.
[0169] Alternatively, in some embodiments, the toehold segment
links the 3' terminus of the activation segment and the 5' terminus
of the displacement segment, and is arranged as a loop between the
activation segment and the displacement segment, (see e.g. FIGS. 3,
4, 5 and 7. In particular, FIG. 3, panel C shows a schematic
illustration of the signal activatable construct in an inactive
conformation, where the toehold segment (segment 7) is arranged at
the 3' terminus of protection segment (segment 6) and the 5'
terminus of the displacement segment (segment 5), and is located in
the loop of a stem-loop structure formed by the activation segment
and the displacement segment. FIG. 3, panel D shows schematic
illustration of an activated conformation of the signal activatable
construct according to the embodiment as shown in FIG. 3, panel C.
In this activated conformation, portions (1) and (3) form a
double-stranded duplex through base paring, portions (2), (4) form
a double-stranded base pairing, and together they form the
activated targeting domain.
[0170] In some embodiments, in absence of a signal polynucleotide,
the displacement segment and the protection segment form a
double-stranded duplex. In particular, the double-stranded duplex
formed by the displacement segment and the protection segment can
have up to 30 consecutive base pairs, if the duplex comprises only
unmodified ribonucleotides. In other embodiments, the
double-stranded duplex formed by the displacement segment and the
protection segment can be longer than 30 base pairs, if the duplex
comprises mismatches and/or modified ribonucleotides. In
particular, mismatches and/or modifications are expected to
contribute to preventing activation of innate immune system and/or
increase stability. Exemplary modifications to the first and the
second segments include but are not limited to 2'-O-methylation,
2'-Fluoro modifications, 2'-amino modifications, and inclusion of
LNA or PNA nucleotides. In particular 2'-O-methylation can be used
to passivate against innate immune activation. In some embodiments,
the displacement segment is at least 12 nucleotides in length. In
some embodiments, the displacement segment can be at least 14
nucleotides in length.
[0171] In some embodiments, the construct is configured to minimize
immune responses. In these embodiments, each consecutive 30 base
pairs duplex can have at least 5% 2'-O-methyl modifications
(Molecular Therapy (2006) 13, 494-505, herein incorporated by
reference in its entirety) or one or two mismatches. In other
embodiments, the construct is configured to stimulate immune
responses. In these embodiments, the construct can comprises at
least one consecutive 30 base-pair duplex with no 2'-O-methyl
modifications when the construct is in the activated conformation.
For example, the total length of the toehold segment and the
displacement segment can be at least 30 nucleotides without
2'-O-methyl modifications, and will be perfectly base paired with
the signal polynucleotide sequence.
[0172] In some embodiments an activated conformation of the
activatable construct herein described or related component (e.g.
locking sensor), a DNA portion comprised in the activation segment
(herein also DNA activation sequence or portion) binds to an RNA
portion comprised in the protection segment (herein RNA activation
sequence) through complementary base paring to form a RNAase H
binding site.
[0173] In human cells, RNAse H commonly cleaves the RNA sequence of
a DNA:RNA duplex at a position that is 5 nucleotides from the 5'
end of the RNA sequence forming the duplex. If the duplex is longer
than 7 base pairs, RNAse H can cleave at additional positions to
the 3' of the first cleavage site. Accordingly, the DNA:RNA duplex
formed in the activated conformation according to the current
disclosure can be at least 5 nucleotides, and in particular 7-8
nucleotides. In particular, in some embodiments, the DNA activation
sequence is no longer than 10 nucleotides. In particular, in
several embodiments, an RNAase H cleavage site comprises a DNA: RNA
duplex of at least 5 consecutive base pairs, in particular, the
DNA: RNA duplex has 7 consecutive base pairs. In some embodiments,
cleavage rate is expected to increase if 8 or more consecutive base
pairs are present in the duplex, but there will be multiple
cleavage sites. Higher Tm of the DNA:RNA duplex is expected to
generally improve cleavage efficiency. In some embodiments, Tm can
be greater than or equal to the expected operating temperature. For
example, when working at room temperature, Tm can be about
25.degree. C. or more. In another example when operating in human
cells, Tm can be 37.degree. C. or more. In particular, Tm can be
not lower than about 15.degree. C. In the DNA:RNA duplex,
deoxyribonucleotides can be replaced with phosphorothioate
deoxyribonucleotides. The nucleotides flanking the DNA activation
sequence in the activation segment can be unmodified
ribonucleotides to keep the highest RNAase cleavage efficiency.
Alternatively, flanking nucleotides can also be modified
ribonucleotides, such as 2'-O-methyl ribonucleotides, 2'-Fluoro
ribonucleotides, or LNA.
[0174] FIG. 16 shows the products of RNAse H cleavage of the
activated signal activatable construct according to the embodiment
shown in FIG. 16. A double-stranded RNA molecule is released from a
remanent. The released double-stranded RNA molecule is bound to the
signal molecule on one terminus and has at least 2-base
single-stranded overhang at the 3' of the toher terminus and
therefore can be used as a siRNA or a suitable substrate for Dicer.
Other exemplary embodiments can be also found in FIGS. 15 and
17.
[0175] In some embodiments, the sensor domain is configured to
avoid immune activation in the cell, wherein the sensor domain
forms a double strand duplex with the signal polynucleotide of no
longer than about 30 bp. In other embodiments, the sensor domain is
configured to induce immune activation in the cell, wherein the
sensor domain forms a double strand duplex with the signal
polynucleotide of longer than about 30 bp.
[0176] In some embodiments where the double-stranded duplex formed
by the displacement segment and the protection segment is longer
than 16 base pairs, and in particular, the exposed 3' terminus of
the double-stranded duplex comprises modifications configured to
block processing of Dicer.
[0177] In some embodiments, the guide strand is configured to
interfere with a target intracellular process of the cells through
RNAi in presence of the signal polynucleotide. Accordingly suitable
targeting domain include siRNA, microRNA and additional duplex
structure suitable to be used in connection with RNA
interfering.
[0178] The term "RNA interfering" or "RNAi" as used herein refers
to a mechanism or pathway of living cells that controls level of
gene expression that has been found in many eukaryotes, including
animals. The RNAi pathway has many important roles, including but
not limited to defending cells against parasitic genes such as
viral and transposon genes, directing development and regulating
gene expression in general. The enzyme Dicer, which is an
endoribonuclease in the RNAse III family, initiates the RNAi
pathyway by cleaving double-stranded RNA (dsRNA) molecules into
short fragments of dsRNAs about 20-25 nucleotides in length. Dicer
contains two RNase III domains and one PAZ domain; the distance
between these two regions of the molecule is determined by the
length and angle of the connector helix and determines the length
of the siRNAs it produces. Dicer cleaves with the highest
efficiency dsRNA substrates 21 bp and longer with a two-base
overhang at the 3' end.
[0179] The small fragments of dsRNAs produced by Dicer are known as
small interfering RNA (siRNA). The term "small interfering RNA" or
"siRNA", sometimes also known as short interfering RNA or silencing
RNA, refers to a class of dsRNA molecules which is typically 20-25
nucleotides in length and plays a variety of roles in biology. The
most notable role of siRNA is its involvement in the RNAi pathway.
In addition to its role in the RNAi pathway, siRNA also acts in
RNAi-related pathways, including but not limited to several
antiviral pathways and shaping chromatin structure of a genome.
[0180] Each siRNA is unwound into two single-stranded (ss) ssRNAs,
namely the passenger strand and the guide strand. The passenger
strand is degraded, while the guide strand is incorporated into a
multiprotein complex, known as the RNA-induced silencing complex
(RICS). RICS uses the incorporated ssRNA as a template for
recognizing a target messenger RNA (mRNA) molecule that has
complementary sequence to the ssRNA. Upon binding to the target
mRNA, the catalytic component of RICS, Argonaute, is activated,
which is an endonuclease that degrades the bound mRNA molecule.
[0181] Similar to siRNAs, microRNAs (miRNAs) also mediate the RNAi
pathway. The term "microRNA" or "miRNA" as used herein indicates a
class of short RNA molecules of about 22 nucleotides in length,
which are found in most eukaryotic cells. miRNAs are generally
known as post-transcriptional regulators that bind to complementary
sequences on target mRNA transcripts, usually resulting in
translational repression and gene silencing.
[0182] miRNAs are encoded by miRNA genes and are initially
transcribed into primary miRNAs (pri-miRNA), which can be hundreds
or thousands of nucleotides in length and contain from one to six
miRNA precursors in hairpin loop structures. These hairpin loop
structures are composed of about 70 nucleotides each, and can be
further processed to become precursor-miRNAs (pre-miRNA) having a
hairpin-loop structure and a two-base overhang at its 3' end.
[0183] In the cytoplasm, the pre-miRNA hairpin is cleaved by the
RNase III enzyme Dicer. Dicer interacts with the 3' end of the
hairpin and cuts away the loop joining the 3' and 5' arms, yielding
an imperfect miRNA:miRNA duplex about 22 nucleotides in length.
Overall hairpin length and loop size influence the efficiency of
Dicer processing, and the imperfect nature of the miRNA:miRNA base
pairing also affects cleavage. Although either strand of the duplex
can potentially act as a functional miRNA, only one strand is
usually incorporated into RICS where the miRNA and its mRNA target
interact.
[0184] In those embodiments, wherein the guide strand is configured
for interfering a target intracellular process through RNAi, the
double-stranded duplex typically formed by the guide strand and
passenger strands can have a melting temperature (Tm) of at least
about 25.degree. C. In particular, the 5' terminal nucleotide of
the guide strand can be base paired to one of the passenger
strands. In some embodiments, nicked double-stranded duplex formed
by the guide strand and passenger strands are stable under
conditions of the environment where delivery will be performed. In
embodiments where RNAi is performed in mammals the nicked
double-stranded duplex typically formed by the guide strand and
passenger strand can have a melting temperature (Tm) of at least
about 37.degree. C.
[0185] In some embodiments, a double-stranded polynucleotide duplex
with a 3' overhang of 2 nucleotides in length is most efficiently
bound by the PAZ domain of the endonucleases enzyme Dicer (Jin-Biao
Ma, et al, 2004). In human cells, RNAse H commonly cleaves the RNA
sequence of a DNA:RNA duplex at a position that is 5 nucleotides
from the 5' end of the RNA sequence forming the duplex. If the
duplex is longer than 7 base pairs, RNAse H can cleave at
additional positions to the 3' of the first cleavage site.
Accordingly, in embodiments using an RNAse H substrate, the DNA:RNA
duplex formed in the activated conformation according to the
current disclosure is at least 5 nucleotides, and in particular 7-8
nucleotides.
[0186] In those embodiments where the targeting domain is
configured to interfere with a target intracellular process of the
cells through RNAi, the first segment and the second segment are at
least 16 nucleotides in length. In particular, in some embodiments,
they are no short than 22 nucleotides. In particular, in some
embodiments, the second segment is at least 2 nucleotides longer
than the first segment. Accordingly, in some embodiment, the
double-stranded duplex formed by the first segment and second
segment has a 2-base single strand overhang at the 3' terminus of
the second segment.
[0187] In particular, in some embodiments, the double-stranded
duplex formed by the first segment and the second segment are no
longer than 30 consecutive base pairs, if the duplex comprises only
unmodified ribonucleotides. In other embodiments, the
double-stranded duplex formed by the first segment and the second
segment can be longer than 30 base pairs, if the duplex comprises
mismatches and/or modified ribonucleotides. The mismatches and/or
modifications are likely to prevent activation of innate immune
system. Exemplary modifications to the first and the second
segments include but are not limited to 2'-O-methylation, 2'-Fluoro
modifications, 2'-amino modifications, and inclusion of LNA or PNA
nucleotides. In particular, 2'-O-methyl, 2'Fluoro, 2'amino, LNA and
PNA are expected to improve stability of the structure.
[0188] Further, in these embodiments, at least one at least one
strand of the duplex is configured for interfering a target
intracellular process through RNAi. In some embodiments, the at
least one strand is at least partially complementary to a target
gene sequence for silencing that gene through RNAi. In other
embodiments, the at least one strand is at least partially
complementary to a common sequence shared by multiple genes or
members of a gene family. In other embodiments, the at least one
strand is configured to be incorporated into a protein complex to
activate the complex and/or the substrate of the complex or to
initiate a cascade of activation of downstream effectors of the
complex. In some embodiments, from 2 to 8 bases of the at least one
strand incorporated into RISC is complementary with a target gene
forming a "seed region" usually considered particularly important
for RNAi activity as will be understood by a skilled person.
[0189] According to several embodiments, the duplex formed by the
guide strand and the passenger strand has a blunt end at the 3' end
of the guide strand. The duplex formed by the first segment and the
second segment is at least 21 bp long. In particular, the first 21
nucleotide from the 3' terminus of the guide strand is configured
for interfering a target intracellular process through RNAi, and
the 21.sup.st and 22.sup.nd 5' terminus of the first segment and
from the 3' terminus of the second segment are unmodified RNA
nucleotides so as to allow efficient Dicer processing after signal
activation of the signal activatable construct.
[0190] In other embodiments, the 3' terminal region of segments
other than the guide strand comprises modifications to inhibit RNAi
loading pathway enzyme processing from the 3' terminus of the first
segment. In particular, in some embodiments, the last at least 1
base at the 3' terminal region of the first segment is a DNA
modified DNA base. In particular, the last 2 nucleotides at the 3'
terminal region of the first segment is a DNA modified DNA base. In
other embodiments, the 3' terminal region of segment 1 is
chemically modified. Exemplary modifications includes but are not
limited to 3'-O-propanediol modifications, 3'-O-fluorescin
modifications, 3'-puromycin modifications, 3'-inverted dT
modifications, inverted Dideoxy-T modifications and the like that
are identifiable by a skilled person in the art.
[0191] In some embodiments, the double-stranded duplex formed by
the activation and displacement segments have additional
modifications at the 3' terminus of segment 7 and/or the 5'
terminus of segment 5 to further prevent processing of the
inactivate construct by RNAi loading pathway enzymes, such as
Dicer. In some embodiments, (see FIG. 6, panel C) the 3' terminus
of segment 7 has additional secondary structures, such as a
terminal polynucleotide hairpin with 4-15 bp long stem. In some
embodiments, the 3' terminus of segment 7 is connected with a
synthetic polynucleotide structure, such as a DNA or RNA
multi-crossover tile, a DNA or RNA origami, a DNA or RNA crystal,
and other structures identifiable by a person skilled in the
art.
[0192] In particular, in some embodiments, at least one of the
passenger segments and the guide segment comprises a sequence
homologous to an endogenous microRNA sequence. More particularly,
in some embodiment, the first segment and the second segment have
the exact same sequence and structure as a known or predicted
mammalian pre-miRNA. In some embodiments, at least one of the t
passenger segments and the guide segment has the same sequence as a
known or predicted mammalian miRNA. In some embodiments, the
double-stranded duplex formed by the passenger and guide segments
comprises mismatches and/or bulges configured to mimic a known or
predicted mammalian miRNA. In some embodiments at least one of the
passenger segments or guide segment is homologous to the sequence
of a known or predicted mammalian miRNA. The term "homologous" or
"homology" used herein with respect to biomolecule sequences as
indicates sequence similarity between at least two sequences. In
particular, according to the current disclosure, a homologous
sequence of a mammalian miRNA can have the same sequence located at
base position 2-7 from the 5' terminus of the guide strand of the
miRNA.
[0193] In some embodiments, the targeting domain is configured to
deliver a cargo molecule other than a polynucleotide in the
presence of the signal polynucleotide. In these embodiments, the
targeting domain can also comprise a double-stranded polynucleotide
duplex as part of the cargo. Reference is made to the constructs
illustrated in
[0194] The term "aptamers" as used here indicates oligonucleic acid
or peptide molecules that bind a specific target. In particular,
nucleic acid aptamers can comprise, for example, nucleic acid
species that have been engineered through repeated rounds of in
vitro selection or equivalently, SELEX (systematic evolution of
ligands by exponential enrichment) to bind to various molecular
targets such as small molecules, proteins, nucleic acids, and even
cells, tissues and organisms. Aptamers are useful in
biotechnological and therapeutic applications as they offer
molecular recognition properties that rival that of the antibodies.
Peptide aptamers are peptides that are designed to specifically
bind to and interfere with protein-protein interactions inside
cells. In particular, peptide aptamers can be derived, for example,
according to a selection strategy that is derived from the yeast
two-hybrid (Y2H) system. In particular, according to this strategy,
a variable peptide aptamer loop attached to a transcription factor
binding domain is screened against the target protein attached to a
transcription factor activating domain. In vivo binding of the
peptide aptamer to its target via this selection strategy is
detected as expression of a downstream yeast marker gene.
[0195] The term "small molecule" as used herein indicates an
organic compound that is of synthetic or biological origin and
that, although might include monomers and/or primary metabolites,
is not a polymer. In particular, small molecules can comprise
molecules that are not protein or nucleic acids, which play a
biological role that is endogenous (e.g. inhibition or activation
of a target) or exogenous (e.g. cell signaling), which are used as
a tool in molecular biology, or which are suitable as drugs in
medicine. Small molecules can also have no relationship to natural
biological molecules. Typically, small molecules have a molar mass
lower than 1 kgmol.sup.-1. Exemplary small molecules include
secondary metabolites (such as actinomicyn-D), certain antiviral
drugs (such as amantadine and rimantadine), teratogens and
carcinogens (such as phorbol 12-myristate 13-acetate), natural
products (such as penicillin, morphine and paclitaxel) and
additional molecules identifiable by a skilled person upon reading
of the present disclosure.
[0196] The terms "peptide" and "oligopeptide" usually indicate a
polypeptide with less than 50 amino acid monomers, wherein the term
"polypeptide" as used herein indicates an organic linear, circular,
or branched polymer composed of two or more amino acid monomers
and/or analogs thereof. The term "polypeptide" includes amino acid
polymers of any length including full length proteins and peptides,
as well as analogs and fragments thereof. As used herein the term
"amino acid", "amino acidic monomer", or "amino acid residue"
refers to any of the twenty naturally occurring amino acids,
non-natural amino acids, and artificial amino acids and includes
both D an L optical isomers. In particular, non-natural amino acids
include D-stereoisomers of naturally occurring amino acids (these
including useful ligand building blocks because they are not
susceptible to enzymatic degradation). The term "artificial amino
acids" indicate molecules that can be readily coupled together
using standard amino acid coupling chemistry, but with molecular
structures that do not resemble the naturally occurring amino
acids. The term "amino acid analog" refers to an amino acid in
which one or more individual atoms have been replaced, either with
a different atom, isotope, or with a different functional group but
is otherwise identical to original amino acid from which the analog
is derived.
[0197] In an embodiment, a targeting domain can be attached to a
locking sensor herein described with methods and approaches
identifiable by a skilled person. In particular, attachment can be
performed at a portion of the protection domain configured for
binding the targeting domain (e.g. presenting a suitable functional
group) and presented for binding in the sensor domain. The term
"attach" or "attached" as used herein, refers to connecting or
uniting by a bond, link, force or tie in order to keep two or more
components together, which encompasses either direct or indirect
attachment where, for example, a first molecule is directly bound
to a second molecule or material, or one or more intermediate
molecules are disposed between the first molecule and the second
molecule or material. The term "present" as used herein with
reference to a compound or functional group indicates attachment
performed to maintain the chemical reactivity of the compound or
functional group as attached. Accordingly, a functional group
presented on a segment, is able to perform under the appropriate
conditions the one or more chemical reactions that chemically
characterize the functional group. Exemplary target binding portion
herein described comprise a monomer presented in the 5' terminus of
the protection domain. A skilled person will be able to identify
additional suitable portions, including intermediate compound or
functional groups used to covalently attach the targeting domain
with the protection domain at any suitable portion. In particular
the target binding portion of the protection segment and the
activation domain are typically attached of the RNA portion of the
protection segment.
[0198] In some embodiments, a system for intracellular information
processing and controlling of cells is described. The system
comprising two or more signal activatable constructs as described
for simultaneous combined or sequential use in the cells, in which
the targeting domain of at least one construct of the two or more
constructs is configured to release a second signal in the presence
of the signal polynucleotide, and the second signal is configured
to activate one or more construct of the two or more
constructs.
[0199] In some embodiments, one or more signal activatable
constructs and/or component thereof including sensor domains can be
used in a method for XRN1 or RNAse H assisted signal activated
molecular delivery in cells. The method comprises delivering to the
cells an effective amount of one or more of the signal activatable
construct described herein possibly preceded by contacting the
sensor domain with a suitable targeting domain to provide the
construct.
[0200] In some embodiments, RNA and DNA nanostructures herein
described can allow specific biomolecules to trigger specific
changes in their secondary, tertiary and quaternary structure.
These characteristics are comprised in several embodiments of
activatable constructs herein described as will be understood by
the skilled person to develop novel switching mechanisms that work
with endogenous nucleases to activate or release therapeutic
cargo.
[0201] In one embodiment, a sensor gated siRNA can be provided with
selectively activated RNAi activity in cells expressing a specific
RNA sequence. The activating sequence switches ON the siRNA by
binding to its sensor domain and triggering internal conformational
changes that induce processing by endogenous RNAse H or XRN1. The
result is an active Dicer substrate that can direct targeted
RNAi.
[0202] As disclosed herein, the signal activated constructs and
related components herein described can be provided as a part of
systems for enzyme assisted molecule delivery, including any of the
deliveries described herein. The systems can be provided in the
form of kits of parts. In a kit of parts, the signal activated
constructs and related components and other reagents to perform
enzyme-assisted delivery can be comprised in the kit independently.
The signal activated constructs and related components can be
included in one or more compositions, and each construct or
component can be in a composition together with a suitable
vehicle.
[0203] Additional components can include labeled molecules and in
particular, labeled polynucleotides, labeled antibodies, labels,
microfluidic chip, reference standards, and additional components
identifiable by a skilled person upon reading of the present
disclosure. The terms "label" and "labeled molecule" as used herein
as a component of a complex or molecule referring to a molecule
capable of detection, including but not limited to radioactive
isotopes, fluorophores, chemiluminescent dyes, chromophores,
enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors,
dyes, metal ions, nanoparticles, metal sols, ligands (such as
biotin, avidin, streptavidin or haptens) and the like. The term
"fluorophore" refers to a substance or a portion thereof which is
capable of exhibiting fluorescence in a detectable image. As a
consequence, the wording "labeling signal" as used herein indicates
the signal emitted from the label that allows detection of the
label, including but not limited to radioactivity, fluorescence,
chemiluminescence, production of a compound in outcome of an
enzymatic reaction and the like.
[0204] In some embodiments, detection of molecule delivery can be
carried either via fluorescent based readouts, in which the labeled
antibody is labeled with fluorophore, which includes, but not
exhaustively, small molecular dyes, protein chromophores, quantum
dots, and gold nanoparticles. Additional techniques are
identifiable by a skilled person upon reading of the present
disclosure and will not be further discussed in detail.
[0205] In particular, the components of the kit can be provided,
with suitable instructions and other necessary reagents, in order
to perform the methods here described. The kit will normally
contain the compositions in separate containers. Instructions, for
example written or audio instructions, on paper or electronic
support such as tapes or CD-ROMs, for carrying out the assay, will
usually be included in the kit. The kit can also contain, depending
on the particular method used, other packaged reagents and
materials (i.e. wash buffers and the like).
[0206] In some embodiments, one or more signal activated constructs
and/or related components, (e.g. sensor domain) herein described
are comprised in a composition together with a suitable vehicle.
The term "vehicle" as used herein indicates any of various media
acting usually as solvents, carriers, binders or diluents for
signal activated constructs and related components that are
comprised in the composition as an active ingredient. In
particular, the composition including the signal activated
constructs and related components can be used in one of the methods
or systems herein described.
[0207] In some embodiments, a composition for XRN1 and/or RNAse H
assisted signal activated molecular delivery in can comprise one or
more of the signal activatable construct as described together with
a suitable vehicle. In some embodiments, the vehicle is suitable
for delivering the signal activatable construct to cells. Exemplary
suitable vehicles according to the current disclosure include but
are not limited to nanoparticle, such as cyclodextrin, gold
nanoparticle and dendrimer; liposome and liposome analogues;
conjugated aptamer; conjugated antibody; conjugated cell
penetrating peptide or peptide analogue; carbon nanotubes;
conjugated fatty acids and quantum dots.
[0208] In some embodiments, the signal activated constructs and
related components herein described are comprised in pharmaceutical
compositions together with an excipient or diluent.
[0209] The term "excipient" as used herein indicates an inactive
substance used as a carrier for the active ingredients of a
medication. Suitable excipients for the pharmaceutical compositions
herein described include any substance that enhances the ability of
the body of an individual to absorb the signal activated constructs
and related components herein described or combinations thereof.
Suitable excipients also include any substance that can be used to
bulk up formulations with the peptides or combinations thereof, to
allow for convenient and accurate dosage. In addition to their use
in the single-dosage quantity, excipients can be used in the
manufacturing process to aid in the handling of the peptides or
combinations thereof concerned. Depending on the route of
administration, and form of medication, different excipients can be
used. Exemplary excipients include, but are not limited to,
antiadherents, binders, coatings, disintegrants, fillers, flavors
(such as sweeteners) and colors, glidants, lubricants,
preservatives, sorbents.
[0210] The term "diluent" as used herein indicates a diluting agent
which is issued to dilute or carry an active ingredient of a
composition. Suitable diluents include any substance that can
decrease the viscosity of a medicinal preparation.
[0211] In particular, in some embodiments, disclosed are
pharmaceutical compositions which contain at least one signal
activated constructs and related components as herein described, in
combination with one or more compatible and pharmaceutically
acceptable vehicles, and in particular with pharmaceutically
acceptable diluents or excipients. In those pharmaceutical
compositions the signal activated constructs and related components
can be administered as an active ingredient for treatment or
prevention of a condition in an individual.
[0212] The term "treatment" as used herein indicates any activity
that is part of a medical care for, or deals with, a condition,
medically or surgically.
[0213] The term "prevention" as used herein indicates any activity
which reduces the burden of mortality or morbidity from a condition
in an individual. This takes place at primary, secondary and
tertiary prevention levels, wherein: a) primary prevention avoids
the development of a disease; b) secondary prevention activities
are aimed at early disease treatment, thereby increasing
opportunities for interventions to prevent progression of the
disease and emergence of symptoms; and c) tertiary prevention
reduces the negative impact of an already established disease by
restoring function and reducing disease-related complications.
[0214] The term "condition" as used herein indicates a physical
status of the body of an individual (as a whole or as one or more
of its parts), that does not conform to a standard physical status
associated with a state of complete physical, mental and social
well-being for the individual. Conditions herein described include
but are not limited disorders and diseases wherein the term
"disorder" indicates a condition of the living individual that is
associated to a functional abnormality of the body or of any of its
parts, and the term "disease" indicates a condition of the living
individual that impairs normal functioning of the body or of any of
its parts and is typically manifested by distinguishing signs and
symptoms.
[0215] The wording "associated to" as used herein with reference to
two items indicates a relation between the two items such that the
occurrence of a first item is accompanied by the occurrence of the
second item, which includes but is not limited to a cause-effect
relation and sign/symptoms-disease relation.
[0216] The term "individual" as used herein in the context of
treatment includes a single biological organism, including but not
limited to, animals and in particular higher animals and in
particular vertebrates such as mammals and in particular human
beings.
[0217] For example in some embodiments, a multi-stage therapeutic
nanoparticles can be provided that utilize XRN1 and/or RNAaseH
activated release of a cargo in a cell to achieve controlled
step-wise disassembly and cargo release in target environment such
as solid tumor microenvironments.
[0218] A skilled person will be able to identify further
application and in particular therapeutic applications as well as
cargo molecules to be used as active agents in the treatment and
design a corresponding signal activatable construct to be
administered according to the features of the construct and the
desired effect. In particular, in applications wherein signal
activatable construct is desired system administration of the agent
can be performed. In embodiments, where an activated construct is
instead used, topical administration to the specific target cell
and tissue can be performed.
[0219] Further advantages and characteristics of the present
disclosure will become more apparent hereinafter from the following
detailed disclosure by way or illustration only with reference to
an experimental section.
EXAMPLES
[0220] The synthesized signal activatable constructs herein
disclosed are further illustrated in the following examples, which
are provided by way of illustration and are not intended to be
limiting.
[0221] The following material and methods were used in the
experiments illustrated in the following examples.
[0222] Northern blot analysis: HCT116 cells were transfected using
with the indicated Medusa complexes or controls at a final
concentration of 1 nMolar with pBluescript (pBS) as carrier, using
Lipofectamine2000 according to the manufacturer's (Invitrogen)
protocol. The cell medium (American Type Culture Collection,
recommended formulation) was replaced at 18 hours
post-transfection. Total cell RNA was harvested using RNASTAT60
(amsbio) according to the manufacturer's instructions, except for
the addition of a 1:1 phenol:chloroform pH 6.7 extraction prior to
precipitation. For Northern analysis, 15 ug of total RNA in
1.times. formamide loading buffer was run on a 10% urea/PAGE gel,
using .sup.32P-end-labeled Ambion Decade size markers. The RNA was
transferred to Hybond XL (Amersham) using the BioRad TransBlot SD
(semi-dry) cell. Transferred RNA was cross-linked to the membrane
using the UV Stratalinker 2400 (Stratagene) preset conditions.
Membranes were prehybridized 6-10 hours at 37 degrees Celsius with
Perfecthyb Plus (Sigma) and hybridized in the same buffer overnight
at 37 degrees Celsius with 5-10 pmoles of .sup.32P-end-labeled
oligo probes as shown. After 2.times.SSC/1% SDS washes at 37
Celsius, the membranes were exposed using Kodak BioMax film and
intensifying screens at -80 C and developed according to the
manufacturer's instructions.
[0223] Transfections for Luciferase analyses: HCT116 cells were
transfected using with the indicated exemplary activatable
complexes or controls at a final concentration of 1 nMolar with
pBluescript (pBS) as carrier, using Lipofectamine2000 according to
the manufacturer's (Invitrogen) protocol. The cell medium was
replaced at 18 hours post-transfection. One day before
transfection, cells were seeded in growth medium without
antibiotics so that cells would reach 90-95% confluency at the time
of transfection (as recommended by Invitrogen protocols). Each well
was transfected with a final DNA mix consisting of: 40 nanograms
(ng) psiCHECK (Promega) plasmid bearing a Firefly luciferase (Fluc)
control reporter and a Renilla luciferase (Fluc) reporter with the
target in the 3' UTR; 120 ng pBluescript carrier DNA; and an amount
of Medusa complex 10.times. with respect to the final concentration
indicated in the specific experiment in a 20 ul final volume (
1/10th the final) of OptiMEM (Invitrogen). An equal volume of a
1/50 dilution of Lipofectamine2000 in OptiMEM was added (bringing
the volume to 1/5th the final) and incubated according to the
manufacturer's instructions. The liposome/DNA complexes were added,
along with fresh complete medium to the cells to give a final
volume of 200 ul. Total cell RNA was harvested using RNASTAT60
(amsbio) according to the manufacturer's instructions, except for
the addition of a 1:1 phenol:chloroform pH 6.7 extraction prior to
precipitation. To reduce sample to sample variability, the psiCHECK
target mix was made in batch and aliquoted to allow 3 technical
replicates (wells) for each condition. The Medusa complexes were
diluted to the appropriate concentration in OptiMEM with the
Lipofectamine2000 dilution (also made in batch). Two complete sets
of triplicates were run per experiment: one using and psiCHECK
vector without the U5 siRNA target (as a check for non-target
specific knockdown) and a second using the psiCHECK with the U5
siRNA target in the Renilla luciferase 3' UTR. After transactions,
samples were collected for luciferase analysis.
Example 1
Exemplary Activatable Constructs
[0224] Exemplary molecular constructs were provided having the
features summarized in Tables 1 and 2 below
TABLE-US-00001 TABLE 1 RNA Complexes and Component Strands.
Complexes SEQ ID Abbreviation Sequences NO: G Ac Bc G mC mU mU mG C
G U C U G A G G G A U C U 1 C U A G U U A C C U U Ac C C U C A G A
C G C A A G idT 2 Bc G G U A A C U A G A G A U C 3 G RP G mC mU mU
mG C G U C U G A G G G A U C U 1 C U A G U U A C C U U U RP C C U C
A G A C G C A A G G G U A A C mU 4 A mG A mG A U mC G mC mU mU mG C
G U C U G A G G G A U C U 1 C U A G U U A C C U U U A1 C C U C A G
A C G C A A G mC mU mG mA mU 5 mG mA mG mC mU mC mU mU mC mG mU mC
mG mC mU mG mU mU idT B1 mA mA mG mG mU dC dC dC dT dG dA dT C G A
6 C G A A G A G C U C A U C A G G G U A A C U A G A G A U C G mC mU
mU mG C G U C U G A G G G A U C U C 1 U A G U U A C C U U A1 C C U
C A G A C G C A A G mC mU mG mA mU 5 mG mA mG mC mU mC mU mU mC mG
mU mC mG mC mU mG mU mU idT B4 mA mA mG mG mU dC dC dC dT dG dA dT
C G 7 A C G A A G A G C U C A U C A G G G U A A C mU Am G A mG A U
mC G A1 B6b G mC mU mU mG C G U C U G A G G G A U C U C 1 U A G U U
A C C U U A1 C C U C A G A C G C A A G mC mU mG mA mU 5 mG mA mG mC
mU mC mU mU mC mG mU mC mG mC mU mG mU mU idT B6b C G A C G A A G A
G C U C A U C - C3 - 8,9 .sup..sctn. mG * mG * mU A A C mU Am GA mG
A U mC G2 A3 B7 G2 mC mG C G U C U G A G G G A U C U C U A 10 G U U
A C C U U A3 C C C U C A G A C G mC * mG * - 18S - G A 11,12, mU G
mA G - - mC mU U C mG mU C G - 9S - 13 .sup..sctn. G U mC U mC mC G
mC 9S idT B7 C G A C G A A G C U C A U C A - C3 - 14,15 .sup..sctn.
mG * mG * mU A A C U mA G A mG A mU tat activator strand S mA mA mA
mA mA G C G G A G A C A G C G A 16 C G A A G A G C U C A U C A G mA
mA mA mA mA idT G Ac Bc G mC mU mU mG C G U C U G A G G G A U C U 1
C U A G U U A C C U U Ac C C U C A G A C G C A A G idT 2 Bc G G U A
A C U A G A G A U C 3 GRP G mC mU mU mG C G U C U G A G G G A U C U
C 1 U A G U U A C C U U U RP C C U C A G A C G C A A G G G U A A C
mU 4 A mG A mG AU mC G mC mU mU mG C G U C U G A G G G A U C U C 4
U A G U U A C C U U U A1 C C U C A G A C G C A A G mC mU mG mA mU 1
mG mA mG mC mU mC mU mU mC mG mU mC mG mC mU mG mU mU idT B1 mA mA
mG mG mU dC dC dC dT dG dA dT C G A 5 C G A A G A G C U C A U C A G
G G U A A C U A G A G A U C G mC mU mU mG C G U C U G A G G G A U C
U 6 C U A G U U A C C U U A1 C C U C A G A C G C A A G mC mU mG mA
mU 1 mG mA mG mC mU mC mU mU mC mG mU mC mG mC mU mG mU mU idT B4
mA mA mG mG mU dC dC dC dT dG dA dT C G 7 A C G A A G A G C U C A U
C A G G G U A A C mU Am G A mG A U mC G A1 B6b G mC mU mU mG C G U
C U G A G G G A U C U C 1 U A G U U A C C U U A1 C C U C A G A C G
C A A G mC mU mG mA mU mG mA mG mC mU mC mU mU mC mG mU mC mG mC 5
mU mG mU mU idT B6b C G A C G A A G A G C U C A U C - C3 - mG * mG
* mU A A C mU A mG A mG A U mC 8,9 G2 A3 B7 G2 mC mG C G U C U G A
G G G A U C U C U A G 10 U U A C C U U A3 C C C U C A G A C G mC *
mG * - 18S -G A 11,12, mU G mA G - - mC mU U C mG mU C G - 9S - 13
.sup..sctn. G U mC U mC mC G mC 9S idT B7 C G A C G A A G C U C A U
C A - C3 - mG * 14,15 mG * mU A A C U mA G A mG A mU tat activator
strand S mA mA mA mA mA G C G G A G A C A G C G A 16 C G A A G A G
C U C A U C A G mA mA mA mA mA idT .sup..sctn. Sequences connected
by a linker as indicated in the Sequences column
[0225] In particular, Table 1 indicates for each exemplary
molecular construct the specific sequences of the strands that are
complementary bound to provide the molecular constructs. herein
described. The corresponding configuration is illustrated in FIGS.
21-32 each identifying the construct by the corresponding
abbreviation.
[0226] Additional features of the complexes listed in table 1 are
summarized in Table 2
TABLE-US-00002 TABLE 2 Complexes and component strand features
Complex Abbrevi- SEQ ID Name ation # nt Description Sequence 5'
-> 3' Notes NO: Medusa G or G1 29 U5K2 targeting mC mU mU mG C 1
guide 29 mer guide strand G_U C U G A G G for the medusa G A U C U
C U A G U U A C C U U Medusa A or A1 37 Sensor A C C U C A G A C G
5 sensor A version 1 for C A A G mC mU binds to medusa mG mA mU mG
act strand mA mG mC mU mC mU mU mC mG mU mC mG mC mU mG mU mU idT
Medusa B1 44 Sensor B mA mA mG mG over- 6 sensor B version 1 for mU
dC dC dC dT lapping with RNAseH medusa dG dA dT C G A C 586, 585
activation G A A G A G C U C and senB A U C A G G G U A LNA A C U A
G A G A U homo- C logies Medusa Ac 14 Truncated C C U C A G A C G 2
passenger A sensor A C A A G idT control strand for medusa control,
3' end of passenger (together with Bc, homologous to guide) Medusa
Bc 14 Truncated G G U A A C U A G 3 passenger B sensor B A G A U C
control strand for medusa control, homologous to 3' end of guide;
with Ac homologous to entireity of guide Tat 28 base S 39 Tat/Rev
signal mA mA mA mA mA over- 16 act strand activator, 28 G C G G A G
A C A lapping_ nt to fit G C G A C G A A G 179,585 extended A G C U
C A U C A homologies toehold G mA mA mA mA mA idT Medu sen B B4 44
Sensor B with mA mA mG mG over- 7 V4 7 base DNA mU dC dC dC dT
lapping region shifted dG dA dT C G A C 586,585 from 2 and 3 G A AG
AG C U C and senB and extra 2'-O- A U C A G G G U A LNA me to
stabilize A C mU A mG A homologies act stem. mG A U mC RNAseH-
activated, e.g in Ac Bv4 G Medusa RP 27 Ac and Bc C C U C A G A C G
4 reverse linked in C A A G G G U A A linked reverse C mU A mG A mG
passenger topology (3' of A U mC strand Ac is linked to 5' of Bc)
Medusa B B6a 32 Sensor B, C G A C G A A G A over- 17,18 .sup..sctn.
v6a 18 bp XRN1 5' G C U C A U C A G lapping sensor removal, 18 bp -
C3 - mG * mG * 585 and sensor mU A A C mU A senB mG A mG A U mC LNA
homologies Medusa B B6b 30 Sensor B, CGACGAAGA over- 8,9
.sup..sctn. v6b 16 bp XRN1 5' G C U C A U C: lapping sensor
removal, 16 bp C3 - mG * mG * 585 and sensor mU A A C mU A senB mG
A mG A U mC LNA homologies Medusa B Bc6 14 Bc, but with C3 mG * mG
* mU 19 v6 control terminal C3 A A C mU A mG A and mG A U mC
phosphorothio ates, and 2'-O- me to mimic truncated Bv6 products
Medusa H1 H1 67 G A C A G C G A C Clasp, 20,21, G A A G G C G A C
5' end 22,23 .sup..sctn. G G C C3 *mG * homologous mG * U A A C U A
to middle G A G A UC 18S of 179 18S C C U C A G (shown), A C G C A
*mA the 3 5' *mG 18S G C C G end of U C G C A G C U C 585, and A U
C A G idT 3' end of J1 (-6to- 18, inclusive) and 3' end homologous
to bases J1 5', 1-10 Medusa Av2 Av2 36 C C U C A G A C G 24,25
.sup..sctn. C A *mA *mG 18S G A U G A G C U C U U C G U C G C U G U
C U C Medusa G2 G2 27 non- mC mG C G U C U 14 methylated G A G G G
A U C U sequence C U A G U U A C C same as G; G U U had 5' mC mU mU
mG Medusa J2 J2 27 ' 9S mC U mG A U 26,27, mG A 9S mG mC U 28 mC U
mU C mG U 18S_mC G mC U mG U mC U mC mC 18S idT Medusa A3 A3 35
CCCUCAGAC CUGAU 11,12, G mC * mG * 18S GAGCU 13 .sup..sctn. (in G A
mU G mA G CUUCG sequence -- mC mU U C UCGCU 5'->3' mG mU C G_9S
G GUCUC column), U mC U mC mC G CGC 29 (in mC 9S idT -- Notes GAUGA
column) G- CUUCG UCG- GUCUC CGC Bubbled to interrupt continuo us
helix in portion indicate d with double dash Medusa B7 B7 28 C G A
C G A A G C over- U C A U C A C3 lapping 586, mG * mG * mU A A 585
and 14,15 ' C U mA G A mG A senB mU LNA homologies The wording 18S,
9S reported in bold fonts indicates PEG linkers. .sup..sctn. The
sequences are connected by a linker as indicated in the sequence
5'->3' column
[0227] The probes used for detection of the construct are listed in
Table 3 below.
TABLE-US-00003 TABLE 3 Probes for medusa constructs and related
segments Abbrev- SEQ Detected SEQ Name iation Description Sequence
ID NO: sequence ID NO: Sensor A LNA CTT.sub.+GC.sub.+GTCT 30 CCUCAG
31 LNA probe for GAGG ACGCAA Probe Medusa G sensor A Sensor B LNA
probe GA.sub.+TC.sub.+TCTAG 32 GGUAAC 33 LNA for Medusa T.sub.+TACC
UAGAGA Probe sensor B UC RH3'TP1 541 3' toe CAGACTTTGTT 34 UUUCAA
35 passenger GGATTTGAAA AUCCAA strand CAAAGU probe CUG RH3'GP6.31
542 3' Toe CTTCAAGCCA 36 CAAAUC 37 Guide GACTTTGTTGG CAACAA Probe
6-31 ATTTG AUCUGG CUUGAA G RH3'GP51.72 543 3' Toe ACAGCGACGA 38
CUGAUG 39 Guide probe AGAGCTCATC UGCUCU 51-72 AG UCGUCG CUGU
U5K2GPrb 544 Probe for GGTAACTAGA 40 (TCT)GA 41 HIV-1 U5K2
GATCCCTCAG GGGAUC shRNA A UCUAGU guide UACC strand. cf 564 Medi Sen
584 Probe for 3' ACAGCGACGA 42 CUGAUG 43 A3'Pr end of AGAGCTCATC
AGCUCU Medusa 1 AG UCGUCG sensor A CUGU toehold region. Medi Sen
585 Probe for CCCTGATGAG 44 CGACGA 45 BStPr sensor stem CTCTTCGTCG
AGAGCU of Medusa 1 CAUCAG RNAseH GG activation strand (sensor B).
Med1Sen 586 Probe for 5' CTTCGTCGATC 46 AAGGAC 47 B5'Pr end of
AGGGTCCTT CCUGAU Medusa 1 CGACGA RNAseH AG activation strand
(sensor B).
Example 2
Stability of an Exemplary Activatable Construct
[0228] The G A1B4 construct was assembled by combining all three
component strands at 1:1:1 and annealing per standard experimental
procedures. The construct was then transfected at 1 nM
concentration in to HCT 116 cells for 24 hours. After 24 hours, the
cells were lysed and the RNA extracted for Northern Blot. Different
probes are used to prove for the presence of the Guide (left), A1
(middle), and B4 (right) strands.
[0229] The results illustrated in FIG. 29 show the stability of the
component strands of the locked siRNA constructs in human HCT116
cells. In particular, the results of these experiments show that
each strand is present, with little detectable signs of
degradation. This shows that each component strand of the G A1 B4
sensor locked siRNA is stable in human HCT116 cells for at least 24
hours.
Example 3
Confirmation of RNAai Processing of the Guide Strand in Exemplary
Complexes by Luciferase Analysis
[0230] To test successful release and processing of the guide
strand from a targeting domain in an activated conformation of the
exemplary molecular complexes of Example 1 Applicants performed
dual Luciferase assays whose results are illustrated in FIG.
30.
[0231] In particular FIG. 30 shows functioning of the different
implementations of the complexes in dual luciferase assays. In
these dual Luciferase assays the ratio of Renilla Luciferase to
Firefly Luciferase luminosity is compared to a negative control. A
value of 1.0 signifies undetectable RNAi activity. A value of 0.0
constitutes perfect RNAi activity, meaning there is zero activity
from the Renilla luciferase target of RNAi knockdown. In FIG. 30
panel D, a series of positive controls are used to show activity of
the targeting domains.
[0232] Medusa control, also known as G Ac Bc (SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO:3), is shown to have very good RNAi knockdown at
all levels. G Ac B6c (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
19) is a variant of Medusa control with 2'-O-methyl modifications,
phosphoriothioate modifications and a C3 linker. It simulates the
targeting domain of G A1 B6b (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 8 and SEQ ID NO: 9), shown in FIG. 10. This complex has less
RNAi activity compared to Medusa control, but is still able to have
significant and target specific RNAi knockdown of Renilla
luciferase. When a 5' extension is added, as in G Ac B6b (SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 9), there is no
impact on the RNAi activity compared with G Ac B6c (SEQ ID NO: 1,
SEQ ID NO: 2, and SEQ ID NO: 19). This illustrates that the
combination of C3 linker modification and 2'-O-methyl and
phosphorothioate modifications added is effective in preventing the
5' overhang from interfering with correct Dicer processing of the
targeting domain.
[0233] G Ac B4 mimics the targeting domain of G A1 B4. Although not
as effective as G Ac B6b, it still has significant detectable RNAi
activity against the Renilla luciferase target, illustrating that
the RNAase H trimming mechanism of G A1 B1 and G A1 B4 is effective
in allowing for proper processing of the targeting domain.
[0234] In particular, in panels A and B, the inactive and active
forms of two RNAase H activated designs for the sensor locked siRNA
are compared with positive controls. In panel A, compared with the
positive controls (dsiRNA and Medusa Control), G A1 B1 has
significantly reduced RNAi activity. When activated (G A1 B1+
signal), there is a detectable and significant increase in the RNAi
activity. Similarly, in panel B, G A1 B4 has significantly less
RNAi activity than Medusa control, and the activated form (G A1 B4+
signal) has increased RNAi activity.
[0235] In panel C, G A1 B6b (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO:
8 and SEQ ID NO: 9), the XRN1 activated design as illustrated in
FIGS. 10 and 18, is tested. Compared with Medusa control, the
"inactive" G A1 B6b (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8 and
SEQ ID NO: 9) has significantly lower RNAi activity. When a signal
is activated, the RNAi activity increases for the 5.0 nM and 1.0 nM
transfection concentrations.
[0236] In the examples illustrated in FIG. 30, the spurious RNAi
activity of the "INACTIVE" state constructs are likely caused by
spurious opening of the locking sensor in the cellular environment.
This can be ameliorated by increasing the thermodynamic stability
of the locking sensor via incorporation of modified bases such as
LNA.
Example 4
Confirmation of Accessibility and Processing of Medusa Complex
Segments in HCT116 Cells
[0237] To confirm the accessibility of the individual segments of
exemplary molecular Applicants transfected some of the constructs
tested in Example 3 into HCT116 cells, extracted RNA, and performed
a Northern blot with probes specific to the different segments of
the complex to observe presence as well as processing.
[0238] FIG. 31, some of the constructs shown in FIG. 30 are tested
on a Northern blot. In particular, in the illustration of FIG. 31,
Probe (oligo 544) (SEQ ID NO: 40) hybridizes to intact guide strand
G (29 nucleotides) (SEQ ID NO: 1) seen in lanes 1-9 and the
approximately 21 nucleotide Dicer product, indicated by the arrow,
visible in lanes 1,2, 7 and 8. Importantly, Dicer products are
visible for lane 7 (G Ac B6) (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 8 and SEQ ID NO: 9), lane 8 (G Ac Bc6) (G Ac Bc6) (SEQ ID NO:
1, SEQ ID NO: 2, and SEQ ID NO: 19), lane 1 (G Ac B4) (G Ac B4)
(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7), and lane 2 (G Ac Bc) (G
Ac Bc) (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3), indicating the
sensor domains are correctly processed by Dicer. However, lane 5
and lane 6 showed little detectable Dicer product. This indicates
that the 3' overhang on sensor A (SEQ ID NO: 5) or on sensor A (SEQ
ID NO: 5) bound to the signal RNA is inhibitory for Dicer
processing. This is likely caused by the right side of FIG. 19,
where the duplex domain in sensor A (SEQ ID NO: 5) overhang or
sensor A (SEQ ID NO: 5)::signal RNA stem stacks with the targeting
domain, forming a RNA duplex longer than 30 base-pairs. This can
induce binding by the PKR protein, a cellular immune sensor, which
can interfere with processing by Dicer.
[0239] In FIG. 14, Applicants design an implementation, G2 A3 B7
(SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
NO: 14, and SEQ ID 15), to circumvent this effect. Compared with G
A1 B6b (SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 9)
(FIG. 10), G2 A3 B7 (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID 15) uses connecting
triethylene glycols and hexaethylene glycols and mismatches between
the signal binding domain and the signal to create mismatches in
the duplex formed by the signal (SEQ ID NO: 16) and the sensor
strand, and create an unstructured domains separating the targeting
domain and the signal bound sensor domain. This reduced PKR
activation. However, the 14 bp sensor stem, with reduced
2'-O-methyl content, is less thermodynamically stable than the 16
bp and 18 bp sensor domains of G A1 B6b (SEQ ID NO: 1, SEQ ID NO:
5, SEQ ID NO: 8 and SEQ ID NO: 9) and G A1 B1, G A1 B4 (SEQ ID NO:
1, SEQ ID NO: 5, SEQ ID NO: 7). Thus, there are two effects, seen
in FIG. 32.
[0240] The ACTIVE version, G2A3B7S (G A3 B7+ signal), has much
increased RNAi activity on the dual luciferase assay than GAB6bS (G
A1 B6b+ signal), due to the PKR avoidance features. However, the
INACTIVE conformation, G2A3B7 (G2 A3 B7), also has greater baseline
RNAi activity compared with GAB6b* (G A1 B6b). This is due to the
decreased stability of the sensor domain. One can also see,
however, that G2A3B7S is more active than G2A3B7, indicating that
the locking sensor, though unstable, is having some effect.
[0241] To obtain better shut-off, the sensor stem for G2A3B7 can be
increased to as long as 18 bp, contain uniform 2'-O-methyl bases,
or incorporate other stabilizing chemical modifications such as LNA
bases.
[0242] FIG. 32 is a northern blot on the same set of constructs.
Most notably, G2 A3 B7 S (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID 15, and SEQ ID NO: 16) has
a detectable Dicer product. In fact it has more product than the
INACTIVE conformation G2 A3 B7 (SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID 15). On the same
gel, Dicer products are not detected for GAB6b (SEQ ID NO: 1, SEQ
ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 9) or GAB6bs (SEQ ID NO: 1,
SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 16). As
the luciferase test shows, both GA1B6b (SEQ ID NO: 1, SEQ ID NO: 5,
SEQ ID NO: 8 and SEQ ID NO: 9) and GA1B6bS (SEQ ID NO: 1, SEQ ID
NO: 5, SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 16) have some
RNAi activity. However, the amount of Dicer product produced may be
lower than the detection limit of Northern blog.
[0243] Note that in these examples, the spurious Dicer processing
and RNAi activity for the INACTIVE constructs is likely the result
of the spurious dissociation of the sensor domain. Dicer likely has
minimal interaction with the locked siRNA when the targeting domain
is actually folded as shown in FIG. 1.
[0244] FIG. 33 shows a Northern blot of exemplary Medusa complexes
with and without signal strands and controls. Lane M, RNA size
markers, number of nucleotides is indicated. Probe (oligo 544) (SEQ
ID NO: 40) hybridizes to intact guide strand G (29 nucleotides)
(SEQ ID NO:1) seen in all lanes and the approximately 21 nucleotide
Dicer product, indicated by the arrow, seen with G Ac Bc6 (SEQ ID
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 19), G2 A3 B7 (SEQ ID NO: 10,
SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID
15) and G2 A3 B7 S (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID 15, and SEQ ID NO: 16) (lanes
2, 5 and 6, respectively).
Example 5
Structure Assembly and Processing of an Exemplary Molecular
Construct
[0245] An exemplary molecular construct was assembled and tested
for processing as illustrated below.
[0246] FIG. 34 shows an unlocked (G Ac Bc) (SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3) and a locked (G RP) (SEQ ID NO: 1, SEQ ID NO:
4) RNAi targeting domain. G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, SEQ
ID NO: 3) and G RP (SEQ ID NO: 1, SEQ ID NO: 4) have identical
sequences, but in G RP (SEQ ID NO: 1, SEQ ID NO: 4) the 3' of
Passenger A (SEQ ID NO: 5) is directly linked to the 5' of
Passenger B (SEQ ID NO: 6), comprising a single "reversed topology"
passenger strand. This linkage locks the RNAi targeting domain into
a folded conformation that minimizes proper Dicer processing.
[0247] FIG. 35 shows the assembled G RP (SEQ ID NO: 1, SEQ ID NO:
4) product. The individual strands composing GRP (SEQ ID NO: 1, SEQ
ID NO: 4) or G Ac Bc (SEQ ID NO: 1) were ordered from a commercial
company, Thermo Scientific. For assembly the strands composing G Ac
Bc (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) or G RP (SEQ ID NO:
1, SEQ ID NO: 4) or G RP were combined at 1 micromolar
concentration in 1.times.PBS buffer (approximately 150 mM KCl with
other components), heated to .about.90 degrees Celsius, and allowed
to cool to room temperature. During this process the strands
self-assemble into either G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, SEQ
ID NO: 3) or G RP (SEQ ID NO: 1, SEQ ID NO: 4). The resulting G RP
(SEQ ID NO: 1, SEQ ID NO: 4) products were assessed by running
through 8% non-denaturing polyacrylamide gel in 1.times.TBE buffer
following standard practices in the art. The first lane on the left
shows the band corresponding to the Guide strand alone. In the G RP
(SEQ ID NO: 1, SEQ ID NO: 4) lane, there is a clear band showing a
construct corresponding to the G RP (SEQ ID NO: 1, SEQ ID NO: 4)
construct in the correct conformation. In this conformation, Dicer
processing is minimized. In addition, there are a number of higher
molecular weight lanes, corresponding to incorrect, multimeric
assemblies of G (SEQ ID NO: 1) and RP (SEQ ID NO: 4) strands. These
higher molecular weight products can have spurious Dicer processing
and RNAi activity. If desired, these products can be removed by
filtering using HPLC, or filtration membranes with the appropriate
molecular weight cutoff, or by extracting them using native
polyacrylamide gel electrophoresis.
[0248] FIG. 36 shows dual luciferase assay of G Ac Bc (SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3) compared with G RP (SEQ ID NO: 1,
SEQ ID NO: 4). The Y axis indicates relative strength of the
Renilla luciferase: Fire fly luciferase ratio compared with a
negative control. 1.0 is the level at which there is no detectable
RNAi activity. 0.0 would constitute perfect RNAi knockdown. (Since
this is a biology experiment there is some normal variation of
experimental values in the range of 10% to 20%). For this
experiment, G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3)
transfected into HCT 116 cells at 5 nm, 1 nm, or 0.2 nm
concentration reduced Renilla luciferase activity to near or below
20% of the nominal value. In contrast, cells transfected with G RP
assemblies had much more Renilla luciferase activity at every
concentration. This shows that there was a significant decrease in
the RNAi activity of the G RP (SEQ ID NO: 1, SEQ ID NO: 4)
assembly.
[0249] Although the RNAi activity of G RP (SEQ ID NO: 1, SEQ ID NO:
4) is reduced, it is still detectable. This is because the G RP
(SEQ ID NO: 1, SEQ ID NO: 4) assemblies were transfected without
filtration or removal of the higher molecular weight complexes seen
in FIG. 35. Thus, while we expect that the correctly assembled G RP
(SEQ ID NO: 1, SEQ ID NO: 4) to have minimal Dicer processing and
RNAi activity, the presence of higher molecular weight complexes in
this assembly is responsible for the detectible amount of RNAi
activity seen in this experiment. As stated previously, if desired,
the incorrect complexes can be removed from the assemblies using
purification methods standard in the art, such as High Performance
Liquid Chromatography, native gel electrophoresis and extraction,
or molecular separation using filtration membranes with the correct
molecular weight cutoff.
[0250] FIG. 37 shows the definition of Dicer processing. For a
duplex RNAi targeting domain with a guide strand, such as the one
shown (G Ac Bc (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) from FIG.
34), correct processing occurs when PAZ domain of Dicer binds to 3'
of the Guide strand (SEQ ID NO: 1) and the endonuclease domain of
Dicer cleaves the 5' end of the guide strand (SEQ ID NO: 1) in the
position indicated. This will produce a 20 to 26 nucleotide long
product, depending on base-pairing. If the targeting domain was
perfectly base paired, Dicer processing will produce a 20 to 23
nucleotide long product, highlighted in gray. For an imperfect
duplex, the product can be up to 26 nucleotides long.
[0251] FIGS. 31 and 33 show Northern blots that include G RP (SEQ
ID NO: 1, SEQ ID NO: 4), and a positive control, G Ac Bc6, which is
nearly identical in structure with G Ac Bc (SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 8, SEQ ID NO: 9). A correct Dicer product is
clearly seen for G Ac Bc6 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
19) in both Northern blots, while the Dicer product for G RP (SEQ
ID NO: 1, SEQ ID NO: 4) in undetectable.
Example 6
Testing and Measuring of the Melting Temperature (Tm) of the Three
Way Function of an Activated RNAaseH Based Construct
[0252] Exemplary experimental procedures for testing/measuring the
melting temperature of the three-way activation formed between the
portions of the activation segment in constructs herein described
having an RNAaseH based design.
[0253] Applicants first synthesized oligonucleotides comprising
sequences of designed for the guide strand passenger strand,
displacement segment and activation segment respectively using
standard methods for oligonucleotide synthesis well establish in
the art. Then the synthesized oligonucleotides were then purified
based on their expected lengths. The purified oligonucleotides were
mixed together in an RNAase free buffer containing PBS. To allow
proper annealing of the oligonucleotides, the mixture was heated to
about 90.degree. C. for 1 minutes and then cooled to a desired
melting temperature of about 15.degree. C. at the rate of 1.degree.
C. every 10 seconds. After annealing, RNAse H was added to the
buffer and incubated according to manufacturer's instructions to
allow cleavage of the construct by RNAse H. The cleavage products
were then loaded onto a denaturing polyacrylamide gel (SDS-PAGE)
following by electrophoresis to examine whether a proper DNA: RNA
duplex of at least 5 consecutive base pairs have formed during
annealing and whether the construct was cleaved at the expected
RNAse H cleavage site.
[0254] To examine whether an activation junction formed among the
segments (e.g. a three-way activation junction) is formed properly,
Applicants attached pairs of fluorophore/quencher to nucleotides
that are expected to form base pairs between opposing strands when
the activation junction is properly formed, and examined whether
significant quenching of the fluorescence signal can be observed at
the minimum melting temperature using fluorescent microcopy.
Additionally, the fluorophore/quencher pairs can be attached to
pairs of neighboring nucleotides near the junction. Alternatively,
in the above experiments, the fluorophore/quencher pairs can be
replaced by pairs FRET acceptor/donor fluorophores, and examine
significant FRET can be observed at the minimum melting
temperature.
[0255] As a complimentary approach, Applicants used a standard set
of procedures known to the art to establish the secondary structure
of the construct.
[0256] First, Applicants used single stranded RNA endonucleases to
digest the construct, and examined whether RNA portions of the
segments that are expected to form double strands were protected
from the cleavage by the endonuclease by formation of proper
secondary structures.
[0257] Second, Applicants used single stranded DNA endonucleases to
digest the construct, and examine whether the construct is
protected from the cleavage by formation of the secondary structure
of the duplex.
[0258] Third, Applicants tested whether the expected duplex regions
of the junction is protected from RNA modifying and RNA cleaving
chemical probes using 5' or 3' radionucleotide labeling or primer
extension analysis.
[0259] After the above procedures of examining the structure of the
activation junction, the construct was exposed to gradual
temperature increasing, and the melting temperature of the properly
formed activation junction was determined by the inflection points
in the UV absorption at 260 nm during the gradual temperature
increasing.
[0260] The above described experiments can also be performed
according to commonly used experimental protocols and procedures,
such as the one described in Keril J. Blight et al., Journal Of
Virology, Oct. 1997, vol 71, p. 7345-7352 herein incorporated by
reference in its entirety.
Example 7
Testing and Measuring of the Melting Temperature (Tm) of the
Construct-Locking Sensor Duplex Stem
[0261] Exemplary experimental procedures for testing/measuring the
melting temperature (Tm) of the double-stranded duplex formed by
the activation segment and the displacement segment are described
below:
[0262] Applicants first synthesized oligonucleotides comprising
sequences of designed for the guide strand passenger strand,
activation segment, displacement segment and toehold segment
respectively using standard methods for oligonucleotide synthesis
well establish in the art. Then an internal fluorophore was
attached to the 3' end of the displacement segment, and a quencher
was attached to the 5' end of the protection segment opposing the
base carrying the fluorophore. Alternative, the quencher can be
attached to the 3' end of the displacement segment, while the
internal fluorophore was attached to the 5' end of the protection
segment opposing the base carrying the fluorophore. Also, a FRET
donor/acceptor fluorophore pairs can be used instead of the
fluorophore/quencher pair.
[0263] Then the oligonucleotides were purified based on their
expected lengths and are mixed together in an RNAse free buffer
containing PBS. To allow proper annealing of the oligonucleotides,
the mixture was heated to about 90.degree. C. for 1 minutes and
then cooled to a desired melting temperature of about 25.degree. C.
at the rate of 1.degree. C. every 10 seconds. During the annealing,
the fluorescence signal was observed using a spectrofluorometer to
examine whether a proper double-stranded duplex is formed between
the protection segment and the displacement segment. At the melting
temperature of 25.degree. C., the fluorescence signal was quenched
(if a FRET pair was used instead of the fluorophore/quencher pair,
significant FRET signal between the FRET pairs is expected to be
observed), which indicated that a double-stranded duplex has been
formed properly between the protection segment and the displacement
segment.
[0264] In addition, Applicants used the standard panel of enzymatic
digest and chemical probe tests to further examine the melting
temperature of the construct. Applicants used single strand
endonuclease to digest the construct at or below the expected
melting temperature (e.g. 25.degree. C.) to examine whether the
double-stranded portion of the displacement segment and the
protection segment was protected from the endonuclease
cleavage.
[0265] After the above procedures of examining the structure of the
activation junction, the construct was exposed to gradual
temperature increasing, and the melting temperature of the properly
formed activation junction was determined by the inflection points
in the UV absorption at 260 nm during the gradual temperature
increasing.
Example 8
Testing and Measuring of the Strand Displacement of the
Construct
[0266] Exemplary experimental procedures for testing and measuring
the strand displacement of the construct are described below:
[0267] Applicants first synthesized oligonucleotides comprising
sequences of designed for activation segment, displacement segment
and toehold segment (locking sensor) using standard methods for
oligonucleotide synthesis well establish in the art. Then an
internal fluorophore was attached to the terminus of the
displacement segment that is further away from the toehold segment.
A quencher was attached to the terminus of the activation segment
that is further away from the toehold segment. Alternative, the
internal fluorophore can be attached to the terminus of the
protection segment that is further away from the toehold segment,
while a quencher was attached to the terminus of the displacement
segment that is further away from the toehold segment. Also, a FRET
donor/acceptor fluorophore pairs can be used instead of the
fluorophore/quencher pair. Also synthesized was a corresponding
signal polynucleotide designed for the sensor domain described
above.
[0268] Then the synthesized oligonucleotides were purified based on
their expected lengths and were incubated with an equal amount of
the signal polynucleotide under the operating condition (e.g.
1.times.PBS buffer) at the expected operating temperature (e.g.
37.degree. C.).
[0269] The change in the fluorescent signal during the process of
strand displacement was monitored and recorded using a
spectrofluorometer. The recorded signal was then plotted as a
function of time and the kinetic rate of the displacement reaction
was determined from the plot.
[0270] To examine whether the attachment of the
fluorophore/quencher introduces artifacts to the displacement
kinetics and whether the entire protection segment is displaced
during the process, the fluorophore/quencher pair was then attached
to a different pair of nucleotides selected respectively from the
protection segment and the displacement segment at positions closer
to the toehold segment, and the above procedures were repeated.
Example 9
Process of Designing, Synthesis and Testing the Activity of a
Signal Activated Construct
[0271] Exemplary processes are described below for the designing,
synthesis and testing the activity of a signal activated construct,
which comprise a targeting domain configured for interfering a
target intracellular process through RNAi.
[0272] To design a construct, Applicants started with the analysis
of a RNA sequence that was to be targeted (interference) by RNAi,
such as a target mRNA or a set of target mRNAs. According to the
RNA sequence to be targeted, applicants selected the sequences for
the targeting domain of the construct that were known in the
art.
[0273] For example, in the G2 A3 B7 (SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15)
construct shown in FIG. 15, applicants started with the Dicer
substrate 27/29 mers duplex with the 29 nucleotide guide strand
sequence 5'-C U U G C G U C U G A G G G A U C UC U A G U U A C C U
U-3'(SEQ ID NO: 60), where the underlined portion is fully
complementary to a conserved HIV-1 target RNA transcript.
[0274] Applicants introduce a nick in the passenger 27 nucleotide
passenger strand complementary to this guide strand, so that the
passenger strand is divided into a 13 nucleotide and a 14
nucleotide piece.
[0275] Applicants then selected a conserved portion of the HIV-1
Tat mRNA sequence as a signal polynucleotide. The signal sequence
selected was long enough so that there are very few spurious
matches to possible RNA transcripts from the organism's genome. In
particular, the signal sequence was selected to comprise at least a
difference of 4 nucleotides between the signal sequence and its
nearest homologous sequence. For using in human cells, the signal
polynucleotide can have a minimum length of about 14 nucleotides,
but in this case a longer signal was used. Further, the signal
polynucleotide was designed to have at least 4 nucleotides that
complementarily bind to the toehold segment. In total, the signal
nucleotide selected for the use in human cells should be at least
18 nucleotides in length.
[0276] In the construct shown in FIG. 13, the applicants chose to
have a 16 bp sensor stem and a 5 nucleotide long toehold. Thus,
corresponding signal polynucleotide was 21 nucleotides long.
[0277] The 5' of this signal polynucleotide was complementary to
the 3' sensor toehold illustrated in FIG. 13 and the rest was
complementary to the signal binding side of the 16 bp sensor
stem.
[0278] At this point, the sensor domain was fully specified. The
applicants then connected the signal binding strand (left side of
the 16 bp sensor stem) to the 3' passenger strand and the displaced
segment to the 5' piece of the passenger strand. This allows the
sensor domain to lock the targeting domain into the folded,
inactive conformation.
[0279] In order to ensure sufficient geometric slack to allow
formation of the construct, a 2 nucleotide spacer was introduced on
the signal binding side and a C3 linker was introduced between the
displaced strand and the passenger strand.
[0280] The C3 linker also serves to prevent the 5' sensor overhang
from interfering with Dicer processing of the ACTIVE RNAi targeting
domain.
[0281] To further prevent the possible processive exonucleolytic
degradation of the 5' passenger strand by XRN1, two 2'-O-methyl
base modifications and 2 phosphorothioate backbone modifications
were placed immediately to the 3' side of the C3 linker
[0282] At this stage all segments in the sensor domain (i.e. the
protection segment, displacement segment, activation segment and
the toehold segment) have been specified. Using the above
algorithm, Applicants designed the sensor domains for the every
possible 21-nucleotides sequence of the chosen signal
polynucleotide (in this case a conserved portion of the HIV-1
Tat-Rev RNA transcript. Then Applicants examined each candidate
design by running the sequences through an RNA secondary structure
calculation code to examine the predictions for secondary structure
conformation and stability. Based on the result, applicants chose
one or more candidate designs with the best stability, and the
least complicated secondary structure in the toehold, and added
chemical modifications to regulate base pair stability.
[0283] In particular, for increased stability, Applicants applied
added 2'-O-methyl modifications to the entire signal binding side
of the sensor duplex. Applicants also changed the 4 bases at the 5'
terminus of the guide strand to 2'-O-methyl, and applicants changed
some bases in the 3' piece of the passenger strand (the one with 14
base-pairs to the guide strand) to 2'-O-methyl. In addition, an
inverted dT base was added to the 3' terminus of the sensor toehold
to prevent Dicer binding.
[0284] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the constructs, complexes,
sensors, arrangements, devices, compositions, systems and methods
of the disclosure, and are not intended to limit the scope of what
the inventors regard as their disclosure. All patents and
publications mentioned in the specification are indicative of the
levels of skill of those skilled in the art to which the disclosure
pertains.
[0285] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference. All references cited in this disclosure are
incorporated by reference to the same extent as if each reference
had been incorporated by reference in its entirety individually.
However, if any inconsistency arises between a cited reference and
the present disclosure, the present disclosure takes precedence.
Further, the computer readable form of the sequence listing of the
ASCII text file P1210-US-Sequence-Listing_ST25 is incorporated
herein by reference in its entirety.
[0286] The terms and expressions which have been employed herein
are used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the disclosure claimed. Thus, it
should be understood that although the disclosure has been
specifically disclosed by exemplary embodiments and optional
features, modification and variation of the concepts herein
disclosed can be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this disclosure as defined by the appended claims.
[0287] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting. As used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. The term "plurality" includes two or more referents
unless the content clearly dictates otherwise. Unless defined
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which the disclosure pertains.
[0288] When a Markush group or other grouping is used herein, all
individual members of the group and all combinations and possible
subcombinations of the group are intended to be individually
included in the disclosure. Every combination of components or
materials described or exemplified herein can be used to practice
the disclosure, unless otherwise stated. One of ordinary skill in
the art will appreciate that methods, device elements, and
materials other than those specifically exemplified can be employed
in the practice of the disclosure without resort to undue
experimentation. All art-known functional equivalents, of any such
methods, device elements, and materials are intended to be included
in this disclosure. Whenever a range is given in the specification,
for example, a temperature range, a frequency range, a time range,
or a composition range, all intermediate ranges and all subranges,
as well as, all individual values included in the ranges given are
intended to be included in the disclosure. Any one or more
individual members of a range or group disclosed herein can be
excluded from a claim of this disclosure. The disclosure
illustratively described herein suitably can be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0289] A number of embodiments of the disclosure have been
described. The specific embodiments provided herein are examples of
useful embodiments of the disclosure and it will be apparent to one
skilled in the art that the disclosure can be carried out using a
large number of variations of the devices, device components,
methods steps set forth in the present description. As will be
obvious to one of skill in the art, methods and devices useful for
the present methods can include a large number of optional
composition and processing elements and steps.
[0290] In particular, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
REFERENCES
[0291] 1. Wu, H. et al., "Properties of cloned and expressed human
RNase H1", The Journal of Biological Chemistry, Vol. 274, pp. 28270
(1999). [0292] 2. Zamaratski, E. et al., "A critical survey of the
structure function of the antisense oligo/RNA heteroduplex as
substrate for RNase H", Journal of Biochemical and Biophysical
Methods, Vol. 48, pp. 189 (2001). [0293] 3. Cazenave, C. et al.,
"Characterization and subcellular localization of ribonuclease H
activities from Xenopus laevis oocytes", The Journal of biological
chemistry, Vol. 269, pp. 25185 (1994). [0294] 4. Nowotny, M. et
al., "Crystal structures of RNase H bound to an RNA/DNA hybrid:
substrate specificity and metal-dependent catalysis", Cell, Vol.
121, pp. 1005 (2005). [0295] 5. Song, J. J. et al., "The crystal
structure of the Argonaute2 PAZ domain reveals an RNA binding motif
in RNAi effector complexes", Nature Structural Biology, Vol. 10,
pp. 1026 (2003). [0296] 6. Ma, J. B. et al., "Structural basis for
overhang-specific small interfering RNA recognition by the PAZ
domain", Nature, Vol. 429, pp. 318 (2004). [0297] 7. Yan, K. S. et
al., "Structure and conserved RNA binding of the PAZ domain",
Nature, Vol. 426, pp. 468 (2003). [0298] 8. Lingel, A. et al.,
"Structure and nucleic-acid binding of the Drosophila Argonaute 2
PAZ domain", Nature, Vol. 426, pp. 465 (2003). [0299] 9. Behlke, M.
A. et al., "Chemical modification of siRNAs for in vivo use",
Oligonucleotides, Vol. 18, pp. 305 (2008). [0300] 10. Rose, S. D.
et al., "Functional polarity is introduced by Dicer processing of
short substrate RNAs", Nucleic Acids Research, Vol. 33, pp. 4140
(2005). [0301] 11. Tomari, Y., et al., "A Protein Sensor for siRNA
Asymmetry", Science, Vol. 306, pp. 1377, (2004). [0302] 12. Susan
M. Freier and Karl-Heinz Altman, The ups and downs of nucleic acid
duplex stability: structure-stability studies on
chemically-modified DNA:RNA duplexes, Nucleic Acids Research, 1997,
Vol. 25, No. 22 4429-4443 [0303] 13. Nucleic Acids Research, 1998,
Vol. 26, No. 9,2224-2229 [0304] 14. Nucleic Acids Research, 2005,
Vol. 33, No. 16,5082-5093 [0305] 15. 564-574 Nucleic Acids
Research, 2006, Vol. 34, No. 2 [0306] 16. Sequence-specific
recognition of double helical RNA and RNA.DNA by triple helix
formation, PNAS May 1, 1993 vol. 90 no. 9 3806-3810 [0307] 17.
Burge S, Parkinson G N, Hazel P, Todd A K, Neidle S (2006).
"Quadruplex DNA: sequence, topology and structure". NAR 34 (19):
5402-5415. doi:10.1093/nar/gk1655 [0308] 18. J. N. Zadeh, C. D.
Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M.
Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid
systems. J Comput Chem, 32, 170-173, 2011. [0309] 19. R. M. Dirks,
J. S. Bois, J. M. Schaeffer, E. Winfree, and N. A. Pierce. (2007)
Thermodynamic analysis of interacting nucleic acid strands. SIAM
Rev, 49, 65-88. [0310] 20. R. M. Dirks and N. A. Pierce. (2003) A
partition function algorithm for nucleic acid secondary structure
including pseudoknots. J Comput Chem, 24, 1664-1677. [0311] 21. R.
M. Dirks and N. A. Pierce. (2004) An algorithm for computing
nucleic acid base-pairing probabilities including pseudoknots. J
Comput Chem, 25, 1295-1304. [0312] 22. J. N. Zadeh, B. R. Wolfe, N.
A. Pierce. Nucleic acid sequence design via efficient ensemble
defect optimization. J Comput Chem, 32, 439-452, 2011. [0313] 23.
M. Zuker. Mfold web server for nucleic acid folding and
hybridization prediction. Nucleic Acids Res. 31 (13), 3406-3415,
2003. [0314] 24. Waugh, P. Gendron, R. Altman, J. W. Brown, D.
Case, D. Gautheret, S. C. Harvey, N. Leontis, J. Westbrook, E.
Westhof, M. Zuker & F. Major. RNAML: A standard syntax for
exchanging RNA information. RNA 8 (6), 707-717, 2002. [0315] 25. M.
Zuker & A. B. Jacobson. Using Reliability Information to
Annotate RNA Secondary Structures. RNA 4, 669-679, 1998. [Abstract]
[Preprint] Note: Explains color annotation of secondary structure.
[0316] 26. N. R. Markham & M. Zuker. UNAFold: Software for
Nucleic Acid Folding and Hybridization. In Data, Sequence Analysis,
and Evolution, J. Keith, ed., Bioinformatics: Volume 2, Chapter 1,
pp 3-31, Humana Press Inc., 2008. [0317] 27. M. Zuker, D. H.
Mathews & D. H. Turner. Algorithms and Thermodynamics for RNA
Secondary Structure Prediction: A Practical Guide In RNA
Biochemistry and Biotechnology, 11-43, J. Barciszewski and B. F. C.
Clark, eds., NATO ASI Series, Kluwer Academic Publishers,
Dordrecht, N L, 1999. [0318] 28. M. Zuker. Prediction of RNA
Secondary Structure by Energy Minimization. In Computer Analysis of
Sequence Data A. M. Griffin and H. G. Griffin eds. Methods in
Molecular Biology, Humana Press Inc., 267-294, 1994. [0319] 29. J.
A. Jaeger, D. H. Turner & M. Zuker. Predicting Optimal and
Suboptimal Secondary Structure for RNA. In Molecular Evolution:
Computer Analysis of Protein and Nucleic Acid Sequences, R. F.
Doolittle ed. Methods in Enzymology 183, 281-306, 1990. [0320] 30.
M. Zuker. On Finding All Suboptimal Foldings of an RNA Molecule.
Science 244, 48-52, 1989. [0321] 31. D. H. Mathews, J. Sabina, M.
Zuker & D. H. Turner. Expanded Sequence Dependence of
Thermodynamic Parameters Improves Prediction of RNA Secondary
Structure J. Mol. Biol. 288, 911-940, 1999. [0322] 32. E. Walter,
D. H. Turner, J. Kim, M. H. Lyttle, P. Muller, D. H. Mathews &
M. Zuker. Coaxial stacking of helixes enhances binding of
oligoribonucleotides and improves predictions of RNA folding. Proc.
Natl. Acad. Sci. USA 91, 9218-9222, 1994. [0323] 33. D. H. Mathews,
W. N. Moss and D. H. Turner Folding and Finding RNA Secondary
Structure in Cold Spring Harb Perspect Biol. 2010. [0324] 34. D. H.
Mathews, D. H. Turner & M. Zuker. RNA Secondary Structure
Prediction. In Current Protocols in Nucleic Acid Chemistry S.
Beaucage, D. E. Bergstrom, G. D. Glick, and R. A. Jones eds., John
Wiley & Sons, New York, 11.2. 1-11.2. 10, 2007. [0325] 35. D.
H. Mathews, S. J. Schroeder, D. H. Turner & M. Zuker.
Predicting RNA Secondary Structure. In The RNA World, R. F.
Gesteland, T. R. Cech and J. F. Atkins eds., 3rd edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Chapter
22, 2006. [0326] 36. D. H. Mathews & M. Zuker. Predictive
Methods Using RNA Sequences. In Bioinformatics: A Practical Guide
to the Analysis of Genes and Proteins, A. Baxevanis and F.
Ouellette eds., 3rd edition, John Wiley & Sons, New York,
Chapter 7, 2005. [0327] 37. D. H. Mathews, M. D. Disney, J. L.
Childs, S. J. Schroeder, M. Zuker & D. H. Turner. Incorporating
chemical modification constraints into a dynamic programming
algorithm for prediction of RNA secondary structure. Proc. Natl.
Acad. Sci. USA 101 (19), 7287-7292, 2004. [0328] 38. M. Zuker &
D. Sankoff. RNA Secondary Structures and their Prediction. Bull.
Mathematical Biology 46, 591-621, 1984. [0329] 39. M. Zuker &
P. Stiegler. Optimal computer folding of large RNA sequences using
thermodynamics and auxiliary information. Nucleic Acids Res. 9,
133-148, 1981. [0330] 40. J.-M. Rouillard, M. Zuker & E.
Gulari. OligoArray 2. 0: design of oligonucleotide probes for DNA
microarrays using a thermodynamic approach. Nucleic Acids Res. 31
(12), 3057-3062, 2003. [0331] 41. J.-M. Rouillard, C. J. Herbert
& M. Zuker. OligoArray: Genome-scale oligonucleotide design for
microarrays. Bioinformatics 18 (3), 486-487, 2002. [0332] 42. RNA
secondary structure prediction by centroids in a Boltzmann weighted
ensemble, Ye Ding, Chi Yu Chan, and Charles E. Lawrence, R N A
2005. 11: 1157-1166 [0333] 43. Oligonucleotide synthesis: methods
and applications, Volume 288 of Methods in molecular biology, Piet
Herdewijn, Humana Press, 2005 [0334] 44. Principles of Nucleic Acid
Structure, Stephen Neidle, 2008 Elsevier Inc, ISBN:
978-O-12-369507-9 [0335] 45. RNA Interference in Mammalian Cells by
Chemically-Modified RNA, Biochemistry 2003, 42, 7967-7975 [0336]
46. Modified Nucleosides: in Biochemistry, Biotechnology and
Medicine, Piet Herdewijn (Editor), Wiley-VCH, 2008 [0337] 47.
in-Biao Ma, Keqiong Ye & Dinshaw J. Patel Structural basis for
overhang specific small interfering RNA recognition by the PAZ
domain, Nature, 429, 318 (2004) [0338] 48. Nature Reviews Drug
Discovery 8, 129-138 (February 2009) |doi:10.1038/nrd2742, Knocking
down barriers: advances in siRNA delivery [0339] 49. Simeoni, F.
"Insight into the mechanism of the peptide based gene delivery
system MPG: implications for delivery of siRNA into mammalian
cells." Nucleic acids research 31.11 (2003):2717. [0340] 50. Liu,
Z., Winters, M., Holodniy, M. and Dai, H. (2007), siRNA Delivery
into Human T Cells and Primary Cells with Carbon-Nanotube
Transporters. Angewandte Chemie, 119: 2069-2073. doi:
10.1002/ange.200604295 [0341] 51. Aptamer mediated siRNA delivery
Nucl. Acids Res. 34(10): e73 doi:10.1093/nar/gk1388 [0342] 52.
Dynamic PolyConjugates for targeted in vivo delivery of siRNA to
hepatocytes PNAS 2007 104 (32) 12982-12987 [0343] 53. Bioconjugate
Chem., 2007, 18 (5), pp 1391-1396, DOI: 10.1021/bc060367e [0344]
54. T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in
Humanized Mice, Cell, Volume 134, Issue 4, 22 Aug. 2008, Pages
577-586 [0345] 55. A universal RNAi-based logic evaluator that
operates in mammalian cells, Nature Biotechnology 25, 795-801
(2007) [0346] 56. Molecular Therapy (2010) 18 4, 796-802.
doi:10.1038/mt.2009.321, RNA (2010), 16:1275-1284 [0347] 57.
Molecular Therapy (2006) 13,494-505 [0348] 58. Hong-Wei Wang,
Cameron Noland, Bunpote Siridechadilok, David W Taylor, Enbo Ma,
Karin Felderer, Jennifer A Doudna & Eva Nogales Structural
insights into RNA processing by the human RISC-loading complex
Nature Structural & Molecular Biology 16, 1148-1153 (2009)
Sequence CWU 1
1
60129RNAArtificial sequenceSynthetic polynucleotide 1cuugcgucug
agggaucucu aguuaccuu 29214DNAArtificial sequenceSynthetic
polynucleotide 2ccucagacgc aagt 14314RNAArtificial
sequenceSynthetic polynucleotide 3gguaacuaga gauc
14427RNAArtificial sequenceSynthetic polynucleotide 4ccucagacgc
aaggguaacu agagauc 27537DNAArtificial sequenceSynthetic
polynucleotide 5ccucagacgc aagcugauga gcucuucguc gcuguut
37644DNAArtificial sequenceSynthetic polynucleotide 6aagguccctg
atcgacgaag agcucaucag gguaacuaga gauc 44744DNAArtificial
sequenceSynthetic polynucleotide 7aagguccctg atcgacgaag agcucaucag
gguaacuaga gauc 44816RNAArtificial sequenceSynthetic polynucleotide
8cgacgaagag cucauc 16914RNAArtificial sequenceSynthetic
polynucleotide 9gguaacuaga gauc 141027RNAArtificial
sequenceSynthetic polynucleotide 10cgcgucugag ggaucucuag uuaccuu
271112RNAArtificial sequenceSynthetic polynucleotide 11cccucagacg
cg 121216RNAArtificial sequenceSynthetic polynucleotide
12gaugagnncu ucgucg 16138RNAArtificial sequenceSynthetic
polynucleotide 13gucuccgc 81415RNAArtificial sequenceSynthetic
polynucleotide 14cgacgaagcu cauca 151513RNAArtificial
sequenceSynthetic polynucleotide 15gguaacuaga gau
131639DNAArtificial sequenceSynthetic polynucleotide 16aaaaagcgga
gacagcgacg aagagcucau cagaaaaat 391718RNAArtificial
sequenceSynthetic polynucleotide 17cgacgaagag cucaucag
181814RNAArtificial sequenceSynthetic polynucleotide 18gguaacuaga
gauc 141914RNAArtificial sequenceSynthetic polynucleotide
19gguaacuaga gauc 142021RNAArtificial sequenceSynthetic
polynucleotide 20gacagcgacg aaggcgacgg c 212114RNAArtificial
sequenceSynthetic polynucleotide 21gguaacuaga gauc
142213RNAArtificial sequenceSynthetic polynucleotide 22ccucagacgc
aag 132319DNAArtificial sequenceSynthetic polynucleotide
23gccgucgcag cucaucagt 192413RNAArtificial sequenceSynthetic
polynucleotide 24ccucagacgc aag 132523RNAArtificial
sequenceSynthetic polynucleotide 25gaugagcucu ucgucgcugu cuc
23267RNAArtificial sequenceSynthetic polynucleotide 26cugauga
7279RNAArtificial sequenceSynthetic polynucleotide 27gcucuucgu
92810RNAArtificial sequenceSynthetic polynucleotide 28cgcugucucc
102953RNAArtificial sequenceSynthetic polynucleotide 29cugaugagcu
cuucgucgcu gucuccgcng augagncuuc gucgngucuc cgc 533015DNAArtificial
sequenceSynthetic polynucleotide 30cttngcngtc tgagg
153113RNAArtificial sequenceSynthetic polynucleotide 31ccucagacgc
aag 133217DNAArtificial sequenceSynthetic polynucleotide
32gantcntcta gtntacc 173314RNAArtificial sequenceSynthetic
polynucleotide 33gguaacuaga gauc 143421DNAArtificial
sequenceSynthetic polynucleotide 34cagactttgt tggatttgaa a
213521RNAArtificial sequenceSynthetic polynucleotide 35uuucaaaucc
aacaaagucu g 213626DNAArtificial sequenceSynthetic polynucleotide
36cttcaagcca gactttgttg gatttg 263725RNAArtificial
sequenceSynthetic polynucleotide 37caaauccaac aaaucuggcu ugaag
253822DNAArtificial sequenceSynthetic polynucleotide 38acagcgacga
agagctcatc ag 223922RNAArtificial sequenceSynthetic polynucleotide
39cugaugugcu cuucgucgcu gu 224021DNAArtificial sequenceSynthetic
polynucleotide 40ggtaactaga gatccctcag a 214121DNAArtificial
sequenceSynthetic polynucleotide 41tctgagggau cucuaguuac c
214222DNAArtificial sequenceSynthetic polynucleotide 42acagcgacga
agagctcatc ag 224322RNAArtificial sequenceSynthetic polynucleotide
43cugaugagcu cuucgucgcu gu 224420DNAArtificial sequenceSynthetic
polynucleotide 44ccctgatgag ctcttcgtcg 204520RNAArtificial
sequenceSynthetic polynucleotide 45cgacgaagag cucaucaggg
204620DNAArtificial sequenceSynthetic polynucleotide 46cttcgtcgat
cagggtcctt 204720RNAArtificial sequenceSynthetic polynucleotide
47aaggacccug aucgacgaag 204813RNAArtificial sequenceSynthetic
polynucleotide 48ccucagacgc aag 134923RNAArtificial
sequenceSynthetic polynucleotide 49gaugagcucu ucgucgcugu cuc
235038DNAArtificial sequenceSynthetic polynucleotide 50aaaaagcgga
gacagcgacg aagagcucau cgaaaaat 385181DNAArtificial
sequenceSynthetic polynucleotide 51aagguccctg atcgacgaag agcucaucag
gguaacuaga gaucccucag acgcaagcug 60augagcucuu cgucgcuguu t
815281DNAArtificial sequenceSynthetic polynucleotide 52aagguccctg
atcgacgaag agcucaucag gguaacuaga gaucccucag acgcaagcug 60augagcucuu
cgucgcuguu t 815328RNAArtificial sequenceSynthetic polynucleotide
53cugaugagcu cuucgucgcu gucuccgc 285412RNAArtificial
sequenceSynthetic polynucleotide 54cccucagacg cg
125517RNAArtificial sequenceSynthetic polynucleotide 55gcagagcgac
gaagagc 175619RNAArtificial sequenceSynthetic polynucleotide
56ggagacagcg cgcucugca 195713RNAArtificial sequenceSynthetic
polynucleotide 57gguaacuaga gau 135858DNAArtificial
sequenceSynthetic polynucleotide 58aagguccctg atcgacgaag agcucaucag
gguaacuaga gaucccucag acgcaagt 585928DNAArtificial
sequenceSynthetic polynucleotide 59gguaacuaga gaucccucag acgcaagt
286029RNAArtificial sequenceSynthetic polynucleotide 60cuugcgucug
agggaucucu aguuaccuu 29
* * * * *