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Thermodynamically Stable RNA three-way junctions as platform for constructing multi-functional nanoparticles for delivery of therapeutics

Dan Shu, Yi Shu, Farzin Haque, Sherine Abdelmawla, and Peixuan Guo Supplementary information Synthesis and purification of pRNA The pRNAs were synthesized by enzymatic methods as described previously 1. RNA oligos were synthesized chemically by IDT.

2’-deoxy-2’-Fluoro (2’-F) modified RNAs were synthesized by in vitro transcription with the mutant Y639F T7 RNA polymerase 2 using the 2’-F modified dCTP and dUTP 3 (Trilink). Construction and purification of pRNA complexes

Dimers were constructed by mixing equal molar ratios of pRNA Ab’ and Ba’ in presence of 5 mM magnesium. Similarly, trimers were assembled by mixing pRNA Ab’, Bc’, and Ca’. The 3WJ domain was constructed using three pieces of RNA oligos, denoted as a3wj, b3wj, and c3wj that were mixed at 1:1:1 molar ratio in DEPC treated water or TMS buffer (89 mM Tris, 5 mM MgCl2, pH 7.6) (Fig. 1d).

To purify the complex, the band corresponding to the dimer, trimer and 3WJ domain was excised from 8-15% native PAGE gel, running in TBM (89 mM Tris, 200 mM Boric Acid, 5 mM MgCl2, pH 7.6) buffer, eluted in 0.5 M NH4OAC, 0.1 mM EDTA, 0.1% SDS, and 5 mM MgCl2 for ~4 hrs at 37°C, followed by ethanol precipitation overnight. The dried pellet was then rehydrated in DEPC treated water or TMS buffer

The formation of the complexes were then analyzed by 8-15% native PAGE or 8M urea PAGE gel in TBM running buffer, as specified in the Results Section. After running at 4°C for 3 hrs, the RNA was visualized by ethidium bromide or SBYR Green II staining.

Stability assay in serum

RNA nanoparticles were synthesized in the presence 2’-F dCTP and dUTP 3, and incubated in RPMI-1640 medium containing 10% fetal bovine serum (Sigma). 200 ng of RNA were taken at 10 mins, 1 hr, 12 hr, and 36 hr time points after incubation at 37°C, followed by analysis using 8% native PAGE gel. Flow cytometry analysis of folate mediated cell binding

Human nasopharyngeal carcinoma KB cells [American Type Culture Collection (ATCC)] were maintained in folate-free RPMI-1640 medium (Gibco), then trypsinized and rinsed with PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). 200 nM Cy3 labeled 3WJ-pRNA/siSur-Rz-FA and the folate-free control 3WJ-pRNA/siSur-Rz-NH2 were each incubated with 2 x 105 KB cells at 37°C for 1 hr. After washing with PBS, the cells were resuspended in PBS buffer. Flow Cytometry (Beckman Coulter) was used to observe the cell binding efficacy of the Cy3 3WJ-RNA nanoparticles. Confocal microscopy

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2011.105

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KB cells were grown on glass coverslides in folate free medium overnight. Cy3 labeled 3WJ-pRNA/siSur-Rz-FA and the folate-free control 3WJ-pRNA/siSur-Rz-NH2 were each incubated with the cells at 37°C for 2 hrs. After washing with PBS, the cells were fixed by 4% paraformaldehyde and stained by Alexa Fluor® 488 phalloidin (Invitrogen) for cytoskeleton and TO-PRO®-3 iodide (642⁄661) (Invitrogen) for nucleus. The cells were then assayed for binding and cell entry by Zeiss LSM 510 laser scanning confocal microscope.

Assay for the silencing of genes in cancer cell model. Two 3WJ-RNA constructs were assayed for the subsequent gene silencing effects: one

harboring folate and survivin siRNA; and, the other harboring folate and survivin siRNA scramble control.

KB cells were transfected with 25nM of the individual 3WJ-RNAs and a positive Survivin siRNA control (Ambion, Inc.) using Lipofectamine 2000 (Invitrogen). After 48 hrs treatment, cells were collected and target gene silencing effects were assessed by both qRT-PCR and Western Blot assays.

Cells were processed for total RNA using illustra RNAspin Mini kits (GE healthcare). The first cDNA strand was synthesized on mRNA (500 ng) from KB cells with the various 3WJ-RNAs treatment using SuperScriptTM III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed using Roche Universal Probe Library Assay. All reactions were carried out in a final volume of 10 µl and assayed in triplicate.

Primers for human GAPDH and survivin are: GAPDH left: 5′-AGCCACATCGCTCAGACAC-3′; GAPDH right: 5′-GCCCAATACGACCAAATCC-3′; Survivin left: 5′-CACCGCATCTCTACATTCAAGA-3′; Survivin right: 5′-CAAGTCTGGCTCGTTCTCAGT-3′. PCR was performed on LightCycler 480 (Roche) for 45 cycles. The data was analyzed by

the comparative CT Method (ΔΔCT Method). For Western Blot assays, cells were lysed by RIPA lysis buffer (Sigma) and the cell total

protein was extracted for the assay. Equal amounts of proteins were then loaded onto 15% SDS-PAGE and electrophoretically transferred to Immun-Blot PVDF membranes (Bio-rad). The membrane was probed with survivin antibody (R&D) (1:4000 diluted) and β–actin antibody (Sigma) (1:5000 diluted) overnight, followed by 1:10000 anti-rabbit secondary antibody conjugated with HRP (Millipore) for 1 hr. Membranes were blotted by ECL kits (Millipore) and exposed to film for autoradiography.

AFM imaging

RNA was imaged using specially modified mica surfaces (APS mica)4 with MultiMode AFM NanoScope IV system (Veeco/Digital Instruments, Santa Barbara, CA), operating in tapping mode. Stability and systemic pharmacokinetic analysis in animals Each mouse was injected via the tail vein with 150 µg (6 mg/kg) of 2’-F U/C modified AlexaFluor647-labeled pRNA nanoparticles that contain the 3WJ domain as a scaffold. DY647-labeled siRNA with equal molar concentration (40 µg/mice or 1.6 mg/kg) was used as a control. Up to 20 µl of blood samples were collected via the lateral saphenous vein at 1, 4, 8, 12, 16, and

24 hours post injection. Blood samples were collected in BD Vacutainer® SST™ Serum Separation Tubes, and samples were mixed by inverting the tubes five times, and kept for 30-min at room temperature. After clotting, serum was collected by centrifugation at 1000-1300xg for 10 min. Each 2 µl of serum was mixed with 1 µl of proteinase-K and 37 µl of water, and incubated at 37°C for 30 minutes before loading for capillary gel electrophoresis (CGE-Beckman Coulter, dsDNA 1000 Kit), for measuring the fluorescence intensity using a P/ACE MDQ capillary electrophoresis system, equipped with a 635 nm laser (Beckman Coulter, Inc., Fullerton, California). Samples were loaded into a 32 cm x 100 µm capillary, by voltage injection for 40 second at 4 kV, and ran for 25 mins at 7.8 kV. The results were integrated using the software “32 Karat” version 7 (Beckman Coulter). The pRNA concentration was calculated from a standard curve. The serum concentration profiles were fitted with an IV bolus non-compartmental model using the Kinetica program (Fisher Scientific, Inc.) to deduce the key secondary pharmacokinetic parameters including t1/2, AUC (area under the curve), Vd (volume of distribution), Cl (clearance), and MRT (mean residence time). Targeting tumor xenograft by systemic injection in animals

For biodistribution, stability assay and specific tumor targeting studies on pRNA nanoparticles with the 3WJ domain as a scaffold, 6-week-old male nude mice (nu/nu) (NCI/Frederick) were fed a folate-free diet for a total of 2 weeks before the experiment. The mice were injected with cancer cells (KB cells ~3 x 106 cells per mouse) in a 40% matrigel in a folate free RPMI-1640 medium. When the tumors grew up to about 500 mm3, the mice were injected intravenously through the tail vein with a single dose of 600 µg of 2’-F U/C modified FA-AlexaFluor647-labeled pRNA nanoparticle (about 15 nmol in PBS buffer, equal to 24 mg/kg). After 24 hours post injection, the mouse was euthanized by CO2 asphyxiation. Whole-body imaging was carried out with an IVIS® Lumina station with the body of the mouse lying sideways in the imaging chamber. After whole-body imaging, the tumors, liver, spleen, heart, lung, intestine, kidney, and skeleton muscle of the mice were dissected and individually imaged.

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SUPPLEMENTARY INFORMATION DOI: 10.1038/NNANO.2011.105


KB cells were grown on glass coverslides in folate free medium overnight. Cy3 labeled 3WJ-pRNA/siSur-Rz-FA and the folate-free control 3WJ-pRNA/siSur-Rz-NH2 were each incubated with the cells at 37°C for 2 hrs. After washing with PBS, the cells were fixed by 4% paraformaldehyde and stained by Alexa Fluor® 488 phalloidin (Invitrogen) for cytoskeleton and TO-PRO®-3 iodide (642⁄661) (Invitrogen) for nucleus. The cells were then assayed for binding and cell entry by Zeiss LSM 510 laser scanning confocal microscope.

Assay for the silencing of genes in cancer cell model. Two 3WJ-RNA constructs were assayed for the subsequent gene silencing effects: one

harboring folate and survivin siRNA; and, the other harboring folate and survivin siRNA scramble control.

KB cells were transfected with 25nM of the individual 3WJ-RNAs and a positive Survivin siRNA control (Ambion, Inc.) using Lipofectamine 2000 (Invitrogen). After 48 hrs treatment, cells were collected and target gene silencing effects were assessed by both qRT-PCR and Western Blot assays.

Cells were processed for total RNA using illustra RNAspin Mini kits (GE healthcare). The first cDNA strand was synthesized on mRNA (500 ng) from KB cells with the various 3WJ-RNAs treatment using SuperScriptTM III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed using Roche Universal Probe Library Assay. All reactions were carried out in a final volume of 10 µl and assayed in triplicate.

Primers for human GAPDH and survivin are: GAPDH left: 5′-AGCCACATCGCTCAGACAC-3′; GAPDH right: 5′-GCCCAATACGACCAAATCC-3′; Survivin left: 5′-CACCGCATCTCTACATTCAAGA-3′; Survivin right: 5′-CAAGTCTGGCTCGTTCTCAGT-3′. PCR was performed on LightCycler 480 (Roche) for 45 cycles. The data was analyzed by

the comparative CT Method (ΔΔCT Method). For Western Blot assays, cells were lysed by RIPA lysis buffer (Sigma) and the cell total

protein was extracted for the assay. Equal amounts of proteins were then loaded onto 15% SDS-PAGE and electrophoretically transferred to Immun-Blot PVDF membranes (Bio-rad). The membrane was probed with survivin antibody (R&D) (1:4000 diluted) and β–actin antibody (Sigma) (1:5000 diluted) overnight, followed by 1:10000 anti-rabbit secondary antibody conjugated with HRP (Millipore) for 1 hr. Membranes were blotted by ECL kits (Millipore) and exposed to film for autoradiography.

AFM imaging

RNA was imaged using specially modified mica surfaces (APS mica)4 with MultiMode AFM NanoScope IV system (Veeco/Digital Instruments, Santa Barbara, CA), operating in tapping mode. Stability and systemic pharmacokinetic analysis in animals Each mouse was injected via the tail vein with 150 µg (6 mg/kg) of 2’-F U/C modified AlexaFluor647-labeled pRNA nanoparticles that contain the 3WJ domain as a scaffold. DY647-labeled siRNA with equal molar concentration (40 µg/mice or 1.6 mg/kg) was used as a control. Up to 20 µl of blood samples were collected via the lateral saphenous vein at 1, 4, 8, 12, 16, and

24 hours post injection. Blood samples were collected in BD Vacutainer® SST™ Serum Separation Tubes, and samples were mixed by inverting the tubes five times, and kept for 30-min at room temperature. After clotting, serum was collected by centrifugation at 1000-1300xg for 10 min. Each 2 µl of serum was mixed with 1 µl of proteinase-K and 37 µl of water, and incubated at 37°C for 30 minutes before loading for capillary gel electrophoresis (CGE-Beckman Coulter, dsDNA 1000 Kit), for measuring the fluorescence intensity using a P/ACE MDQ capillary electrophoresis system, equipped with a 635 nm laser (Beckman Coulter, Inc., Fullerton, California). Samples were loaded into a 32 cm x 100 µm capillary, by voltage injection for 40 second at 4 kV, and ran for 25 mins at 7.8 kV. The results were integrated using the software “32 Karat” version 7 (Beckman Coulter). The pRNA concentration was calculated from a standard curve. The serum concentration profiles were fitted with an IV bolus non-compartmental model using the Kinetica program (Fisher Scientific, Inc.) to deduce the key secondary pharmacokinetic parameters including t1/2, AUC (area under the curve), Vd (volume of distribution), Cl (clearance), and MRT (mean residence time). Targeting tumor xenograft by systemic injection in animals

For biodistribution, stability assay and specific tumor targeting studies on pRNA nanoparticles with the 3WJ domain as a scaffold, 6-week-old male nude mice (nu/nu) (NCI/Frederick) were fed a folate-free diet for a total of 2 weeks before the experiment. The mice were injected with cancer cells (KB cells ~3 x 106 cells per mouse) in a 40% matrigel in a folate free RPMI-1640 medium. When the tumors grew up to about 500 mm3, the mice were injected intravenously through the tail vein with a single dose of 600 µg of 2’-F U/C modified FA-AlexaFluor647-labeled pRNA nanoparticle (about 15 nmol in PBS buffer, equal to 24 mg/kg). After 24 hours post injection, the mouse was euthanized by CO2 asphyxiation. Whole-body imaging was carried out with an IVIS® Lumina station with the body of the mouse lying sideways in the imaging chamber. After whole-body imaging, the tumors, liver, spleen, heart, lung, intestine, kidney, and skeleton muscle of the mice were dissected and individually imaged.




SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1: Comparison of 3WJ-5S rRNA and 3WJ-Alu SRP cores. a, Assembly and urea denaturation assay of the 3WJ-5S rRNA and 3WJ- Alu SRP cores using three pieces of small RNA oligos (denoted a3WJ, b3WJ, and c3WJ) assayed in 16% native (upper) and 16% 8M Urea (lower) PAGE gel. b, Melting curves showing the assembly of the 3WJ-5S rRNA and 3WJ- Alu SRP cores using the three pieces of RNA oligos under the physiological buffer TMS. Supplementary Figure 2: Assembly and stability of 25 3WJ-3pRNA constructs. Three individual phi29 pRNAs subunits with three protruding ends were used as modules. The sequences of each of the three RNA oligos of the respective 3WJs were placed at the 3’-end of the pRNA monomer Ba’ and the complex was assembled into a three-way branched nanoparticles harboring one pRNA at each arm, as demonstrated by 8% native (upper) and 8M Urea (lower) PAGE gel. The bands corresponding to the assembled 3WJ structures are boxed in red. Supplementary Figure 3: RNase and serum stability assay of trivalent RNA nanoparticles 3WJ-3pRNA (unmodified and 2’-F modified). RNA nanoparticles were incubated in RPMI-1640 medium containing 10% fetal bovine serum (Sigma). 200 ng of RNA samples were taken at 10 mins, 1 hr, 12 hr, and 36 hr time points after incubation at 37°C, followed by analysis using 8% native PAGE gel. Supplementary Figure 4: Folate labeled particle specifically target folate receptor positive cell lines. The KB cell line (FR+) is folate receptor positive, and HT29 cell line (FR-) is the folate receptor negative control. Free folate served as the competitor in this assay. Red: Cy3-negative cells; Purple: Cy3-positive cells.




Supplementary Table 1: Sequences used for constructing the RNA nanoparticles with functionalities

Fragment Name Sequence (5’→ 3’) Folate-DNA oligo folate-CTCCCGGCCGCCATGGCCGCGGGATT

siRNA: UGACAGAUAAGGAACCUGCUU siRNA antisense strand

Scramble: AUAGUGGGACCAAUCAAGCUU

Fragment-1 (F1) GGCCAUGUGUAUGUGGGAAAAAAAACAAAUUCUUUACUGAUGAGUCCGUGAGGACGAAACGGGUCAAAAAAAACCCACAUACUUUGUUGAUCCAAUGACAGAUAAGGAACCUGCUUU

3WJ-

pRN

A

siSu

r-FA

-Rz-

FA

Fragment-2 (F2) GGCAGGUUCCUUAUCUGUCAAAGGAUCAAUCAUGGCCAAUCCCGCGGCCAUGGCGGCCGGGAG

Fragment-1 (F1) GGCCAUGUGUAUGUGGGGGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCCCCCACAUACUUUGUUGAUCCAAUGACAGAUAAGGAACCUGCUUU

3WJ-

pRN

A

siSu

r-FA

-MG

-FA


Fragment-1 (F1) ggCACCAGCGUUCCGGGAAAAAAAACAAAUUCUUUACUG(A)AUGAGUCCGUGAGGACGAAACGGGUCAAAAAAAACCCGGUUCGCCGCCAAAUGACAGAUAAGGAACCUGCUUU

3WJ-

5S r

RN

A

siSu

r-FA

-Rz-

FA

Fragment-2 (F2) GGCAGGUUCCUUAUCUGUCAAAAGGCGGCCAUAGCGGUGccAAUCCCGCGGCCAUGGCGGCCGGGAG

Fragment-1 (F1) ggCACCAGCGUUCCGGGGGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCCCCCGGUUCGCCGCCAAAUGACAGAUAAGGAACCUGCUUU

3WJ-

5S r

RN

A

siSu

r-FA

-Rz-

FA


Supplemental Table 2: Comparison of assembly and stability of the other 14 3WJ cores that was not practical for thorough investigations.

Assembly of 3WJ-pRNA harboring three pRNA monomers

Family Name Sequence

5’-3’

Native Gel 8M Urea Denaturing Gel

16s H22-H23-H23a a) GGA ACG CCG AUG GCG b) GAG AGG GUG GUG GAA U c) GGC AGC CAC CUG GU

No No

16s H25-H25-H26a a) CGC GUU AAG CGC b) GGG CCG AAG CU c) GCG CUA GGU CU

No No

23s H48-Hx-H60 a) GCC UAA UGG AU b) AAG CCA AGG C c) GUC CAU GGC GG

No No

A

23s H99-H100-H101 a) GAC GCG GUC GAU AGA CU b) UCC CGC GUA C c) AGC ACU AAC AGA

No No

16s H33-H33a-H33b a) AGG GAA CCC GG GU b) GGG AGC CCU c) GCC UGG GGU GCC C

Yes No

23s H33-H34-H35 a) UGU GUA GGG GUG b) GAC GAU CUA CGC A c) GGC CCA UCG AGU C

No No

16s H28-H29-H43 a) AUC GCU AGU AAU b) GUG AAU ACG UUC CCG GG c) CCC GCA CAA GCG GU

No No

B

16S H32-H33-H34 a) UCA GCA UGG CCC UUA CGG CCU GGG C b) CAC AGG UGC UGC AUG G c) UUA CCA GGC CUU GAC AUG

No No

RNase P B-type

a) AGG GCA GGA b) GGU AAA CCC CU c) UCC UUG AAA GUG CC

No No C

L11-rRNA

a) GCC AGG AUG UAG GCU b) AGC UCA CUG GU c) GCA GCC AUC AUU UAA AGA AAG CG

No No

M2/NF

a) UAG UAU GGC ACA UGA UUG GG b) CCC ACA UGU CAC GGG G c) CCC UCU UAC UA

Yes No

SF5

a) UAA UGU AUG UGU GUC GG b) CCG ACA GCA GGG GAG c) CUC UUG CAU UA

Yes No

B103

a) UAGUAUGGUGCGUGAUUGGG b) CCCACACGCCACGGGG c) CCCUCUUACUA

Yes No

Unknown

GA1

a) AUA UAU GGC UGU GCA ACG G b) CCG UUG ACA GGU UGU UGC c) GCA AUA CUA UAU AU

No No

Note: The sequences of the 3WJ cores were obtained from References 5-9. Families A, B and C are based on Lescoute and Westhof classification 5.




Supplementary Table 1: Sequences used for constructing the RNA nanoparticles with functionalities

Fragment Name Sequence (5’→ 3’) Folate-DNA oligo folate-CTCCCGGCCGCCATGGCCGCGGGATT

siRNA: UGACAGAUAAGGAACCUGCUU siRNA antisense strand

Scramble: AUAGUGGGACCAAUCAAGCUU

Fragment-1 (F1) GGCCAUGUGUAUGUGGGAAAAAAAACAAAUUCUUUACUGAUGAGUCCGUGAGGACGAAACGGGUCAAAAAAAACCCACAUACUUUGUUGAUCCAAUGACAGAUAAGGAACCUGCUUU

3WJ-

pRN

A

siSu

r-FA

-Rz-

FA


Fragment-1 (F1) GGCCAUGUGUAUGUGGGGGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCCCCCACAUACUUUGUUGAUCCAAUGACAGAUAAGGAACCUGCUUU

3WJ-

pRN

A

siSu

r-FA

-MG

-FA


Fragment-1 (F1) ggCACCAGCGUUCCGGGAAAAAAAACAAAUUCUUUACUG(A)AUGAGUCCGUGAGGACGAAACGGGUCAAAAAAAACCCGGUUCGCCGCCAAAUGACAGAUAAGGAACCUGCUUU

3WJ-

5S r

RN

A

siSu

r-FA

-Rz-

FA


Fragment-1 (F1) ggCACCAGCGUUCCGGGGGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCCCCCGGUUCGCCGCCAAAUGACAGAUAAGGAACCUGCUUU

3WJ-

5S r

RN

A

siSu

r-FA

-Rz-

FA


Supplemental Table 2: Comparison of assembly and stability of the other 14 3WJ cores that was not practical for thorough investigations.

Assembly of 3WJ-pRNA harboring three pRNA monomers

Family Name Sequence

5’-3’

Native Gel 8M Urea Denaturing Gel

16s H22-H23-H23a a) GGA ACG CCG AUG GCG b) GAG AGG GUG GUG GAA U c) GGC AGC CAC CUG GU

No No

16s H25-H25-H26a a) CGC GUU AAG CGC b) GGG CCG AAG CU c) GCG CUA GGU CU

No No

23s H48-Hx-H60 a) GCC UAA UGG AU b) AAG CCA AGG C c) GUC CAU GGC GG

No No

A

23s H99-H100-H101 a) GAC GCG GUC GAU AGA CU b) UCC CGC GUA C c) AGC ACU AAC AGA

No No

16s H33-H33a-H33b a) AGG GAA CCC GG GU b) GGG AGC CCU c) GCC UGG GGU GCC C

Yes No

23s H33-H34-H35 a) UGU GUA GGG GUG b) GAC GAU CUA CGC A c) GGC CCA UCG AGU C

No No

16s H28-H29-H43 a) AUC GCU AGU AAU b) GUG AAU ACG UUC CCG GG c) CCC GCA CAA GCG GU

No No

B

16S H32-H33-H34 a) UCA GCA UGG CCC UUA CGG CCU GGG C b) CAC AGG UGC UGC AUG G c) UUA CCA GGC CUU GAC AUG

No No

RNase P B-type

a) AGG GCA GGA b) GGU AAA CCC CU c) UCC UUG AAA GUG CC

No No C

L11-rRNA

a) GCC AGG AUG UAG GCU b) AGC UCA CUG GU c) GCA GCC AUC AUU UAA AGA AAG CG

No No

M2/NF

a) UAG UAU GGC ACA UGA UUG GG b) CCC ACA UGU CAC GGG G c) CCC UCU UAC UA

Yes No

SF5

a) UAA UGU AUG UGU GUC GG b) CCG ACA GCA GGG GAG c) CUC UUG CAU UA

Yes No

B103

a) UAGUAUGGUGCGUGAUUGGG b) CCCACACGCCACGGGG c) CCCUCUUACUA

Yes No

Unknown

GA1

a) AUA UAU GGC UGU GCA ACG G b) CCG UUG ACA GGU UGU UGC c) GCA AUA CUA UAU AU

No No

Note: The sequences of the 3WJ cores were obtained from References 5-9. Families A, B and C are based on Lescoute and Westhof classification 5.




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6. Chen, C., Zhang, C. & Guo, P., Sequence requirement for hand-in-hand interaction in formation of pRNA dimers and hexamers to gear phi29 DNA translocation motor. RNA 5, 805-818 (1999).

7. Honda, M., Beard, M. R., Ping, L. H. & Lemon, S. M., A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J. Virol. 73, 1165-1174 (1999).

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9. Kulshina, N., Edwards, T. E. & Ferre-D'Amare, A. R., Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch. RNA. 16, 186-196 (2010).




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