RNA-DNA Hybrid Origami: Folding a Long RNA … · RNA-DNA Hybrid Origami: Folding a Long RNA Single Strands into Complex Nanostructures ... Forward primer (P1): 5'-ttctaatacgactcactataggtctgacagttaccaat-3

# Supplementary Material (ESI) for Chemical Communications # This journal is (c) The Royal Society of Chemistry 2010

RNA-DNA Hybrid Origami: Folding a Long RNA Single

Strands into Complex Nanostructures Pengfei Wang,a Seung Hyeon Ko,b± Cheng Tian,b Chenhui Hao b & Chengde Mao*b

Supplementary Information

Materials and Methods

Oligonucleotides:

Forward primer (P1): 5'-ttctaatacgactcactataggtctgacagttaccaat-3' (the T7 promoter sequence is underlined.); Reverse primer (P2): 5'-gacgaaagggcctcgt-3'. The sequences for RNA scaffold and DNA staples are given in Figure S1. Both primers and DNA staple strands were purchased from Integrated DNA technology (IDT) and used without further purification.

PCR amplification of DNA template:

The dsDNA template containing T7 promoter sequence was prepared by polymerase chain reaction (PCR) with a DNA plasmid pUC19 (New England Biolabs), primers (P1 and P2), and GoTaq Flexi DNA polymerase (Promega).

In vitro RNA transcription:

AmpliScribe™ T7-Flash™ Transcription Kit (Epicentre) was used to transcribe single-stranded RNA from DNA template, following the protocol provided with the kit. Briefly, a total of 20 uL mixture, including DNA template, ribonucleotides, RNase inhibitor together with T7-Flash RNA polymerase, was prepared in the provided reaction buffer, and then incubated at 42 °C for 120 mins. Then, transcription product was treated by DNase I to remove DNA template by incubating at 37 °C for 15 mins.

Quantification of RNA scaffold:

Purified RNA strands were quantified by a UV-Vis spectrophotometer. RNA strands without purification after in vitro transcription were indirectly quantified through agarose gel electrophoresis by using purified RNA as the calibrator. 1 μg of purified RNA was loaded into the gel, and unpurified RNA with known volume was loaded into the same gel. To compare the intensity of the two bands after staining with EtBr, the concentration of unpurified RNA could be obtained relatively based on the purified RNA with known concentration.

Regular assembly of RNA-DNA hybrid origami:

10 nM of RNA scaffold was mixed with DNA staples (at a ratio of 1:10 or specifically indicated value) in 1×TAE/Mg2+ buffer, which contains 40 mM Tris base (pH 8.0), 20 mM Acetic acid, 2 mM ethylenediamine tetraacetic acid (EDTA), and 12.5 mM Mg(OAc)2. The mixed aqueous solution was incubated under the following protocol: 10 mins each at 65, 50, 37 and 25 °C. All RNA-DNA hybrid origami structures were assembly by this method unless specifically indicated by isothermal assembly.

Isothermal assembly of RNA-DNA hybrid origami:

10 nM of RNA scaffold was mixed with DNA staples (at a ratio of 1:10) in 1×TAE/Mg2+ buffer, which contains 40 mM Tris base (pH 8.0), 20 mM Acetic acid, 2 mM ethylenediamine tetraacetic acid (EDTA), and 12.5 mM Mg(OAc)2. The mixed aqueous solution was incubated at one specified temperature for a specified duration. The assembly process was cryogenically stopped by being quenched into dry ice.

RNase H treatment

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

50 μL RNA-DNA hybrid origami sample (assembled from 10 nM RNA and 100 nM DNA staples) was incubated with 5 units of RNase H (New England Biolabs) in the provided RNase H buffer for 30 mins at 37 °C.

Agarose gel electrophoresis:

1% (for PCR amplification and RNA transcription characterization) and 2.5% (for RNA-DNA hybrid origami characterization) agarose gels were prepared with 1×TBE buffer (89 mM Tris base, 2 mM EDTA, 89 mM Boric acid) and run in the same buffer under constant voltage 8 V/cm. The gels were stained with EtBr solution and then visualized under a UV illuminator.

AFM imaging:

5 uL of RNA-DNA hybrid origami sample was deposited onto freshly cleaved mica for 20 seconds, washed with 30 uL of water and then dried with compressed air. A MultiMode 8 AFM (Bruker) was used to image the samples under ScanAsyst-Air mode, using a ScanAsyst-Air probe (Bruker).


TATCGTAGT TATCTACA CGACGGGG AGTCAGGC AACTATGGA TGAACGAA ATAGACAG ATCGCTGA GATAGGTGC CTCACTGA TTAAGCATTGG

CCAAUGCUUAA UCAGUGAG GCACCUAUC UCAGCGAU CUGUCUAU UUCGUUCA UCCAUAGUU GCCUGACU CCCCGUCG UGUAGAUA ACUACGAUA

CGGGAGGGC UUACCAUC UGGCCCCA GUGCUGCA AUGAUACCG CGAGACCC ACGCUCAC CGGCUCCA GAUUUAUCA GCAAUAAA CCAGCCAG CCGGAAGG GCCGAGCGCAGAAGUGG

TGCGCTCGGC CCTTCCGG CTGGCTGG TTTATTGC TGATAAATC TGGAGCCG GTGAGCGT GGGTCTCG CGGTATCAT TGCAGCAC TGGGGCCA GATGGTAA GCCCTCCCG

UCCUGCAACUUUAUCCG CCUCCAUC CAGUCUAU UAAUUGUU GCCGGGAAG CUAGAGUA AGUAGUUC GCCAGUUA AUAGUUUGC GCAACGUU GUUGCCAU UGCUACAG GCAUCGUGG C

CACGATGC CTGTAGCA ATGGCAAC AACGTTGC GCAAACTAT TAACTGGC GAACTACT TACTCTAG CTTCCCGGC AACAATTA ATAGACTG GATGGAGG CGGATAAAGT

5’

Rib

b1-

4R

ibb

1-3

Rib

b1-

1R

ibb

1-2

Rib

b1-

8R

ibb

1-6

Rib

b1-

7R

ibb

1-5

UGGUAUGGCUUCAUUCA

UGUCACGCUCGUCGUU

Rib

b1-

9

CAATGATGAGC ACTTTTAAA GTTCTGCT ATGTGGCG CGGTATTA TCCCGTATT

GACGCCGG GCAAGAGC AACTCGGT CGCCGCATACACTATTC

AUGGUUAUGGC AGCACUGC AUAAUUCU CUUACUGUC AUGCCAUC CGUAAGAU GCUUUUCU GUGACUGGU GAGUACUC AACCAAGU CAUUCUGA

GAAUAGUGUAUGCGGCG ACCGAGUU GCUCUUGC CCGGCGUC AAUACGGGA UAAUACCG CGCCACAU AGCAGAAC UUUAAAAGU GCUCAUCAUUG

TTCTGACAACG ATCGGAGG ACCGAAGG AGCTAACCG CTTTTTTG CACAACAT GGGGGATC ATGTAACTC GCCTTGAT CGTTGGGA ACCGGAGC

TCAGAATG ACTTGGTT GAGTACTC ACCAGTCAC AGAAAAGC ATCTTACG GATGGCAT GACAGTAAG AGAATTAT GCAGTGCT GCCATAACCAT

GCUCCGGU UCCCAACG AUCAAGGC GAGUUACAU GAUCCCCC AUGUUGUG CAAAAAAG CGGUUAGCU CCUUCGGU CCUCCGAU CGUUGUCAGAA

Rib

b2-

1

Rib

b2-

5

AGUGUUA UCACUC

GUAAGU UGGCCGC

Rib

b2-

4R

ibb

2-3

Rib

b2-

2

Rib

b2-

7R

ibb

2-6

Rib

b2-

8

3’

H

I

AUCUUUUAC UUUCACCA GCGUUUCU GGGUGAGC AAAAACAGG AAGGCAAA AUGCCGCA AAAAAGGG AAUAAGGGC GACACGGA AAUGUUGAAUA U

AUUGAAGCAU UUAUCAGG GUUAUUGUC UCAUGAGC GGAUACAU AUUUGAAU GUAUUUAGA AAAAUAAA CAAAUAGG GGUUCCGC GCACAUUUC CCCGAAAAGUGCCACCUGACGU

GAAAACGUUCU UCGGGGCG AAAACUCUC AAGGAUCU UACCGCUG UUGAGAUC CAGUUCGAU GUAACCCA CUCGUGCA CCCAACUG AUCUUCAGC

GAAATGTGC GCGGAACC CCTATTTG TTTATTTT TCTAAATAC ATTCAAAT ATGTATCC GCTCATGA GACAATAAC CCTGATAA ATGCTTCAATA

TATTCAACATT TCCGTGTC GCCCTTATT CCCTTTTT TGCGGCAT TTTGCCTT CCTGTTTTT GCTCACCC AGAAACGC TGGTGAAA GTAAAAGAT

GCTGAAGAT CAGTTGGG TGCACGAG TGGGTTAC ATCGAACTG GATCTCAA CAGCGGTA AGATCCTT GAGAGTTTT CGCCCCGA AGAACGTTTTC

Rib

b3-

3R

ibb

3-2

Rib

b3-

1

Rib

b3-

6R

ibb

3-4

Rib

b3-

5

CUCAU ACUCUUC

CUUUUUC AAUAU

Figure S1. Detailed design of the ribbon origami. Colored strand is the RNA scaffold strand and black strands are DNA staple strands.


GGUCUGACAGUUACCAA UGCUUAAUCAGUGAGG CACCUAUCUCAGCGAUC UGUCUAUUUCGUUCAU CCAUAGUUGCCUGACUC CCCGUCGUGUAGAUAA CUACGAUA CGGGAGGGC

AAAUAAACA AAUAGGGG UUCCGCGCACAUUUCC CCGAAAAGUGCCACCUG ACGUCUAAGAAACCAU UAUUAUCAUGACAUUAA CCUAUAAAAAUAGGCG UAUCACGAGGCCTUUUC GUC

GGCAAAAUGCCGCAAAA AAGGGAAUAAGGGCGA CACGGAAAUGUUGAAUA CUCAUACUCUUCCUUU UUCAAUAUUAUUGAAGC AUUUAUCAGGGUUAUU GUCUCAUG AGCGGATAC

AAACGUUCU UCGGGGCG AAAACUCUCAAGGAUC UUACCGCUGUUGAGAUC CAGUUCGAUGUAACCC ACUCGUGCACCCAACUG AUCUUCAGCAUCUUUU ACUUUCACCAGCGUUUC

CUUUUCUGUGACUGGUG AGUACUCAACCAAGUC AUUCUGAGAAUAGUGUA UGCGGCGACCGAGUUG CUCUUGCCCGGCGUCAA UACGGGAUAAUACCGC GCCACAUA GCAGAACUU

CCCCCAUGU UGUGCAAA AAAGCGGUUAGCUCCU UCGGUCCUCCGAUCGUU GUCAGAAGUAAGUUGG CCGCAGUGUUAUCACUC AUGGUUAUGGCAGCAC UGCAUAAUUCUCUUACU

GCUAGAGUAAGUAGUUC GCCAGUUAAUAGUUUG CGCAACGUUGUUGCCAU UGCUACAGGCAUCGUG GUGUCACGCUCGUCGUU UGGUAUGGCUUCAUUC AGCUCCGG UUCCCAACG

UGCAAUGAU ACCGCGAG ACCCACGCUCACCGGC UCCAGAUUUAUCAGCAA UAAACCAGCCAGCCGG AAGGGCCGAGCGCAGAA GUGGUCCUGCAACUUU AUCCGCCUCCAUCCAGU

GCCCTCCCG TATCGTAG TTATCTACACGACGGG GAGTCAGGCAACTATGG ATGAACGAAATAGACA GATCGCTGAGATAGGTG CCTCACTGATTAAGCA TTGGTAACTGTCAGACC A

CTGGATGGAGGCGGAT AAAGTTGCAGGACCAC TTCTGCGCTCGGCCCTT CCGGCTGGCTGGTTTA TTGCTGATAAATCTGGA GCCGGTGAGCGTGGGT CTCGCGGT ATCATTGCA C

GTTGGGAA CCGGAGCT GAATGAAGCCATACCA AACGACGAGCGTGACAC CACGATGCCTGTAGCA ATGGCAACAACGTTGCG CAAACTATTAACTGGC GAACTACTTACTCTAGC A

GTAAGAGAATTATGCA GTGCTGCCATAACCAT GAGTGATAACACTGCGG CCAACTTACTTCTGAC AACGATCGGAGGACCGA AGGAGCTAACCGCTTT TTTGCACA ACATGGGGG A

AGTTCTGC TATGTGGC GCGGTATTATCCCGTA TTGACGCCGGGCAAGAG CAACTCGGTCGCCGCA TACACTATTCTCAGAAT GACTTGGTTGAGTACT CACCAGTCACAGAAAAG G

AAACGCTGGTGAAAGT AAAAGATGCTGAAGAT CAGTTGGGTGCACGAGT GGGTTACATCGAACTG GATCTCAACAGCGGTAA GATCCTTGAGAGTTTT CGCCCCGA AGAACGTTT G

TATCCGCT CATGAGAC AATAACCCTGATAAAT GCTTCAATAATATTGAA AAAGGAAGAGTATGAG TATTCAACATTTCCGTG TCGCCCTTATTCCCTT TTTTGCGGCATTTTGCC G

AAAGGGCCTCGTGATA CGCCTATTTTTATAGG TTAATGTCATGATAATA ATGGTTTCTTAGACGT CAGGTGGCACTTTTCGG GGAAATGTGCGCGGAA CCCCTATT TGTTTATTT

UUACCAUCUG

GCCCCAGUGC

AUCAAGGCGA

GUUACAUGAU

UAAAAGUGCU

CAUCAUUGGA

AUAUUUGAAU

GUAUUUAGAA

CUAUUAAUUG

UUGCCGGGAA

GUCAUGCCAU

CCGUAAGAUG

UGGGUGAGCA

AAAACAGGAA

Re

ct

-1

.1,34nt

Re

ct

-1

.2,16nt

Re

ct

-1

.3,34nt

Re

ct

-1

.4,16nt

Re

ct

-1

.5,34nt

Re

ct

-1

.6,16nt

Re

ct

-1

.7,25nt

Re

ct

-3

.1,34nt

Re

ct

-3

.2,32nt

Re

ct

-3

.3,34nt

Re

ct

-3

.4,32nt

Re

ct

-3

.5,34nt

Re

ct

-3

.6,32nt

Re

ct

-3

.7,34nt

Re

ct

-5

.1,34nt

Re

ct

-5

.2,32nt

Re

ct

-5

.3,34nt

Re

ct

-5

.4,32nt

Re

ct

-5

.5,34nt

Re

ct

-5

.6,32nt

Re

ct

-5

.7,34nt

Re

ct

-7

.1,34nt

Re

ct

-7

.2,32nt

Re

ct

-7

.3,34nt

Re

ct

-7

.4,32nt

Re

ct

-7

.5,34nt

Re

ct

-7

.6,32nt

Re

ct

-7

.7,34nt

Re

ct

-8

.2,16nt

Re

ct

-8

.4,16nt

Re

ct

-8

.6,16nt

5’

3’

Figure S2. Detailed design of the rectangle origami. The red strand is the RNA scaffold strand and black strands are the DNA staple strands.


Figure S3. Detailed design of the triangle origami. Colored strand is the RNA scaffold strand and black strands are DNA staple strands.

TATCGTAGT TATCTACA CGACGGGG AGTCAGGC AACTATGGA TGAACGAA ATAGACAG ATCGCTGA GATAGGTGC CTCACTGA TTAAGCATTGG

CCAAUGCUUAA UCAGUGAG GCACCUAUC UCAGCGAU CUGUCUAU UUCGUUCA UCCAUAGUU GCCUGACU CCCCGUCG UGUAGAUA ACUACGAUA

CGGGAGGGC UUACCAUC UGGCCCCA GUGCUGCA AUGAUACCG CGAGACCC ACGCUCAC CGGCUCCA GAUUUAUCA GCAAUAAA CCAGCCAG CCGGAAGG GCCGAGCGCAGAAGUGG

CCACTTCTGCGCTCGGC CCTTCCGG CTGGCTGG TTTATTGC TGATAAATC TGGAGCCG GTGAGCGT GGGTCTCG CGGTATCAT TGCAGCAC TGGGGCCA GATGGTAA GCCCTCCCG

UCCUGCAACUUUAUCCG CCUCCAUC CAGUCUAU UAAUUGUU GCCGGGAAG CUAGAGUA AGUAGUUC GCCAGUUA AUAGUUUGC GCAACGUU GUUGCCAU UGCUACAG GCAUCGUGG UGUCACGCUCGUCGUU

AACGACGAGCGTGACA CCACGATGC CTGTAGCA ATGGCAAC AACGTTGC GCAAACTAT TAACTGGC GAACTACT TACTCTAG CTTCCCGGC AACAATTA ATAGACTG GATGGAGG CGGATAAAGTTGCAGGA

CAATGATGAGC ACTTTTAAA GTTCTGCT ATGTGGCG CGGTATTA TCCCGTATTGACGCCGG GCAAGAGC AACTCGGT CGCCGCATACACTATTC

AGUGUUA UCACUCAUG GUUAUGGC AGCACUGC AUAAUUCU CUUACUGUC AUGCCAUC CGUAAGAU GCUUUUCU GUGACUGGU GAGUACUC AACCAAGU CAUUCUGA

GAAUAGUGUAUGCGGCG ACCGAGUU GCUCUUGC CCGGCGUC AAUACGGGA UAAUACCG CGCCACAU AGCAGAAC UUUAAAAGU GCUCAUCAUUG

GCGGCCA ACTTACTTCTG ACAACG ATCGGAGG ACCGAAGG AGCTAACCG CTTTTTTG CACAACAT GGGGGATC ATGTAACTC GCCTTGAT CGTTGGGA ACCGGAGC TGAATGAAGCCATACCA

TCAGAATG ACTTGGTT GAGTACTC ACCAGTCAC AGAAAAGC ATCTTACG GATGGCAT GACAGTAAG AGAATTAT GCAGTGCT GCCATAAC CATGAGTGA TAACACT

UGGUAUGGCUUCAUUCA GCUCCGGU UCCCAACG AUCAAGGC GAGUUACAU GAUCCCCC AUGUUGUG CAAAAAAG CGGUUAGCU CCUUCGGU CCUCCGAU CGUUGU

CAGAAGUAAGU UGGCCGC

AUCUUUUAC UUUCACCA GCGUUUCU GGGUGAGC AAAAACAGG AAGGCAAA AUGCCGCA AAAAAGGG AATAAGGGC GACACGGA AAUGUUGA AUACUCAU ACUCUUC

CUUUUUC AAUAUUAU UGAAGCAU UUAUCAGG GUUAUUGUC UCAUGAGC GGAUACAU AUUUGAAU GUAUUUAGA AAAAUAAA CAAAUAGG GGUUCCGC GCACAUUUC CCCGAAAAGUGCCACCUGACGU

GAAAACGUUCU UCGGGGCG AAAACUCUC AAGGAUCU UACCGCUG UUGAGAUC CAGUUCGAU GUAACCCA CUCGUGCA CCCAACUG AUCUUCAGC

ACGTCAGGTGGCACTTTTCGGG GAAATGTGC GCGGAACC CCTATTTG TTTATTTT TCTAAATAC ATTCAAAT ATGTATCC GCTCATGA GACAATAAC CCTGATAA ATGCTTCA ATAATATT GAAAAAG

GAAGAGT ATGAGTAT TCAACATT TCCGTGTC GCCCTTATT CCCTTTTT TGCGGCAT TTTGCCTT CCTGTTTTT GCTCACCC AGAAACGC TGGTGAAA GTAAAAGAT

GCTGAAGAT CAGTTGGG TGCACGAG TGGGTTAC ATCGAACTG GATCTCAA CAGCGGTA AGATCCTT GAGAGTTTT CGCCCCGA AGAACGTTTTC

T10

3’

5’

T5

Tri 1

-1Tr

i 1-3

Tri 1

-2

Tri 1

-4Tr

i 1-6

Tri 1

-5Tr

i 1-7

Tri 2-1

Tri 2-3

Tri 2-2

Tri 2-4

Tri 2-5

Tri 2-7

Tri 2-6

Tri 2-8

Tri 3-1

Tri 3-2

Tri 3-4

Tri 3-6

Tri 3-5

T6

T7

Tri 3-3


Figure S4. Agarose gel analysis of the preparation of DNA template, RNA scaffold strand, and RNA-DNA hybrid origami structures. The RNase H experiments further confirm that the origami structures are hybrid assembly of RNA scaffold and DNA staples.

1.0 kb

1.5 kb

2.0 kb

3.0 kb

0.5 kb

1.0 kb

1 kb

DN

A m

arke

r

pUC

19

DN

A te

mpl

ate

from

PC

R

1 kb

DN

A m

arke

r

DN

A te

mpl

ate

from

PC

R

Tran

scrip

tion

mix

true

with

RN

A p

olym

eras

e

Tran

scrip

tion

mix

ture

w

ithou

t RN

A p

olym

eras

e

Purif

ied

RN

A


Figure S5. Length distribution of the ribbon origami measured from AFM images. Note that ribbons can stack onto each other and leads to ribbons longer than the expected value, 77 nm.

Table S1. Yield quantification of three origami structures (All counts are the observed objects in AFM images)

77177.8NATriangle

64260.1NARectangle

40167.370.3±11.4Ribbon

Total count (N)Yield (%)Length (nm)Origami structures


Figure S6. A negative control: assemblies from the transcription mixture without T7 RNA polymerase. No origami structure has been observed. This experiment confirms that the reported origami structures are indeed from RNA scaffolds instead of potential DNA template contaminations.

300 nm

Ribbon Rectangle Triangle

2 nm

-1 nm

300 nm 300 nm

Figure S7. RNA origami assembled from purified RNA strand.

Ribbon Rectangle Triangle

2 nm

-1 nm

300 nm 300 nm 300 nm


400 nm

25 °C

2 nm

-1 nm

37 °C 45 °C

55 °C 65 °C 70 °C

Figure S8. Isothermal assembly of the triangle origami at different temperature for 1 hour incubation (RNA:DNA = 1:10).


Figure S9. Isothermal assembly of triangle origami at 65 ºC for different incubation time (RNA:DNA = 1:10).

2 nm

-1 nm

1.5 min 3 min 5 min

10 min 15 min 30 min

1 hrs 2 hrs 4 hrs

8 hrs 16 hrs

400 nm


Figure S10. Assembly the triangle origami with different molecular ratio (RNA:DNA). For each condition, the assembly yield was calculated based on the observed objects in AFM imaging and given in parentheses.

1:10 (77.8%) 1:5 (73.5%) 1:2.5 (73.7%)

1:1 (70%) 1:0.5 (59.1%) 1:0.25 (12.1)

300nm

2 nm

-1 nm


Documents

RNA-DNA Hybrid Origami: Folding a Long RNA … · RNA-DNA Hybrid Origami: Folding a Long RNA Single Strands into Complex Nanostructures ... Forward primer (P1): 5'-ttctaatacgactcactataggtctgacagttaccaat-3