Supporting Information: Self-assembly of functionalized RNA nanoparticles
demonstrating potential advancements in automated nanomedicine
Kirill A. Afonin1,2*, Wade W. Grabow1*, Faye M. Walker1, Eckart Bindewald3, Marina A.
Dobrovolskaia4, Bruce A. Shapiro2#, and Luc Jaeger1#
1
Department of Chemistry and Biochemistry, Biomolecular Science and Engineering Program,
University of California, Santa Barbara, CA 93106-9510, USA
2
Center for Cancer Research Nanobiology Program, NCI-Frederick, Frederick, MD 21702, USA
3
Basic Science Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702, USA
4
Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick
Inc., NCI-Frederick, Frederick, MD 21702, USA
* - these authors contributed equally to this project
# - to whom correspondence should be addressed: Luc Jaeger, phone 805-893-3628; fax 805893-4120; e-mail: jaeger@chem.ucsb.edu; Bruce Shapiro, phone 301-846-5536; fax 301-8465598; email: shapirbr@mail.nih.gov
RNA sequences used in this project
Non-concatenated cube and ring scaffold sequences are used from previously published works1, 2.
Sense and antisense concatenated cube sequences:
Letter sequences below the RNA strands indicate the desired interactions between different
strands. Dots indicate the parts of the strands that are per design single-stranded. Sequence
characters in upper case represent the cube scaffold; siRNA-related sequence regions are denoted
in lower case. RNA sequences were obtained regions related to the EGFPS1 siRNA by in vitro
transcription of PCR generated DNA templates.
3` antisense siRNA (EGFPS1) modifications of RNA cubes with 10 bp per side
and 3Us at the corners
siA
GGCAACUUUGAUCCCUCGGUUUAGCGCCGGCCUUUUCUCCCACACUUUCACGuucgguggugcagaugaacuucaggguca
MMMMMM...KKKKKKLLLL...FFFFFFGGGG...BBBBBBCCCC...NNNN..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
siB
GGGAAAUUUCGUGGUAGGUUUUGUUGCCCGUGUUUCUACGAUUACUUUGGUCuucgguggugcagaugaacuucaggguca
QQQQQQ...PPPPPPPPPP...MMMMMMNNNN...EEEEEEEEEE...RRRR..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
siC
GGACAUUUUCGAGACAGCAUUUUUUCCCGACCUUUGCGGAUUGUAUUUUAGGuucgguggugcagaugaacuucaggguca
IIIIII...OOOOOOOOOO...QQQQQQRRRR...DDDDDDDDDD...JJJJ..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
siD
GGCGCUUUUGACCUUCUGCUUUAUGUCCCCUAUUUCUUAAUGACUUUUGGCCuucgguggugcagaugaacuucaggguca
FFFFFF...HHHHHHHHHH...IIIIIIJJJJ...AAAAAAAAAA...GGGG..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
siE
GGGAGAUUUAGUCAUUAAGUUUUACAAUCCGCUUUGUAAUCGUAGUUUGUGUuucgguggugcagaugaacuucaggguca
BBBBBB...AAAAAAAAAA...DDDDDDDDDD...EEEEEEEEEE...CCCC..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
siF
GGGAUCUUUACCUACCACGUUUUGCUGUCUCGUUUGCAGAAGGUCUUUCCGAuucgguggugcagaugaacuucaggguca
KKKKKK...PPPPPPPPPP...OOOOOOOOOO...HHHHHHHHHH...LLLL..ZZZZZZZZZZZZZZZZZZZZZZZZZ..
sense
acccugaaguucaucugcaccaccg
ZZZZZZZZZZZZZZZZZZZZZZZZZ
5` sense siRNA (EGFPS1) modifications of RNA cubes with 10 bp per side and
3Us at the corners
siA
ggcaagcugacccugaaguucauccaaGGCAACUUUGAUCCCUCGGUUUAGCGCCGGCCUUUUCUCCCACACUUUCACG
ZZZZZZZZZZZZZZZZZZZZZZZZZ..MMMMMM...KKKKKKLLLL...FFFFFFGGGG...BBBBBBCCCC...NNNN
siB
ggcaagcugacccugaaguucauccaaGGGAAAUUUCGUGGUAGGUUUUGUUGCCCGUGUUUCUACGAUUACUUUGGUC
ZZZZZZZZZZZZZZZZZZZZZZZZZ..QQQQQQ...PPPPPPPPPP...MMMMMMNNNN...EEEEEEEEEE...RRRR
siC
ggcaagcugacccugaaguucauccaaGGACAUUUUCGAGACAGCAUUUUUUCCCGACCUUUGCGGAUUGUAUUUUAGG
ZZZZZZZZZZZZZZZZZZZZZZZZZ..IIIIII...OOOOOOOOOO...QQQQQQRRRR...DDDDDDDDDD...JJJJ
siD
ggcaagcugacccugaaguucauccaaGGCGCUUUUGACCUUCUGCUUUAUGUCCCCUAUUUCUUAAUGACUUUUGGCC
ZZZZZZZZZZZZZZZZZZZZZZZZZ..FFFFFF...HHHHHHHHHH...IIIIIIJJJJ...AAAAAAAAAA...GGGG
siE
ggcaagcugacccugaaguucauccaaGGGAGAUUUAGUCAUUAAGUUUUACAAUCCGCUUUGUAAUCGUAGUUUGUGU
ZZZZZZZZZZZZZZZZZZZZZZZZZ..BBBBBB...AAAAAAAAAA...DDDDDDDDDD...EEEEEEEEEE...CCCC
siF
ggcaagcugacccugaaguucauccaaGGGAUCUUUACCUACCACGUUUUGCUGUCUCGUUUGCAGAAGGUCUUUCCGA
ZZZZZZZZZZZZZZZZZZZZZZZZZ..KKKKKK...PPPPPPPPPP...OOOOOOOOOO...HHHHHHHHHH...LLLL
antisense (As)
ggaugaacuucagggucagcuugccuu
ZZZZZZZZZZZZZZZZZZZZZZZZZ..
3` antisense siRNA (HIV-1) modifications of RNA cubes with 10 bp per side and
3Us at the corners
The names of corresponding siRNAs 3, 4 are indicated for each concatenated cube strand.
siA_ldr
GGCAACUUUGAUCCCUCGGUUUAGCGCCGGCCUUUUCUCCCACACUUUCACGuugacggacucgcacccaucucucuccuu
MMMMMM...KKKKKKLLLL...FFFFFFGGGG...BBBBBBCCCC...NNNN..SSSSSSSSSSSSSSSSSSSSSSSSS..
siB_nef
GGGAAAUUUCGUGGUAGGUUUUGUUGCCCGUGUUUCUACGAUUACUUUGGUCuuggaggaaauuagcccuuccagucccuu
QQQQQQ...PPPPPPPPPP...MMMMMMNNNN...EEEEEEEEEE...RRRR..TTTTTTTTTTTTTTTTTTTTTTTTT..
siC_pol
GGACAUUUUCGAGACAGCAUUUUUUCCCGACCUUUGCGGAUUGUAUUUUAGGuucuaagacuguaucaucugcuccuguuu
IIIIII...OOOOOOOOOO...QQQQQQRRRR...DDDDDDDDDD...JJJJ..UUUUUUUUUUUUUUUUUUUUUUUUU..
siD_r/t
GGCGCUUUUGACCUUCUGCUUUAUGUCCCCUAUUUCUUAAUGACUUUUGGCCuucgcugacuccgcuucuuccugccauuu
FFFFFF...HHHHHHHHHH...IIIIIIJJJJ...AAAAAAAAAA...GGGG..VVVVVVVVVVVVVVVVVVVVVVVVV..
siE_gag
GGGAGAUUUAGUCAUUAAGUUUUACAAUCCGCUUUGUAAUCGUAGUUUGUGUuuccuggaaugcugucaucauuucuucuu
BBBBBB...AAAAAAAAAA...DDDDDDDDDD...EEEEEEEEEE...CCCC..WWWWWWWWWWWWWWWWWWWWWWWWW..
siF_pol47
GGGAUCUUUACCUACCACGUUUUGCUGUCUCGUUUGCAGAAGGUCUUUCCGAuuucuugaauuacuacugccccuucacuu
KKKKKK...PPPPPPPPPP...OOOOOOOOOO...HHHHHHHHHH...LLLL..XXXXXXXXXXXXXXXXXXXXXXXXX..
Corresponding siRNA sense strands
ldr
ggagagagaugggugcgaguucguc
SSSSSSSSSSSSSSSSSSSSSSSSS
nef
gggacuggaagggcuaauuuucucc
TTTTTTTTTTTTTTTTTTTTTTTTT
pol
acaggagcagaugauacaguuuuag
UUUUUUUUUUUUUUUUUUUUUUUUU
r/t
auggcaggaagaagcggaguuagug
VVVVVVVVVVVVVVVVVVVVVVVVV
gag
gaagaaaugaugacagcauuucagg
WWWWWWWWWWWWWWWWWWWWWWWWW
pol47
gugaaggggcaguaguaauuuaaga
XXXXXXXXXXXXXXXXXXXXXXXXX
Sense and antisense concatenated ring sequences:
Bold letter sequences indicate kissing loop regions. siRNA sequences used for NR modifications
are identical to those in the case of cube NPs shown above. RNA sequences were obtained by in
vitro transcription of PCR generated DNA templates.
3` antisense siRNA (EGFPS1) modifications of RNA rings
((((((((.......))))))))(((((((.......))))))).............................
siA
GGGAAUCCGUCCACUGGAUUCCCGUCACAGAGCCUGCCUGUGACuucgguggugcagaugaacuucaggguca
siB
GGGAAUCCGCAGGCUGGAUUCCCGUCACAGAGAACGCCUGUGACuucgguggugcagaugaacuucaggguca
siC
GGGAAUCCGCGUUCUGGAUUCCCGUCACAGACGUCUCCUGUGACuucgguggugcagaugaacuucaggguca
siD
GGGAAUCCGAGACGUGGAUUCCCGUCACAGUCGUGGUCUGUGACuucgguggugcagaugaacuucaggguca
siE
GGGAAUCCACCACGAGGAUUCCCGUCACAGAACCAUCCUGUGACuucgguggugcagaugaacuucaggguca
siF
GGGAAUCCGAUGGUUGGAUUCCCGUCACAGAGUGGACCUGUGACuucgguggugcagaugaacuucaggguca
sense
acccugaaguucaucugcaccaccg
5` sense siRNA (EGFPS1) modifications of RNA rings
...........................((((((((.......))))))))(((((((.......)))))))
siA
ggcaagcugacccugaaguucauccaaGGGAAUCCGUCCACUGGAUUCCCGUCACAGAGCCUGCCUGUGAC
siB
ggcaagcugacccugaaguucauccaaGGGAAUCCGCAGGCUGGAUUCCCGUCACAGAGAACGCCUGUGAC
siC
ggcaagcugacccugaaguucauccaaGGGAAUCCGCGUUCUGGAUUCCCGUCACAGACGUCUCCUGUGAC
siD
ggcaagcugacccugaaguucauccaaGGGAAUCCGAGACGUGGAUUCCCGUCACAGUCGUGGUCUGUGAC
siE
ggcaagcugacccugaaguucauccaaGGGAAUCCACCACGAGGAUUCCCGUCACAGAACCAUCCUGUGAC
siF
ggcaagcugacccugaaguucauccaaGGGAAUCCGAUGGUUGGAUUCCCGUCACAGAGUGGACCUGUGAC
antisense (As)
ggaugaacuucagggucagcuugccuu
3` antisense siRNA (HIV-1) modifications of RNA rings
The names of corresponding siRNAs 3, 4 are indicated for each concatenated ring strand.
((((((((.......))))))))(((((((.......))))))).............................
siA_ldr
GGGAAUCCGUCCACUGGAUUCCCGUCACAGAGCCUGCCUGUGACuugacggacucgcacccaucucucuccuu
siB_r/t
GGGAAUCCGCAGGCUGGAUUCCCGUCACAGAGAACGCCUGUGACuucgcugacuccgcuucuuccugccauuu
siC_gag
GGGAAUCCGCGUUCUGGAUUCCCGUCACAGACGUCUCCUGUGACuuccuggaaugcugucaucauuucuucuu
siD_pol47
GGGAAUCCGAGACGUGGAUUCCCGUCACAGUCGUGGUCUGUGACuuucuugaauuacuacugccccuucacuu
siE_pol
GGGAAUCCACCACGAGGAUUCCCGUCACAGAACCAUCCUGUGACuucuaagacuguaucaucugcuccuguuu
siF_nef
GGGAAUCCGAUGGUUGGAUUCCCGUCACAGAGUGGACCUGUGACuuggaggaaauuagcccuuccagucccuu
Corresponding siRNA sense strands
ldr
ggagagagaugggugcgaguucguc
nef
gggacuggaagggcuaauuuucucc
pol
acaggagcagaugauacaguuuuag
r/t
auggcaggaagaagcggaguuagug
gag
gaagaaaugaugacagcauuucagg
pol47
gugaaggggcaguaguaauuuaaga
References
1.
2.
3.
4.
Afonin, K.A. et al. In vitro assembly of cubic RNA-based scaffolds designed in silico.
Nat Nanotechnol 5, 676-682 (2010).
Grabow, W.W. et al. Self-assembling RNA nanorings based on RNAI/II inverse kissing
complexes. Nano letters 11, 878-887 (2011).
Berkhout, B. & Sanders, R.W. Molecular strategies to design an escape-proof antiviral
therapy. Antiviral research (2011).
Liu, Y.P. et al. Combinatorial RNAi against HIV-1 using extended short hairpin RNAs.
Mol Ther 17, 1712-1723 (2009).