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Assembly of Novel DNA Cycles with Rigid Tetrahedral Linkers.

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Assembly of Novel DNA Cycles
with Rigid Tetrahedral Linkers**
Jufang Shi and Donald E. Bergstrom*
The chemical synthesis of components for the assembly of
supramolecular structures is likely to play a significant role in
the field of molecular nanotechnology.['l Assembly of complex
and highly differentiated supramolecular structures presents a
formidable task for synthetic chemists. If each unique component of a supramolecular assembly is to be positioned at a precise location in order to create devices capable of complex functions at the molecular level, it will be necessary to construct each
component with unique recognition elements as a code for selfassembly. Association of the natural nucleic acid components
adenine (A) with uracil (U) o r thymine (T), and cytosine (C)
with guanine (G) provides a precise and well-defined code for
molecular recognition. Sequences of the bases as components of
DNA and RNA associate through duplex formation with high
specificity. DNA and its structural analogues are attractive as
components for the coded self-assembly of molecular devices
because 1) they can be fabricated readily on automated synthesizers; 2) bases can be modified by chromophores, fluorophores,
redox-active groups, metal-complexing ligands, and other useful moieties without compromising base-pairing properties; 3)
sequences can be designed to bind to specific proteins, which in
turn could be structurally modified to function as molecular
electronic/photonic components ;and 4) self-assembly can occur
under mild conditions to give well-defined structures.
Seeman and co-workers have elegantly demonstrated the capacity of DNA for the construction of geometrical objects, including a cube and a truncated octahedron by a combination of
enzymatic and synthetic techniques.[**31 The polynucleotide
strands totally define the structures, serving both as edges and
vertices. By clever use of restriction enzymes and D N A ligase
completely closed structures are obtained. Potential uses of
DNA-based assemblies for applications in nanotechnology
have been de~cribed!~
- 'I
In a strategy complementary t o that devised by Seeman and
co-workers we have designed DNA-containing subunits in
which the D N A functions as connectors between rigid vertices.
The vertices were designed such that up to four oligonucleotide
arms are attached through rigid spacers to a single tetrahedral
hub. In principal one could construct the hubs (or vertices) with
different numbers of arms each containing different oligonucleotide sequences as a means to direct the construction of unique
supramolecular assemblies. A similar concept, but using shorter
spacers has been recently outlined by Stephan Jordan in collaboration with von Kiedrowski.r81
For proof of concept we chose a simple two-arm vertex consisting of two p-(2-hydroxyethyl)phenyIethynylphenyl spacers
attached to a single tetrahedral carbon atom (Figure 1). As
described below we have been able to demonstrate that oligonucleotide-conjugated vertices will self-assemble by hybridization
into a series of discrete cyclic supramolecular structures, which
['I
[**I
Prof. D E Bergztrom, J. Shi
Department of Medicinal Chemistry and Molecular Pharmacology
School of Pharmacy and Pharmacdf Sciences, Purdue University
West Lafayette, IN 47907.1333 (USA)
and
Walther Cancer Institute, Indrdnapohs, IN 46208 (USA)
This work was supported in part by a grant from the National Institutes of
Health (ROLA136601). The assistance of Norman Gerry (HPLC, gel electrophoresis, and phosphorimaging) is gratefully acknowledged.
Angex.. Cham. In!. E d Engl. 1997, 36, N o . 1 i Z
L2
d
4
51
O-~O-S-d(ATCGCATGCGAT,)-S
OH
4b in=3)
4~ in=5)
s
O-p-O-3-d(ATCGCATGCGAT,)-5
OH
Figure 1. Self-assembly of DNA cycles based on a rigid vertices attached to two
self-complementary oligonucleotides. The nonhybridized thymidine units at the
5'-ends of the oligonucleotides are symbolized by small rectangles
can be separated by gel electrophoresis under nondenaturing
conditions. Other synthetic D N A cycles have been constructed
but they differ significantly in both structure and concept.['In order to construct vertices containing two oligonucleotide
arms it was necessary to design a precursor that could be attached to a controlled pore glass (CPG) solid support while
leaving two free aliphatic hydroxyl groups for oligonucleotide
extension. Compound 1,L121
which contains two side chains terminating in trityl-protected alcohols and a third short arm attached to the central carbon atom, appeared ideally suited for
this application. Compound 1 was allowed to react with oxalyl
chloride (1 equiv) to yield intermediate 2, which was subsequently coupled to long-chain aminoalkyl controlled-pore glass
(LCAA-CPG) in situ (Figure 2). A similar strategy was reported
for the synthesis of a variety of base-sensitive oligonucleotide
derivatives." 31
Three different oligonucleotide sequences were constructed
on the two-arm linker derivatized LCAA-CPG by means of
standard phosphoramidite chemistry on a n automated synthesizer. Two modifications in the standard protocol were necessary to optimize the yield of the oligonucleotide conjugates. The
reaction time of the first detritylation step was increased to
insure complete cleavage of the more stable phenethyl trityl
ethers, and the reaction time of the first phosphoramidite coupling step was increased. In subsequent cycles the trityl release
was consistent and the overall yield varied ranged from 70 to
95 YO.The oligonucleotide conjugates were cleaved smoothly
from the solid support under standard conditions (see Experimental Section).
G VCH Verlagsgesellsrhaft mhH. 0-69451
Weinhrim, 1997
0570-0833i97i36of-of 11 $ fS.OO+ .ZS,(I
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TI0
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2
X=CI
3 x=NH-CPG
R
a
S-d(GCGCATATGCGC)-Y
b
S-d(TITAGCGTACGCTA)-3’
C
S-dil-ITlTAGCGTACGCTA)-Y
Figure 2 Synthesis of conjugates 4a-c. a) Oxalyl chloride, tetrazole, pyridine,
b) CPG-NH,; c) sequential addition of nucleoside phosphoramidites; d) NH,OH,
55 “ C , 5-8 h.
The solutions containing oligonucleotide conjugates 4a-4c
were treated with concentrated NH,OH to remove the protecting groups, and then diluted with H,O and Iyophilized. The
crude products were purified directly by polyacrylamide geI
electrophoresis (PAGE). Typically the major band (see Experimental Section for method of detection) corresponded to the
desired conjugates; for a two-arm 12-mer (24 nt) that band was
just below that of xylyl-cyanol (XC, 20% denatured PAGE,
corresponding to approximately a 28-mer) while bands of the
two-arm 14- and 16-mers (28 and 32 nt) ran above that of XC.
After denaturing gel electrophoresis, the desired bands were cut
and eluted in H,O, desalted either by C18 reverse-phase chromatography or filtration to yield
pure products. The structures of
the conjugates were confirmed
by matrix-assisted laser desorption ionization (MALDI) mass
spe~trometry.[’~]
Native PAGE was used to
characterize the hybridized
products from the oligonucleotide conjugates. The purified
two-arm
self-complementary
oligonucleotide conjugates 4a4c formed distinct “ladders” of
bands, which ranged in size continuously from 24 base-pairs to
98 base-pairs. Conjugate 4b,
which consists of a 12-mer selfcomplementary oligonucleotide
segment to which two additional
thymine (T) residues were attached, gave a relatively clean
“ladder”, in which at least eight
bands are clearly discernible
Figure 3. Polyacrylamide gel elec(Figure 3). Conjugate 4c with
trophoresis (PAGE; 2oy0,
ti&). A : Zn buffer, 4”C, 3 h ; B:
four extra T residues generally
TAE-Mg buffer, 4 ° C 40 h.
gave slightlv more diffuse bands.
(see Figure 1 ) are indicated.
112
tures.
Q VCH Verlagsgesellschaft mbH, D-69451 Weinheim.1997
DNA sequences to
rigid
hydrocarbon
0 ,, , , . , . , . ,
, , / ,
, . . , , , , . ,
Experimental Section
3: Oxalyl chloride (25 pL, 0.05 mM) was added to a solution of 1,2,4-triazole (20 mg,
0.29 mM) in acetonitrile (1 mL). The small amount ofprecipitate that formed disappeared after addition of pyridine (25 pL). A solution of vertex l(48.5 mg, 0.05 mM)
in acetonitrile (250 pL) and pyridine (25 pL) was added, the mixture stirred for 1 h,
and then LCAA-CPG (500 mg) was added. The mixture was allowed lo stand for
15 min. Then the liquid was removed and the solid was washed with acetonitrile. dry
methanol, acetonitrile, and a 1 :1 mixture of dimethylaminopyridine (0.3 M) and
acetic anhydride in THF. Unreacted 1 was recovered by chromatography (silica gel
60 PF254 containing gypsum; chromatotron (Harrison Research)). The support
was then washed with anhydrous DMF and anhydrous ether, and then air-dried
before it was loaded onto an ABI DNA synthesis column. The loading yield was
determined by carrying out a standard oligonucleotide synthesis using phosphoramidite chemistry with the derivatized CPG as described above. Coupling yields
were measured from the effluent after detritylation (dimethoxytrityl (DMTr)cation)
by using the standard protocol. The effluent from the detritylation following
the first phosphoramidite coupling from 7.5 mg solid support showed 0.95 A,,,
(30 mL dilution), which corresponded to a loading of 54 pmolg-’ DMTr Since
each linker has two hydroxyl groups, the actual loading of the linker was
27 pmol g- ’ .
Oligonucleotide synthesis: The oligonucleotide conjugates were synthesized on an
Applied Biosystem 381A synthesizer (0.2 pmol scale, pulsed-delivery cycle In the
“trityl off’ and “manual cleavage” mode). The syntheses were evaluated by UV
spectroscopic quantitation of trityl cation released during the trichloroacetic acid
0570-0833/97/3601-0112 3 15.00+ ,2510
Angew. Chem. Inl. Ed.
Engi. 1997, 36, N o . 1/2
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treatment step. The reaction time of the detritylation was extended by 4 min. that
of the first phosphoramidite coupling by 5 min.
Standard cleavage conditions (conc. NH,OH, room temperature, 5-20 min) were
employed to Iiherate the oligomer conjugates from the solid support. Deprotection
was accomplished by treatment with conc NH,OH at 5S'C for 5-8 h. The oligonucleotide conjugatc solutions were then diluted with fivefold deionized H,O, frozen
in dry ice'acetone. and lyophilized. The lyophilized oligomer conjugates were redissolved in aqueous solution and the amount of material quantitated by the UV
absorbance at i. = 260 nm. The oligomer conjugates were purified by PAGE ( I 5 or
20%) under denaturing conditions (8.3 M urea). Following electrophoresis the gels
were covered with plastic wrap and illuminated with a hand-held UV lamp; the
shadow o n a fluorescent TLC plate (Merck, EM Sciences) was photographed.
Subsequently. the oligomers were extracted from the crushed gel by autoclaved H,O
at room temperature overnight. and subsequently desalted either by reverse-phase
chromatography (C18 SEP-PAK) or by centrifugal concentration using an Amicon
Centricon-SRi (3000 MW cutoff) Concentrator The solutions of purified oligomer
conjugates were diluted with fivefold deionized H,O, separated into aliquots
(0.1 O D per aliquot), frozen. lyophilized, and stored in the dark at -20'C.
MALDI-TOF MS analysis wds carried out on a PerSeptives Voyager Biospectrometry Workstation Samples were prepared for mass spectrometric analysis by
combining 5 -30 pm of oligonucleotide with 0.5 pL of a matrix solution containing
0 0184 M M ammonium citrate, 0.0338 M picolinic acid, and 0.279 M 3-hydroxypicolinic acid: 4b: calcd 9136 [ M + l ] , found. 9131: 4c: calcd 10360 [ M +1], found:
I0 360
Hybridization and characterization of oligonucleotide conjugates. A solution of the
oligonucleotide conjugates (0.05 OD) in 15-30 pL of hybridization buffer (Zn
buffer: 12 5 mM ZnCI,, 50 mM HEPES; TMS buffer: 50 mM Tris, 10 mM MgCI,.
100 mM NaCI; TAE-Mg buffer: 40 mM Tris-acetate, 20 mM NaOAc, 2 mM EDTA,
12.5 mM MgCI,) was allowed to hybridize at three different temperatures (4,24, and
50 C) and varying times (from 10 min to >24 h). The effect of concentration on
hybridization was examined by dissolving 0 05 O D units of the oligonucleotide
conjugate in varking amounts of TAE-Mg buffer (12.2, 36.6, and 244 pL) and by
comparing the PAGE results
Native PAGE. In most cases I S or 20% native polyacrylamide gels (15x 17cm)
were cast. allowed to polymerize for a t least 45 min (not more than 2 h), and then
subjected to electrophoresis for 30 min before the samples were loaded. The gels
were electrophoresed for 8- 12 h at 8- 10 Vcm-' in TBE buffer (89 mM Tris, 89 mM
boric acid. 2.5 mM Na,EDTA. adjusted t o pH 8.0 with conc. HCI) at 4'C. The
migration distances were measured from the respective loading wells for each
lane by means of an ethidium bromide stained transillummat~ng photograph
(7.5xlOcm).
Received. August 15, 1996 [296271E]
German version: Angew Chem. 1997, 109. 70-72
Keywords: DNA nucleotides * supramolecular chemistry
[l] K. E. Drexler. Nanosysrems: Molecular Machinery, Munufacturing, and Computarion, Wiley, New York, 1992.
[2] J. Chen. N. C. Seeman, Nature 1991, 350, 631.
131 Y. Zhang. N C. Seeman. J. Am. Chem. Soc. 1994, 116, 1661.
[4] J. B Tucker. High Technology 1984, 4, 36.
[5] N. C. Seeman, Nunolechnology 1991, 2, 149.
[6] N. C. Seeman. J. Biomol. Sfrurf.Dynamics 1985, 3, 11.
[7] M. J. Heller. R . H Tullis, Nuno/echnology 1991, 2, 165.
[8] S Jordan. Dissertation, Universitit Gottingen, 1993.
[9] S. 1. Rumney. E. T. Kool. Angel?. Chem. 1992. 104, 1686; Angew. Chem. Inr.
Ed. Engl. 1992. 31. 1617.
[lo] H. Gao, N. Chidamabdram, B. C. Chen, D. E. Pelham, R. Patel, R. Yang. A.
Zhou. A. Cook, J S. Cohen, Bioconjugak Chem. 1994,5,445.
[ l l ] JLH Chen. N . R Kallenbach, N. C. Seeman, J. Am. Chem. SOC.1989, 111,
6402
1121 Compound 1 was prepared in two steps from 2.2-diphenylethanol by iodination followed by palladium-catalyzed coupling with p-(trityloxyethy1)phenylethyne.
[I 31 R. H. Alul. C. N . Singman, G . Zhang, R. L. Letsinger, Nucleic Acids Res 1991.
19. 1527.
1141 M C. Fitzgerdld. L M. Smith, Annu. Rev. Biophys. Biomol. Slruci. 1995, 24,
I 1 7.
[IS] Y Wang. 1. E Mueller. B. Kemper, N. C. Seeman, Biochemistry 1991,30,5667.
Angel<,
Clicm. In(. Ed E n d 1991, 36, No. 112
[Me(PhMe,Si),SiLi] and [Ph(Me,Si),SiLi] :
Preparation, Characterization, and Evidence for
an Intramolecular Li-Ph Interaction**
Akira Sekiguchi,* Masato Nanjo, Chizuko Kabuto,
and Hideki Sakurai*
Despite numerous reports on syntheses of lithiosilanes far
less attention has been devoted to their structures in the solid
state."] Up to now, a few crystal structures of lithiosilanes containing chelating solventsI2' and only two examples of unsolvated hexameric lithiosilanes Me,SiLiI3] and tetrameric Me,Si,Li,[41 have been reported. We report here on the isolation and
characterization of the first unsolvated dimeric lithiosilanes, 1a
and 1b. The evidence for an intramolecular lithium-phenyl
interaction is also described.
Bis( 1,3-diphenylpentamethyItrisilyl)mercury was treated with
lithium metal in toluene to give yellow crystals of 1a [Eq. (a)].[51
By the same procedure, 1b was also obtained as yellow crystals.[']
he ri
the
he ii he
la: R'
I
Me, R2 = Ph
lb R'=Ph,R2=Me
Lithiosilane 1 a is dimeric in the solid state and its molecular
structure has a crystallographic inversion center (Figure 1).I7]
The two lithium and two anionic silicon centers constitute a
planar four-membered ring with Li-Si distances of 2.664(5) and
2.778(7) A, a Li-Si-Li bond angle of 58.1(2)', and a Li-Li
distance of 2.65(1) A. The two Si2-Si bond lengths are 2.336(1)
and 2.361(1) A. The Si2-CH3 bond length of 1.923(4) 8, is considerably longer than normal (1.88 A) due to the negative charge
on the silicon center. The bond angles around the anionic silicon
are highly contracted (sum of the angles: 312.1(1)'). The most
striking point of the structure is that each lithium atom is surrounded by the four phenyl groups. Lil and Lil' are not located
directly over the centers of the benzene rings, but are in rather
close contact with the ips0 and ortho carbon atoms. These Li-C
distances (e.g. Li1'-C12 2.348(6), Lil'-C13 2.483(7), Li1'-C17
2.730(8) A) are typical for x-complexed organolithiums.181Such
interactions are apparently caused by electrostatic attraction
between the lithium ion and the benzene rings.
The dimeric structure of lithiosilane 1b was also established
by X-ray diffraction (Figure 2) .I7]The structural parameters are
[*I Prof. Dr. A. Sekiguchi
Department of Chemistry, University of Tsukuba
Tsukuba. Ibaraki 305 (Japan)
Fax: Int. code +(298) 53-4314
e-mail : sekiguch(a staff chem.tsukuba.ac.jp
Prof. Dr. H. Sakurai
Department of Industrial Chemistry, Faculty of Science and Technology
Science University of Tokyo
Noda. Chiba 278 (Japan)
Dipl.-Chem. M. Nanjo. Dr. C. Kabuto
Department of Chemistry and Organosilicon Research Laboratory
Faculty of Science, Tohoku University
Aoba-ku, Sendai 980-77 (Japan)
[**I This work was supported by Grants-in-Aid for Specially Promoted Research
(No. 02102004), for Scientific Research on Priority Area of Reactive Organometallics (No. 05236102), and for Scientific Research (No. 07454159) from the
Ministry of Education, Science and Culture. M. N. thanks the Japan Society
for Promotion of Science for a fellowship
&? VCH Verlagsgesellschaft mbH, 0-6945f Weinheim, 1997
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113
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