Design and construction of arbitrary two-dimensional DNA shapes
The design, synthesis, and proof of new DNA motifs having novel
geometries is a central endeavor in DNA nanotechnology. It typically
involves much computer design, sometimes requires extensive DNA
manipulation, and sometimes requires extensive characterization of the
new motif. Two previously successful methods for designing DNA motifs
are (1) Seeman's use of parallel DNA helices joined by antiparallel
DNA crossovers to create a variety of small multi-stranded motifs and
(2) Shih's use of paranemic crossover interactions to fold a long
single strand of DNA into a large three-dimensional octahedron.
Here, we present a new method for folding single-stranded DNA into
arbitrary two-dimensional shapes. The basic method draws on Seeman's
technique of engineering DNA nanostructures using parallel double helices
and antiparallel double crossovers. We have developed a computer program
that allows the quick design of DNA motifs having arbitrary shapes. We
experimentally demonstrate the folding of approximately seven kilobase
single-stranded DNAs into several shapes --- a square, a triangle and a
five-pointed star. The resulting DNA structures are approximately ten
times larger (in linear dimension) than the double crossovers often used
in DNA nanotechnology --- each with widths of approximately 100
nanometers. Proof of the structures is by correspondence of high
resolution AFM imaging with the structural design. These structures
represent the largest molecular structures ever constructed. Every atom
occurs in a unique and specific position in a structure whose mass is 5
megadaltons, approximately twice the mass of a ribosome. The size of the
structures generated appears limited only by the length of highly pure
single-stranded DNA that may be practically used, currently limiting the
size of the shapes created to approximately 10000 square nanometers. The
resolution with which the shape is rendered is 6 nanometers in one
direction and 3 nanometers in the other. It does not appear that this
resolution may be easily improved.
Our work adds new understanding to the process of designing and
constructing new motifs for DNA nanotechnology. First, it helps
clarify when and how much computer design must be performed on DNA
sequences for nanotechnology. Many of the sequences used in our DNA
nanoconstruction fail sequence design constraints previously used (and
held to be important) in the design of DNA nanostructures, and yet our
structures fold correctly anyway. This does not mean that in general
DNA design may be ignored, but in the specific instance or our
designs, certain features of the design (i.e. intramolecular folding)
weaken the need to apply some stringent constraints on the
design. Second, we show that the space of sequences that may be
assigned to antiparallel DNA crossovers is significantly larger than
previously reported. Our designs employ hundreds of previously
unexplored sequences for immobilized DNA crossovers. Third, a number
of helper strands are employed to fold our designs. In a normal DNA
nanoconstruction, these DNA strands would be purified and subjected to
a combinatorially exhaustive analysis of the possible complexes that
could be formed. We show that the purification of these strands and
subsequent combinatorial characterization is unnecessary for this
method of design.