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`
`DNA assembly using bis-peptide nucleic acids (bisPNAs)
`
`Article  in  Nucleic Acids Research · August 2002
`
`DOI: 10.1093/nar/gkf389 · Source: PubMed
`
`CITATIONS
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`2 authors, including:
`
`David Reid Corey
`University of Texas Southwestern Medical Center
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`2782–2789 Nucleic Acids Research, 2002, Vol. 30, No. 13
`
`© 2002 Oxford University Press
`
`DNA assembly using bis-peptide nucleic acids
`(bisPNAs)
`Christopher J. Nulf and David R. Corey*
`
`Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas,
`5323 Harry Hines Boulevard, Dallas, TX 75390-9041, USA
`
`Received March 21, 2002; Revised and Accepted April 30, 2002
`
`ABSTRACT
`DNA nanostructures are ordered oligonucleotide
`arrangements that have applications for DNA
`computers, crystallography, diagnostics and mater-
`ial sciences. Peptide nucleic acid (PNA) is a DNA/
`RNA mimic that offers many advantages for hybrid-
`ization, but its potential for application in the field of
`DNA nanotechnology has yet to be thoroughly exam-
`ined. We report the synthesis and characterization of
`tethered PNA molecules (bisPNAs) designed to
`assemble two individual DNA molecules through
`Watson–Crick base pairing. The spacer regions link-
`ing the PNAs were varied in length and contained
`amino acids with different electrostatic properties.
`We observed that bisPNAs effectively assembled
`oligonucleotides that were either the exact length of
`the PNA or that contained overhanging regions that
`projected outwards. In contrast, DNA assembly was
`much less efficient if the oligonucleotides contained
`overhanging regions that projected inwards. Surpris-
`ingly, the length of the spacer region between the
`PNA sequences did not greatly affect the efficiency
`of DNA assembly. Reasons for inefficient assembly
`of inward projecting DNA oligonucleotides include
`non-sequence-specific intramolecular
`interactions
`between the overhanging region of the bisPNA and
`steric conflicts that complicate simultaneous binding
`of
`two inward projecting strands. These results
`suggest that bisPNA molecules can be used for self-
`assembling DNA nanostructures provided that the
`arrangement of
`the hybridizing DNA oligonucle-
`otides does not interfere with simultaneous hybrid-
`ization to the bisPNA molecule.
`
`based on subunits of fixed Holliday junctions (2), streptavidin–
`DNA fragment nanoparticle networks (3) and DNA dendrimer
`formations for drug delivery (4; for reviews see 5–8).
`Peptide nucleic acids (PNAs) (Fig. 1A) are a promising
`connector for the assembly of DNA-based nanostructures.
`PNAs are synthetic DNA analogs containing a neutral 2-
`aminoethylglycine backbone (9) and hybridize sequence
`specifically to complementary DNA and RNA oligonucle-
`otides (10). Binding occurs with high affinity (10,11), high
`sensitivity to mismatch discrimination (12) and is unaffected
`by the ionic strength (10). Because PNAs possess a neutral
`amide backbone they bind less to proteins than do oligomers
`with negatively charged linkages (13,14) and are nuclease and
`protease resistant (15). PNAs have an exceptional ability to
`hybridize to sequences within duplex DNA by strand invasion,
`suggesting that PNA molecules should also be superior agents
`for binding to single-stranded DNA at regions that contain
`intramolecular
`structure (16,17). An important practical
`advantage is that methods for PNA synthesis are compatible
`with peptide synthesis, allowing PNAs to be easily modified
`with amino acids and other moieties (18,19). High affinity
`binding by PNAs has already been used for nanostructure
`assembly, with applications for labeling of DNA (20,21) and
`strand invasion into DNA hairpins and tetraloop motifs (22; for
`reviews see 23,24).
`Here we describe the synthesis of bisPNAs that contain
`spacer regions that differ in length and amino acid substitution.
`We characterize the ability of bisPNAs to assemble DNA and
`observe that the capacity of a bisPNA molecule to hybridize to
`two oligonucleotides is primarily dependent on the arrange-
`ment of the DNA oligonucleotides being assembled. These
`results suggest that bisPNA molecules with simple chemical
`modifications can be used in generating DNA:bisPNA:DNA
`‘units’ for nanotechnology and DNA nanostructure assembly,
`but that proper orientation of the assembled DNA oligonucle-
`otides is essential.
`
`INTRODUCTION
`
`MATERIALS AND METHODS
`
`Nanotechnology involves assembly of small molecules into
`complex architectures for higher function (1). The canonical
`Watson–Crick base pairing of adenine to thymine and guanine
`to cytosine is ideal for organizing biomolecules in a highly
`predictable fashion. Numerous reports on using oligonucle-
`otides to build higher order structures include DNA matrices
`
`PNA synthesis
`PNA monomers (Fig. 1), 2-aminoethoxy-2-ethoxy acetic acid
`(AEEA) and other reagents for PNA synthesis were obtained
`from PE Biosystems (Foster City, CA). Fmoc-amino acids
`were obtained from Advanced Chemtech (Louisville, KY) or
`Calbiochem-Novabiochem Corp. (La Jolla, CA). PNAs were
`
`*To whom correspondence should be addressed. Tel: +1 214 648 5096; Fax +1 214 648 5095; Email: david.corey@utsouthwestern.edu
`
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`prepared by automated synthesis using an Expedite 8909
`synthesizer (PE Biosystems) as previously described (25).
`PNAs were analyzed and purified by reverse phase high
`performance liquid chromatography (RP-HPLC) and mass
`spectral analysis by matrix-assisted laser desorption ionization
`time of flight (MALDI-TOF) as previously described (25).
`Briefly, RP-HPLC analysis and purification of bisPNAs
`involved a running buffer of dH2O/0.1% trifluoroacetic acid
`and an elution buffer of acetonitirile/0.1% trifluoroacetic acid
`from a C18 column inside a water jacket maintained at 55°C.
`Elution of PNA was monitored at 260 nm on a Dynamax UV-1
`absorbance detector (Varian, Walnut Creek, CA). After puri-
`fication, PNAs were lyophilized and resuspended in dH2O.
`Mass spectrometry was performed by MALDI-TOF on a
`Voyager-DE Workstation (Applied Biosystems, Foster City,
`CA) using either α-cyano-4-hydroxycinnamic
`acid or
`sinapinic acid solution as a matrix (Sigma-Aldrich, St Louis,
`MO). The concentration of PNA solutions was measured using
`a Cary 100Bio UV-Visible spectrophotometer (Varian) at
`260 nm and room temperature, using an extinction coefficient
`of 241.4 ml µmol–1 cm–1 (all of the bisPNA sequences are the
`same). The extinction coefficients of PNA 24 and PNA 26 are
`127.8 ml µmol–1 cm–1 and 113.6 ml µmol–1 cm–1, respectively.
`Preparation of DNA oligonucleotides
`Technologies,
`DNA oligonucleotides
`(Invitrogen-Life
`Carlsbad, CA) were radiolabeled using [γ-32P]ATP (Amer-
`sham Pharmacia Biotech, Piscataway, NJ) using T4 polynucle-
`otide kinase (Sigma-Aldrich). Unincorporated [γ-32P]ATP was
`removed by passing the oligonucleotides through a Bio-Spin 6
`column (Bio-Rad Laboratories, Hercules, CA) that had been
`pre-equilibrated with distilled water. Equal amounts of un-
`labeled oligonucleotides were treated similarly and purified by
`Bio-Spin 6 column in parallel. These unlabeled oligonucle-
`otides were used to estimate the concentration of the radio-
`labeled oligomers that had been treated similarly. Unlabeled
`oligonucleotides were also used for gel shift experiments. The
`concentration of each unlabeled DNA oligonucleotide was
`calculated on a Cary 100Bio UV-Visible spectrophotometer
`(Varian) at 260 nm using the extinction coefficient given by
`the manufacturer. For each 40mer and 50mer oligonucleotide
`the nucleobases were randomized, except for the complemen-
`tary 12mer target sequences, to minimize the potential for for-
`mation of specific secondary structures.
`
`Melting temperature (Tm) analysis
`Melting temperature (Tm) experiments were performed on a
`Cary 100Bio UV-Visible spectrophotometer (Varian) at 260 nm.
`BisPNA and DNA oligonucleotides were suspended in
`Na2HPO4 buffer (100 µM, pH 7.5) at 2 µM each. The tempera-
`ture was ramped from 95 to 15°C and back up to 95°C at a rate
`of 5°C/min with a 12 s hold at each reading. Cary WinUV soft-
`ware was used to determine the Tm for each combination.
`Polyacrylamide gel analysis of bisPNA:DNA
`hybridizations
`PNAs tend to aggregate upon storage, so each working solu-
`tion of bisPNA was heated to 80°C for 5 min and then cooled
`to room temperature prior to use. bisPNA (150 nM final
`concentration) was mixed with 32P-radiolabeled DNA oligo-
`nucleotides (150 nM final concentration) and unlabeled DNA
`
`Nucleic Acids Research, 2002, Vol. 30, No. 13 2783
`
`Figure 1. (A) Structure of a PNA monomer. (B) Structure of AEEA, the linker
`molecule used to join PNA strands. (C) Model of a bisPNA showing the N- and
`C-terminal PNAs connected by a spacer region that may include amino acids
`or one or more AEEA molecules.
`
`oligonucleotide (0–25 µM) in a 50 µM Tris–HCl buffer
`(pH 8.0) in a 20 µl reaction. Oligonucleotide strands were
`annealed using a PE 9600 Thermocycler (Perkin Elmer,
`Norwalk, CT) (95°C for 5 min, cooled to 4°C over a period of
`60 min). The samples were then flash frozen in an ethanol and
`dry ice bath and stored at –20°C until use. On ice, 10 µl of a
`30% glycerol tracking dye (0.05% bromophenol blue, 0.05%
`xylene cyanol and 0.05% orange G) was added, followed by a
`quick spin, before loading into a non-denaturing 10% (19:1)
`polyacrylamide gel (Bio-Rad). The gel was run at 4°C and
`250 V for 3.5 h. The gel was analyzed by autoradiography
`using a Molecular Dynamics model 425F phosphorimager
`(Sunnyvale, CA).
`
`RESULTS AND DISCUSSION
`
`Design of DNA oligonucleotides and bisPNAs
`We designed bisPNA molecules containing two PNA strands
`linked by spacer regions of varied lengths (Fig. 1A–C).
`Previous
`reports have demonstrated that polypyrimidine
`bisPNAs can form four-stranded complexes capable of
`invading duplex DNA (26). The PNA sequences used in our
`studies, however, contained mixed purine and pyrimidine
`sequences because they were designed to bind and assemble
`two different single-stranded DNA oligonucleotides.
`To test the effects of linker length and amino acid substitu-
`tion within the spacer region between the two PNA strands we
`varied the number of AEEA linker molecules (Fig. 1B and
`Table 1) and number and identity of amino acids. Each AEEA
`molecule is ∼11 Å in length, water soluble and highly flexible.
`Amino acids were included in some of the spacer regions to
`increase the distance between the PNA sequences and to test
`the effect of amino acid charge and steric bulk on the ability of
`the bisPNA to assembly both DNA oligonucleotides.
`We designed DNA oligonucleotides to hybridize to bisPNAs
`in four different arrangements (Fig. 2A–D). The oligonucle-
`otides were either exactly complementary to the PNA strands
`(Fig. 2A) or longer, so that overhanging single-stranded
`
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`2784 Nucleic Acids Research, 2002, Vol. 30, No. 13
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`Table 1. bisPNA sequences, expected and observed masses, and melting temperature values (Tm) for
`hybridization to oligonucleotide complements
`
`bisPNA
`
`Sequence (N→C)
`
`PNA 1
`
`tcttcacctaga-lys
`
`Mass (Da)
`(found/calculated)
`
`3332.70/3332.60
`
`a (°C)
`Tm
`
`50.4
`
`47.8
`
`52.7/48.3
`
`49.5/45.6
`
`49.1/46.3
`
`48.4/46.7
`
`49.1/47.8
`
`50.3/50.5
`
`PNA 2
`AEEA1
`AEEA3
`AEEA6
`AEEA6asp5
`AEEA6lys5
`AEEA6phe5
`
`3412.19/3411.62
`
`gatacatatttg-lys
`tcttcacctaga-(aeea)1-gatacatatttg-lys
`tcttcacctaga-(aeea)3-gatacatatttg-lys
`7476.20/7470.42
`tcttcacctaga-(aeea)6-gatacatatttg-lys
`tcttcacctaga-(aeea-asp)5(aeea)-gatacatatttg-lys 8023.45/8028.92
`tcttcacctaga-(aeea-lys)5(aeea)-gatacatatttg-lys 8092.93/8093.42
`tcttcacctaga-(aeea-phe)5(aeea)-gatacatatttg-lys 8495.29/8496.35
`
`6738.01/6744.42
`
`7032.80/7034.82
`
`aFor bisPNAs, the temperature on the right is the Tm of the N-terminal half of the bisPNA (analogous to
`PNA 1), while the temperature on the left is the Tm of the C-terminal half of the bisPNA (analogous to
`PNA 2).
`
`Figure 2. Models of bisPNA hybridized to DNA oligonucleotides. (A) DNA
`oligonucleotides that are exactly complementary to the PNA and do not have
`overhanging regions. (B) DNA oligonucleotides that have overhanging regions
`projecting outwards. (C) DNA oligonucleotides that have overhanging regions
`partially projecting inwards and partially projecting outwards. (D) DNA oligo-
`nucleotides that have overhanging regions projecting inwards.
`
`regions were created (Fig. 2B–D). The longer oligonucleotides
`could project outwards
`(DNA-1outward and DNA-2outward),
`project partially outwards and partially inwards (DNA-1mid and
`DNA-2mid) or project inwards (DNA-1inward and DNA-2inward)
`relative to the bisPNA (Fig. 2B–D and Table 1). Except for the
`target sequence, each base of the DNA oligonucleotide was
`randomized to minimize secondary structure and DNA:DNA
`interactions. We studied the ability of PNAs to assemble DNA
`oligomers with overhanging bases because such overhangs can
`be used to bind additional nucleic acids and permit complex
`structures to be built up.
`
`Synthesis and characterization of PNA and bisPNA
`molecules
`The automated synthesis of bisPNA molecules involving at
`least 25 couplings required additional precautions to ensure
`complete synthesis of the molecule. Typically, when a PNA
`sequence contains three or more consecutive purines or
`
`Figure 3. (A) RP-HPLC purification of bisPNA-AEEA3. Purification condi-
`tions are described in Materials and Methods. (B) MALDI-TOF analysis of
`bisPNA-AEEA3 with expected and observed molecular weights noted. (C) Tm
`analysis of bisPNA-AEEA3 reversibly hybridizing to complementary DNA-2exact.
`Closed squares represent association during decreasing temperatures. Open
`circles represent dissociation during increasing temperatures.
`
`pyrimidines synthesis efficiency decreases. To minimize this
`problem we repeated coupling steps for addition of the third
`consecutive base and any that followed. We found that the
`spacer molecule AEEA was difficult to couple efficiently, so
`each of these couplings was repeated prior to addition of the
`next molecule. Repeated coupling was also done for each
`amino acid. With these precautions, automated synthesis of
`bisPNAs with extensive spacer regions was routine. We
`analyzed and purified bisPNAs by C18 RP-HPLC (Fig. 3A).
`Full-length PNAs were retained on the C18 column longer than
`
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`Table 2. Sequences of exact complement and overhanging oligonucleotides,
`melting temperatures (Tm) with bisPNA-AEEA3 and differences in Tm
`between exact complements and overhanging oligonucleotides
`
`Nucleic Acids Research, 2002, Vol. 30, No. 13 2785
`
`Name
`Exact complements DNA-1exact
`DNA-2exact
`Projecting outward DNA-1outward
`DNA-2outward
`DNA-1mid
`DNA-2mid
`DNA-1inward
`DNA-2inward
`
`Partial inward/partial
`outward
`
`Projecting inward
`
`Sequence (5′→3′)
`tctaggtgaaga
`
`Tm (°C) ∆Tm (°C)
`49.5
`-
`
`caaatatgtatc
`tctaggtgaaga(n)38
`(n)28caaatatgtatc
`(n)19tctaggtgaaga(n)19
`(n)14caaatatgtatc(n)14
`(n)38tctaggtgaaga
`caaatatgtatc(n)28
`
`45.6
`
`49.3
`
`48.1
`
`54
`52.8
`
`54.4
`
`51.9
`
`-
`
`–0.2
`
`2.5
`
`4.5
`6.2
`
`4.9
`
`5.4
`
`n, a randomized nucleotide.
`
`truncated products from failed syntheses. Correct synthesis
`was confirmed by mass spectrometry (Fig. 3B). Both HPLC
`and mass spectral analysis routinely indicated that the main
`product was the desired one.
`
`Melting temperature (Tm) analysis of exact complement
`DNA oligonucleotides to bisPNAs
`We determined the melting temperature (Tm) values of the
`bisPNAs and complementary DNA oligonucleotides to charac-
`terize their potential for stable and selective hybridization
`(Fig. 3C). The Tm values for hybridization of bisPNAs with
`exactly complementary oligonucleotides were nearly the same
`as that of the individual 12 base PNAs corresponding to each
`half of the bisPNA (Table 1). This similarity in Tm values
`demonstrates that neither the molecules that make up the
`spacer region nor the unbound half of the bisPNA molecule
`significantly affects the temperature dependence of hybridiza-
`tion of the PNA and DNA oligonucleotides that lack an over-
`hanging region. As we describe below, the interactions of
`bisPNAs with DNA oligonucleotides that do contain over-
`hanging regions increase the Tm values in some cases (Table 2).
`Assembly of short, complementary oligonucleotides by
`bisPNA
`We analyzed the hybridization of bisPNAs to DNA oligo-
`nucleotides by monitoring the ability of the PNAs to shift the
`mobility of 32P-radiolabeled DNA upon non-denaturing poly-
`acrylamide gel electrophoresis. In all of the experiments
`described below, we established the mobility of labeled DNA
`alone, the mobility of labeled DNA bound to one PNA strand
`and the mobility of a mixture of two labeled DNA oligonucle-
`otides directed to different PNA strands. 32P-labeled and un-
`labeled oligonucleotides were added to bisPNAs at the same
`time.
`A bisPNA containing three AEEA linkers (PNA-AEEA3,
`Table 1) readily assembled short complementary DNA oligo-
`nucleotides, DNA-1exact and DNA-2exact (Table 2). Surpris-
`ingly, when bisPNA-AEEA3 hybridized to both 12mer DNA
`oligonucleotides migration was faster than a single hybridized
`bisPNA-AEEA3
`(Fig. 4). The
`faster mobility of
`the
`DNA:bisPNA:DNA was not observed with the longer DNA
`
`Figure 4. Non-denaturing polyacrylamide gel electrophoresis of bisPNA-
`AEEA3 with 32P-labeled DNA-1exact and unlabeled DNA-2exact. Left lane,
`bisPNA-AEEA3 and 32P-labeled DNA-1exact are present in a 1:1 ratio (150:150 nM).
`lane, bisPNA-AEEA3 and 32P-labeled DNA-1exact and unlabeled
`Right
`DNA-2exact are present in a 1:1:3.3 ratio (150:150:500 nM).
`
`sequences used in these studies. The increased mobility of
`the larger complex may be due to the dual hybridized
`bisPNA-AEEA3 having twice the negative charge (from the
`second oligonucleotide phosphate backbone) and to formation of
`a more compact structure than the single hybridized bisPNA.
`
`Assembly of oligonucleotides that project beyond the
`bisPNA
`We next tested the ability of bisPNA-AEEA3 (Table 1) to
`hybridize longer DNA oligonucleotides that extended past one
`or both PNA termini. Characterizing assembly of these longer
`oligonucleotides is important because the extended DNA
`sequences provide the potential for additional base pairing
`necessary for formation of higher order structures. When
`bisPNA was incubated with DNA oligonucleotides designed to
`project outwards we observed that the bisPNA readily assem-
`bled both DNA strands with similar results regardless of which
`DNA strand was labeled with 32P (Fig. 5). The efficiency of
`hybridization was not affected by increasing the concentration
`of the unlabeled strands. In contrast to the ability of bisPNAs
`to successfully assemble short DNA oligonucleotides or DNA
`oligonucleotides with overhangs that project outwards (Figs 5
`and 6, lanes 2), assembly of DNA oligonucleotides that project
`inwards upon hybridization was less apparent (Fig. 6, lanes 4
`and 6). To improve binding of inward facing oligomers, we
`varied annealing conditions, but our attempts to bind two DNA
`oligonucleotides with
`inward
`projecting
`overhanging
`sequences invariably yielded only a small fraction of bisPNA
`bound to both DNA strands.
`
`DNA assembly by bisPNAs connected by spacers of
`differing lengths
`Inefficient assembly of the inward facing DNA oligonucle-
`otides suggested that the first oligonucleotide bound to the
`bisPNA blocked binding of the second. One solution for over-
`coming this obstacle was to increase the length of the spacer
`region between the two PNA strands. In theory, this would
`position the PNA strands farther apart, making them less
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`2786 Nucleic Acids Research, 2002, Vol. 30, No. 13
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`Figure 5. Comparison of each half of bisPNA-AEEA3 for hybridization to one
`or two outward projecting DNA oligonucleotides. Lanes 1–7 show hybridization
`of a 1:1 (150:150 nM) ratio of bisPNA-AEEA3 and 32P-labeled DNA-1outward.
`For lanes 2–7, unlabeled DNA-2outward was added in increasing concentrations
`(equivalents relative to bisPNA): 0.33, 1.66, 3.33, 16.6, 33.3 and 166 equiv.
`Lane 8 is 32P-labeled DNA-1outward only. Lanes 9–15 show hybridization of a
`1:1 ratio (150:150 nM) of bisPNA-AEEA3 and 32P-labeled DNA-2outward. For
`lanes 10–15, unlabeled DNA-1outward was added in increasing concentra-
`tions (equivalents relative to bisPNA): 0.33, 1.66, 3.33, 16.6, 33.3 and 166
`equiv. Lane 16 is 32P-labeled DNA-2outward only.
`
`Figure 6. Comparison of bisPNA-AEEA3 hybridization to DNA oligonucleotides
`that project outwards, partially project inwards and project inwards. Lane 1,
`bisPNA-AEEA3 and 32P-labeled DNA-1outward at 1:1 ratios (150:150 nM).
`Lane 2, bisPNA-AEEA3 and 32P-labeled DNA-1outward and unlabeled DNA-2outward
`at 1:1:33 ratios (150:150:5000 nM). Lane 3, bisPNA-AEEA3 and 32P-labeled
`DNA-1mid at 1:1 ratios (150:150 nM). Lane 4, bisPNA-AEEA3 and 32P-labeled
`DNA-1mid and unlabeled DNA-2mid at 1:1:33 ratios (150:150:5000 nM). Lane
`5, bisPNA-AEEA3 and 32P-labeled DNA-1inward at 1:1 ratios (150:150 nM). Lane 6,
`bisPNA-AEEA3 and 32P-labeled DNA-1inward and unlabeled DNA-2inward at 1:1:33
`ratios (150:150:5000 nM).
`
`susceptible to intramolecular interactions that might block
`hybridization.
`To investigate the effect of changing the spacer region on
`PNA assembly we compared bisPNAs with one, three or six
`
`AEEA linkers (bisPNAs AEEA1, AEEA3 and AEEA6, Table 1).
`Spacers also alternated five amino acids (aspartic acid, lysine
`or phenylalanine) within six AEEA molecules (AEEA6asp5,
`AEEA6lys5 or AEEA6phe5, Table 1). Aspartic acid, lysine and
`phenylalanine were selected based on their varied electrostatic
`and steric properties, allowing the effects of substantial chem-
`ical alteration to be investigated. In all cases we observed
`hybridization by DNA oligonucleotides
`that projected
`outwards (Fig. 7A). Hybridization by bisPNA-AEEA6lys5 was
`the least efficient, suggesting that electrostatic interactions
`between lysine and DNA may be blocking hybridization of the
`second DNA strand.
`BisPNA hybridization to partially inward projecting DNA
`oligonucleotides (Fig. 7B) did not occur as readily as to
`outward projecting DNA oligonucleotides. The DNA oligo-
`nucleotides containing a short region projecting inward were
`assembled by the two bisPNAs with the linker
`regions
`containing aspartic acid or phenylalanine (Fig. 7B, lanes 8 and
`12) but not by bisPNA-AEEA6lys5 that contained lysine residues
`(Fig. 7B, lanes 2 and 10). In contrast, DNA oligonucleotides
`with long regions that projected inward exhibited virtually no
`assembly by bisPNAs regardless of the length or chemical
`properties of the linker (Fig. 7C). These same results were
`observed under a variety of annealing conditions.
`In this study we have focused on making increasingly flex-
`ible linkers. It
`is possible that rigid linkers with defined
`geometries might be able to hold the two strands of the PNAs
`far apart and allow more efficient assembly of complex DNA
`oligonucleotides. Shi and Bergstrom have described the
`synthesis of such linkers and have used them to bridge two
`single-stranded DNA oligonucleotides
`for nanostructure
`assembly (27).
`
`Reasons for inefficient assembly of oligonucleotides that
`project inward
`One reason for the inefficient assembly of DNA oligonucle-
`otides containing inward overhanging regions is that once the
`DNA is bound to the complementary PNA strand, the inward
`facing overhanging region may associate with the second PNA
`strand through non-Watson–Crick interactions (Fig. 8A).
`Normally such interactions would be weak, but in this case
`they may be more important because they would be effectively
`intramolecular. If this type of non-specific association were to
`occur, it would be reflected in elevated Tm values for the
`inward facing DNA oligonucleotides relative to DNA oligo-
`nucleotides that are direct complements or that contain regions
`that are outward facing. Support for the belief that non-specific
`PNA–DNA contacts can be important is provided by Raney
`and co-workers, who noted that PNAs have a high propensity
`for interactions with single-stranded DNA that promote aggre-
`gation (28).
`that
`DNA oligonucleotides with overhanging regions
`projected outwards
`(DNA-1outward and DNA-2outward) had
`melting temperatures nearly equal to that of short 12mer DNA
`oligonucleotides (Table 2). However, the DNA oligonucle-
`otides that projected inwards or partially inwards had signifi-
`cantly higher Tm values relative to the exact complement DNA
`increasing by 4.5–6.2°C. These data are
`oligonucleotides,
`consistent with the suggestion that non-Watson–Crick inter-
`actions between PNA and DNA oligonucleotides can form and
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`Nucleic Acids Research, 2002, Vol. 30, No. 13 2787
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`Figure 7. Comparison of oligonucleotide assembly by bisPNAs that contain spacers with varying numbers of AEEA spacers and amino acids. The DNA oligo-
`nucleotides project outwards, partially inwards and inwards. (A) bisPNA hybridization to DNA oligonucleotides that project outwards. (B) bisPNA hybridization
`to DNA oligonucleotides that project partially inwards. (C) bisPNA hybridization to DNA oligonucleotides that project inward. Lanes 1 and 2 represent bisPNA-
`AEEA1, lane 3 and 4 represent bisPNA-AEEA3, lanes 5 and 6 represent bisPNA-AEEA6, lanes 7 and 8 represent bisPNA-AEEA6asp5, lanes 9 and 10 represent
`bisPNA-AEEA6lys5 and lanes 11 and 12 represent bisPNA-AEEA6phe5. For each hybridization reaction, bisPNA and 32P-labeled DNA oligonucleotide are at a 1:1 ratio
`(150:150 nM). Even numbered lanes include an additional 10 equiv. of unlabeled oligonucleotide (1500 nM). Lane 13 is 32P-labeled DNA oligonucleotide only.
`
`may contribute to the inability of the second DNA oligonucle-
`otide to efficiently bind PNA.
`An explanation for inefficient assembly of DNA oligonucle-
`otides that project inwards is that the two overhanging regions
`physically interfere with one another and block dual hybridization
`(Fig. 8B). If this steric conflict were preventing hybridization, it
`should be possible for PNAs to assemble a DNA oligonucle-
`otide with an inward projecting region and a DNA strand that
`is exactly complementary or that contains an overhanging
`region that projects outward. To test this hypothesis we hybrid-
`ized both inward projecting and outward projecting DNA
`oligonucleotides to PNA-AEEA1 or PNA-AEEA6.
`In contrast to the inability of two inward projecting oligo-
`nucleotides
`to hybridize simultaneously (Fig. 7C), we
`observed that PNA-AEEA1 and PNA-AEEA6 were able to
`assembly an inward and an outward projecting oligomer
`(Fig. 8C). Assembly by PNA-AEEA1 (Fig. 8C, lanes 1–7) was
`not as efficient as was assembly by PNA-AEEA6 (Fig. 8C,
`lanes 9–15), suggesting that
`longer spacers can promote
`assembly. The finding that an inward and an outward facing
`DNA can be assembled supports the conclusion that conflicts
`between overhanging strands were one factor contributing to
`the lack of dual hybridization by two inward projecting DNA
`oligonucleotides observed in Figure 7C. It
`is likely that
`assembly is complicated by both steric conflicts between the
`
`overhanging regions and non-sequence-specific interactions
`with the second PNA strand.
`
`Applications of PNAs for the assembly of DNA structures
`PNAs may have many applications in the field of nanotech-
`nology and one of the purposes of this study was to determine the
`rules guiding use of PNAs for the assembly of DNA structures.
`There have been recent reports of the use of DNA oligo-
`nucleotides to assemble nanostructures, most of which
`describe systems that could use PNAs as a facilitator for
`construction. Seeman and co-workers constructed 2- and 3-
`dimensional DNA arrays based on the DNA branched junction
`motif and ‘sticky ends’ to ligate individual ‘units’ together
`(29). bisPNAs could be used similarly, with the tether molecules
`forming the corners between the PNA:DNA duplexes on either
`side in a 2-dimensional arrangement. Furthermore, trisPNAs
`can be synthesized (C.J.Nulf, unpublished results), capable of
`hybridizing to three DNA oligonucleotides and forming the
`vertices of a 3-dimensional cube.
`PNAs could also be used for joining the ends to generate
`circular oligonucleotides or concatamers. Circular DNA oligo-
`nucleotides have unique functions and interesting hybrid-
`ization properties (30). They have been synthesized by ligating
`two ends of a linear oligonucleotide after bringing the two ends
`together with a connecting oligonucleotide (31). The bisPNAs
`used in our studies could be used to stably join DNA ends
`
`MPI EXHIBIT 1016 PAGE 7
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`MPI EXHIBIT 1016 PAGE 7
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`2788 Nucleic Acids Research, 2002, Vol. 30, No. 13
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`Figure 8. (A) Interference with dual hybridization by an inward facing oligo-
`nucleotide that makes Watson–Crick base pairs with a complementary PNA
`strand, and forms non-Watson–Crick interactions with the non-complementary
`PNA strand. (B) Interfence with dual hybridization by steric conflicts between
`two inward facing oligomers. The explanations for the inefficient dual hybrid-
`ization portrayed in (A) and (B) are not mutually exclusive. (C) Comparison of
`bisPNA-AEEA1 and bisPNA-AEEA6 for their ability to hybridize to both an
`inward projecting and an outward projecting DNA oligonucleotide. Lanes 1–7,
`bisPNA-AEEA1 and 32P-labeled DNA-1outward in 1:1 ratios (150:150 nM). For
`lanes 2–7, unlabeled DNA-2inward was added in increasing equivalents (relative to
`bisPNA-AEEA1): 0.33, 1.66, 3.33, 16.6, 33.3 and 166 equiv. Lane 8 is 32P-labeled
`DNA-1outward only. Lanes 9–15, bisPNA-AEEA6 and 32P-labeled DNA-1outward
`in 1:1 ratios (150:150 nM). For lanes 10–15, unlabeled DNA-2inward was added
`in increasing equivalents (relative to bisPNA-AEEA6: 0.33, 1.66, 3.33, 16.6,
`33.3 and 166 equiv. Lane 16 is 32P-labeled DNA-1outward only.
`
`ligation and the resultant circular
`the need for
`without
`constructs could be used to build higher order structures.
`A third possible application of PNAs in nanotechnology is
`the development of electrical-sensitive nanoprobes. Recently,
`Mirkin and co-workers have functionalized gold nanoparticles
`with DNA oligonucleotides that create conductivity changes
`associated with target–probe binding events (32). Similarly,
`Josephson and co-workers have determined by magnetic
`resonance imaging that monodispersed oligonucleotide-modified
`magnetic nanoparticles exhibit a decrease in relaxation time of
`adjacent water molecules when hybridized to a complementary
`oligonucleotide-modified magnetic nanoparticle (33). The
`superior hybridization properties of PNAs might make them
`ideal for these applications, and their neutrality might be useful
`for the probing and optimization of electrical sensors.
`Importantly, all of the applications discussed above involve
`hybridization to the terminal ends of DNA oligonucleotides, an
`ability that our studies indicate is possessed by bisPNAs. Other
`
`applications, especially those that require assembly within
`living cells, may be more challenging. PNAs can efficiently
`invade duplex DNA (21–23), but the assembly of two duplex
`DNAs is likely to be complicated because of the problems that
`we have uncovered with joining inward overhanging
`sequences. We have attempted to use bisPNAs to join two DNA
`plasmids but have not been successful (C.J.Nulf, unpublished
`results). Another interesting biological application would be
`the use of PNAs to bridge RNA sequences to alter splicing or
`expression. This tactic would also require that bisPNAs doubly
`bind RNA overhanging sequences, but since this hybridization
`would be intramolecular (i.e. the PNA strands would bind to
`different sites on the same RNA), it might occur more readily
`than the intermolecular assembly attempted in our studies.
`
`CONCLUSIONS
`We show here that bisPNAs can be used to assemble DNA
`oligomers, but that this assembly depends on the relative
`geometries of the DNAs being assembled. Assembly occurs
`readily when the DNAs are exactly complementary to the PNA
`strands or contain overhanging regions that project outwards.
`Steric conflicts between the DNA oligonucleotides and non-
`Watson–Crick association between DNA and PNA complicate
`assembly of DNA oligonucleotides that contain regions that
`project inwards. Since most applications of DNA for nano-
`structure assembly use oligonucleotides that project outwards
`from a central connection, our results indicate that the bisPNAs
`can be used for nanotechnology applications and that their
`favorable characteristics may lead to improved assemblie