This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.
Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (γPNA) tiles. Each single-stranded tile (SST) is a 12-base γPNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these γPNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.
Successful construction of numerous complex nanostructures1,2,3,4,5,6,7,8,9,10,11,12 in aqueous or substantially hydrated media made using naturally occurring nucleic acids such as DNA1,2,3,4,5,6,7,8,9,10 and RNA11,12 has been shown in previous works. However, naturally occurring nucleic acids undergo duplex helical conformational changes or have reduced thermal stabilities in organic solvent mixtures13,14.
Previously, our lab has reported a method towards the construction of 3-helix nanofibers using gamma-position modified synthetic nucleic acid mimics called gamma-peptide nucleic acids (γPNA)15 (Figure 1A). The need for such a development and potential applications of the synthetic nucleic acid mimic PNA has been discussed within the field16,17. We have shown, through an adaptation of the single-stranded tile (SST) strategy presented for DNA nanostructures18,19,20, that 9 sequentially distinct γPNA oligomers can be designed to form 3-helix nanofibers in select polar aprotic organic solvent mixtures such as DMSO and DMF. The γPNA oligomers were commercially ordered with modifications of (R)-diethylene glycol (mini-PEG) at three γ-positions (1, 4 and 8 base-positions) along each 12-base oligomer based on methods published by Sahu et al.21 These gamma-modifications cause the helical pre-organization that is associated with the higher binding affinity and thermal stability of γPNA relative to unmodified PNA.
This article is an adaptation of our reported work in which we investigate the effects of solvent solution and substitution with DNA on the formation of γPNA-based nanostructures15. The aim of this article is to provide detailed descriptions of the design as well as detailed protocols for solvent-adapted methods that were developed for the self-assembly and characterization of γPNA nanofiber. Thus, we first introduce the modular SST strategy, a general platform for nanostructure design using the synthetic nucleic acid mimic PNA.
The helical pitch for PNA duplexes has been reported to be 18 bases per turn in comparison to DNA duplexes, which undergo one turn per 10.5 bases (Figure 1B). Therefore, the domain-length of the demonstrated γPNA SSTs was set at 6 bases to accommodate one third of a full turn or 120° of rotation to enable interaction between three triangularly arrayed helices. Also, unlike previous SST motifs, each SST contains just 2 domains, effectively creating a 1-dimensional ribbon-like structure that wraps to form a three-helix bundle (Figure 1C). Each 12-base γPNA oligomer is gamma modified at the 1, 4 and 8 positions to ensure uniformly spaced distribution of mini-PEG groups across the overall SST motif. Additionally, within the motif, there are two types of oligomers: “contiguous” strands that exist on a single helix and helix-spanning “crossover” strands (Figure 1D). In addition, oligomers P8 and P6 are labelled with fluorescent Cy3 (green star) and biotin (yellow oval), respectively (Figure 1D), to enable detection of structure formation using fluorescence microscopy. Altogether, the SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers through programmed complementarity of each individual domain to the corresponding domain on a neighboring oligomer (Figure 1E).
1. γPNA sequence design
2. Preparation of γPNA stock strands
3. Melting curve studies of γPNA oligomer subsets
4. Self-assembly protocol for multiple distinct γPNA oligomers
NOTE: To devise a self-assembly thermal ramp protocol for γPNA nanostructures, slow-ramp annealing is desirable.
5. Total internal reflection fluorescence (TIRF) microscopy imaging
6. Transmission electron microscopy (TEM) imaging
7. Different morphologies for γPNA-DNA hybrids based on selective replacement with DNA
8. Different morphologies for γPNA nanofibers in varying concentrations of SDS
The protocols discussed in the sections above describe the design of an adapted SST motif from DNA nanofibers for the robust generation of self-assembled nanofibers structures using multiple, distinct γPNA oligomers. This section describes the interpretation of data obtained from the successful recreation of the protocols described.
Following the protocol described in section 5 for TIRF imaging of samples of γPNA oligomers annealed in 75% DMSO: H2O (v/v) most readily provides evidence of well-organized architectures under microscopic observations as shown in Figure 4A. 75% DMF: H2O (v/v) solvent condition results in spicule-shaped or needle-like nanostructures (Figure 4B) and, in our experience, the 40% 1,4 dioxane: H2O (v/v) condition shows sparse decoration of filamentous nanostructures when viewed using TIRF microscopy (Figure 4C). Furthermore, the samples of γPNA nanotubes formed in 75% DMSO: H2O (v/v) demonstrate bundling of nanofibers at high magnification or nanoscopic resolutions during TEM imaging following steps mentioned in section 6 (Figure 5). Quantitative analyses of the width of the nanostructures showed a median width of 16.3 nm with maximum values beyond 80 nm15.
For devising a scheme towards generating γPNA-DNA hybrid nanostructures in organic solvent mixtures to enable further functionalization using DNA, it is important to consider the oligomeric position in the SST motif being replaced with isosequential DNA oligomers. Adapting steps mentioned in section 7 for the case of the nanotube construct described here, isosequential DNA oligomers that replace the contiguous γPNA oligomers forms straight filamentous structures whereas replacement of the crossover γPNA oligomers forms stellate structures when viewed using TIRF and TEM imaging (Figure 6). Quantitative analyses of the width of the nanostructures that were replaced with contiguous DNA oligomers showed median widths around 19 nm15.
Finally, upon adapting steps from section 8, γPNA nanofibers adopt thinner morphologies consistent with reduced bundling in the presence of SDS concentrations below its critical micelle concentrations (CMC, 8.2 mM). γPNA nanofibers also adopt highly networked morphologies at high SDS concentrations in comparison to its CMC when viewed using TIRF imaging (Figure 7). TEM imaging of γPNA oligomers self-assembled in the presence of SDS indicate that the 5.25 mM SDS condition indicate the most substantial reductions in structural bundling with widths ranging 8‒12 nm as shown in (Figure 7C).
Figure 1: Peptide nucleic acid oligomers as building blocks for complex nanostructures.
(A) Chemical structures of DNA (PNA), PNA and MP-containing γPNA units. (Figure has been reprinted with permission from Ref21.) (B) PNA-PNA helices are less twisted than DNA-DNA helices, having 18 bases per turn instead of 10.5. (C) This three-helix structural single stranded tile (SST) motif consists of 9 distinct 12-base-long γPNA oligomers, each having two six-base domains. (D) Within the structure there are two types of oligomers: “contiguous” strands that exist on a single helix and helix-spanning “crossover” strands. (E) This 18-base-long structural motif can polymerize to form micron-scale filaments. Panels B, C and E have been modified with permission from Ref15. Please click here to view a larger version of this figure.
Figure 2: Representative melt curves of select γPNA oligomers in different solvent conditions.
2-oligomer (p4, p5) subset showing 6-base domains can bind with reasonable thermal stability (black curve) in 1x PBS. 3- oligomer (p4, p5, p6) subset shows increased thermal stability due to cooperativity in 1x PBS (blue curve) and shows little to no instability with respect to Tm when solvent is changed to organic solvents like DMF (red dotted curve). Figure has been modified with permission from Ref15. Please click here to view a larger version of this figure.
Figure 3: Flow chart for fluidic flow channel for sample TIRF imaging.
A step-by-step workflow indicating steps involved in sample preparation for TIRF imaging of γPNA nanofibers after flow channel assembly. Please click here to view a larger version of this figure.
Figure 4: TIRF microscopy imaging of γPNA nanofiber self-assembled in different solvent conditions.
γPNA nanofibers visualized using TIRF microscopy (5 μm scale bar) while monitoring the Cy3 channel when γPNA oligomers are self-assembled in (A) 75% DMSO: water (v/v), (B) 75% DMF: water (v/v) and (C) 40% Dioxane: water (v/v) solvent conditions. (D) When γPNA nanofibers self-assembled in 75% DMSO: water (v/v) bound to the flow channel is washed with 1mM Trolox in water, two-phase microbubbles of DMSO-water form with nanostructures aligning along the interface of the microbubble. Figure has been modified with permission from Ref15. Please click here to view a larger version of this figure.
Figure 5: TEM imaging of γPNA nanofibers self-assembled in 75% DMSO: water. (v/v) using formvar support layer copper 300 mesh grids.
(A) TEM images of γPNA nanofibers visualized under low magnification (15x). (B) TEM images of γPNA nanofibers visualized under high magnification (150x) shows bundling of nanotubes along the width at nanoscopic resolutions. (C) TEM images of γPNA nanofibers visualized under low magnification (10x) using a Formvar-Silicon Monoxide support layer on a Copper grid allows imaging under vigorous specimen conditions. Figure has been modified with permission from Ref15. Please click here to view a larger version of this figure.
Figure 6: Different morphologies for γPNA-DNA hybrids based on selective replacement with DNA.
(A) TIRF (5 μm scale bar) and (C) TEM images (60x magnification) of self-assembly of γPNA-DNA hybrids upon replacing contiguous γPNA oligomers (p3, p5, p9) with isosequential DNA oligomers (D3, D5 D9) show nanotubes adopting a straight filament morphology. (B) TIRF (5 μm scale bar) and (D) TEM images (25x magnification) of self-assembly of γPNA-DNA hybrids upon replacing crossover γPNA oligomers (p1, p4, p7) with isosequential DNA oligomers (D1, D4 D7) show nanofibers adopting a stellate morphology. Figure has been modified with permission from Ref15. Please click here to view a larger version of this figure.
Figure 7: Different morphologies for γPNA nanofibers in varying concentrations of SDS.
TIRF images (5 μm scale bar) of self-assembly of γPNA nanofibers in the presence of SDS at concentrations of (A) 5.25 mM and (B) 17.5 mM. Self-assembly in the presence of SDS concentrations less than the CMC (8.2 mM) shows thinner morphologies from TIRF images based on fluorescence intensity. (C) This is verified by TEM images (100x magnification) of the system where median width for nanofibers lies in the range of 8‒12 nm. At concentrations significantly above the CMC, γPNA nanotubes appear to form higher order assemblies through highly networked nanotube structures. Figure has been modified with permission from Ref15. Please click here to view a larger version of this figure.
MATLAB script string output "thisSeq" | Penalty Score "thisScore" | ||
'acttcgctaaaa cgaagtgaggaa cagtatttcctc aacgagaacacc ctcgttgcccgc ttttaggcgggc ggattgatactg caatcccatagc ggtgttgctatg ' | 0.08 | ||
'ccctcccgaccg ggagggttcagg cacttgcctgaa tggcgttgctat acgccattccaa cggtcgttggaa gagatacaagtg tatctcatttac atagcagtaaat ' | 0.0544 | ||
'tcgcaggagaca ctgcgaggataa aacggcttatcc ccacttgccccg aagtggacgatt tgtctcaatcgt aaatgtgccgtt acattttaccaa cggggcttggta ' | 0.0688 | ||
'gagccccctact gggctcttgata tttcgctatcaa tccacgaccgcc cgtggacaataa agtaggttattg acagatgcgaaa atctgttccttt ggcggtaaagga ' | 0.0528 | ||
'cgggagaaccac ctcccgtttagg cttgaccctaaa gcttacactcgc gtaagccgatga gtggtttcatcg gcaatagtcaag tattgccctgct gcgagtagcagg ' | 0.0656 | ||
'aatgagccgtgc ctcattttatct tagggcagataa ctttcgccacaa cgaaagcagtcc gcacggggactg caatacgcccta gtattggaggaa ttgtggttcctc ' | 0.0592 | ||
'gatggaagcccc tccatcgcctgt ccagagacaggc aagtcaagtagg tgacttttattg ggggctcaataa gcaacgctctgg cgttgctcggtg cctactcaccga ' | 0.0688 | ||
'tagccccccagt gggctatctcgt ttcaggacgaga cttgcggtgtcg cgcaagagtatt actgggaatact gatttacctgaa taaatcaaaggc cgacacgccttt ' | 0.0656 | ||
'taggcaacagac tgcctaccactc tttggagagtgg tagcccctgcgg gggctattcatc gtctgtgatgaa ttcccgtccaaa cgggaaacctta ccgcagtaaggt ' | 0.0736 | ||
'aatgtgtctatc cacattcccttc cgccaagaaggg gtgctgttatgc cagcacgactaa gatagattagtc cctcggttggcg ccgaggtcaaac gcataagtttga ' | 0.0768 | ||
'ttggcgttatcc cgccaaatgctt ggacgaaagcat accgagtgaaca ctcggtggggtc ggataagacccc tctacatcgtcc tgtagagtgccc tgttcagggcac ' | 0.0576 | ||
'ctgtaaggtctc ttacaggtgctt cacgcaaagcac ttcgggatggag cccgaacaatct gagaccagattg gcggactgcgtg gtccgcctattt ctccataaatag ' | 0.072 | ||
'actccaatctat tggagttttgtt ctacccaacaaa cgctcaagtaat tgagcgaacggc atagatgccgtt ctgcgggggtag ccgcaggtcttc attactgaagac ' | 0.064 | ||
'ggttgttccacg acaacctgaagc ttatgtgcttca ttgccccgagcg gggcaatctttc cgtggagaaaga ctactgacataa cagtagacgatg cgctcgcatcgt ' | 0.0688 | ||
'gatttgctgtct caaatcccttct agcgttagaagg cataaaacccag tttatgcctcac agacaggtgagg ctacggaacgct ccgtagtcgtcc ctgggtggacga ' | 0.0688 | ||
'gaaggtctaatg accttcatcctg gcagttcaggat ttggtagcggag taccaaaaatcg cattagcgattt acgacaaactgc tgtcgtaagtgg ctccgcccactt ' | 0.0576 | ||
'tgtagttggtct actacacccttg ggacatcaaggg ttcggcaggcgg gccgaacgctaa agaccattagcg acgagtatgtcc actcgtattttg ccgcctcaaaat ' | 0.0624 | ||
'tggaaccttgta gttccattcgca atcacctgcgaa ccacacttactg gtgtggtcggct tacaagagccga ggcgttggtgat aacgccctatct cagtaaagatag ' | 0.0656 | ||
'agaggtcgtatt acctctgtgagt cggtttactcac actttccagcaa gaaagtccatcc aatacgggatgg tagcccaaaccg gggctaaggcga ttgctgtcgcct ' | 0.0688 | ||
'cggcaaactatg ttgccgatttct acttacagaaat cgtgtcctaaag gacacgggatgg catagtccatcc tgaggcgtaagt gcctcacagcga ctttagtcgctg ' | 0.0704 |
Table 1: 20 sample algorithmic results for potential optimization of γPNA sequence design. 20 sample algorithmic results with sequence outputs in column 1 and their corresponding score in column 2. Repeated iterations of the script are performed to obtain the most minimal score.
γPNA Sequence ID | Sequence | Domain 1 complement | Domain 2 complement |
p1 | N-AATAGCGTTCAC-C | p2 domain 1 | p6-Biotin domain 1 |
p2 | N-GCTATTGAGTAA-C | p1 domain 1 | p3 domain 2 |
p3 | N-GACATCTTACTC-C | p7 domain 2 | p2 domain 2 |
p4 | N-CTGGCGTGCGGA-C | p5 domain 1 | p9 domain 1 |
p5 | N-CGCCAGCCCTCG-C | p4 domain 1 | p6-Biotin domain 2 |
p6-Biotin | N-Biotin-GTGAACCGAGGG-C | p1 domain 2 | p5 domain 2 |
p7 | N-AGTTTTGATGTC-C | p8-Cy3 domain 1 | p3 domain 1 |
p8-Cy3 | N-Cy3-AAAACTACAGAA-C | p7 domain 1 | p9 domain 2 |
p9 | N-TCCGCATTCTGT-C | p4 domain 2 | p8-Cy3 domain 2 |
Table 2: γPNA sequence design results for nanofibers. Individual oligomer sequences were generated as indicated in this table. Underlined bases indicate the gamma-position modifications with mini-PEG. Table has been modified with permission from Ref15.
Temperature Range | Ramp rate |
90 °C | Hold for 3 minutes |
90-80 °C | 0.1°C/minute |
80-70 °C | 0.1°C/minute |
70-60 °C | 0.1°C/3 minutes |
60-50 °C | 0.1°C/3 minutes |
50-40 °C | 0.1°C/3 minutes |
40-30 °C | 0.1°C/minute |
30-20 °C | 0.1°C/minute |
4 °C | Hold indefinite |
Table 3: Anneal ramp protocol for thermal cycler. Table has been modified with permission from Ref15.
Stock concentration | 75% DMSO: water (v/v) anneal sample | 75% DMF: water (v/v) anneal sample | 40% dioxane: water (v/v) anneal sample | Final Concentration | |
γPNA oligomer (x9) | 20 μM | 9 μL (1 μL x 9) | 9 μL (1 μL x 9) | 9 μL (1 μL x 9) | 500 nM |
DMSO | – | 30 μL | – | – | 75 % (v/v) |
DMF | – | – | 30 μL | – | 75 % (v/v) |
1,4-dioxane | – | – | – | 16 μL | 40 % (v/v) |
deionized water | – | 1 μL | 1 μL | 15 μL | 25 or 60% (v/v) |
Total volume | – | 40 μL | 40 μL | 40 μL | – |
Table 4: Protocol for preparing anneal batches of γPNA oligomer in different solvent conditions.
DNA Sequence ID | Sequence |
D1 | 5’-AATAGCGTTCAC-3’ |
D3 | 5’-GACATCTTACTC-3’ |
D4 | 5’-CTGGCGTGCGGA-3’ |
D5 | 5’-CGCCAGCCCTCG-3’ |
D7 | 5’-AGTTTTGATGTC-3’ |
D9 | 5’-TCCGCATTCTGT-3’ |
Table 5: Isosequential DNA sequences as replacement oligomers to γPNA. Table has been modified with permission from Ref15.
Stock concentration | DNA Contiguous strand replacements | DNA Crossover strand replacements | Final Concentration | |
γPNA oligomer (x6) | 20 μM | 6 μL (1 μL x 6) | 6 μL (1 μL x 6) | 500 nM |
DNA oligomer (x3) | 20 μM | 3 μL (1 μL x 3) | 3 μL (1 μL x 3) | 500 nM |
DMSO | – | 30 μL | 30 μL | 75 % (v/v) |
deionized water | – | 1 μL | 1 μL | 25 % (v/v) |
Total volume | – | 40 μL | 40 μL | – |
Table 6: Protocol for preparing anneal batches of γPNA-DNA hybrid nanostructures in 75% DMSO: H2O (v/v) through replacement of contiguous or crossover γPNA oligomers with isosequential DNA oligomers.
Stock concentration | SDS concentrations below CMC | SDS concentrations above CMC | Final Concentration | |
γPNA oligomer (x9) | 20 μM | 9 μL (1 μL x 9) | 9 μL (1 μL x 9) | 500 nM |
6% SDS | 6% (wt/v) | 1 μL | – | 5.25 mM |
20% SDS | 20% (wt/v) | – | 1 μL | 17.5 mM |
DMSO | – | 30 μL | 30 μL | 75 % (v/v) |
deionized water | – | – | – | 25 % (v/v) |
Total volume | – | 40 μL | 40 μL | – |
Table 7: Protocol for preparing anneal batches of γPNA nanostructures in 75% DMSO: H2O (v/v) in the presence of SDS concentrations below and above CMC.
Supplementary Figure 1: Programming script PNA3nanofiber.m for designing oligomer sequences. Please click here to download this figure.
This article focuses on adapting and improving existing nucleic acid nanotechnology protocols towards organic solvent mixtures. The methods described here focus on modifications and troubleshooting within a defined experimental space of select polar aprotic organic solvents. There is yet unexplored potential for other established nucleic acid nanotechnology protocols to be adapted within this space. This could improve potential applications through integration in other fields such as polymer and peptide synthesis which typically are performed in similar organic solvents25,26. Additionally, we focus here on critical steps to be observed while practicing the above-mentioned protocols.
During the preparation of sample for self-assembly, it is important to keep the volume percentages of water at 25% (v/v) for DMSO and DMF conditions. Accordingly, it is important to recognize that the organic solvent used for self-assembly should be from anhydrous stocks as well. The nanofiber structures are hydrophobic and aggregate in increased water content.
Unlike scaffolded DNA origami approaches that can create structures of discrete lengths, the current SST motif design for γPNA nanofibers and other previously established DNA SST nanotubes does not yield structures of discrete lengths. Nanotubes polymerize achieving a range of multi-micron lengths (up to 11 µm). For the same reason, yield associated with structure formation cannot be quantified.
However, due to the uncharged peptide backbone of γPNA, there are no dependencies on counter ion balance in relation to structure formation. Imaging buffers, therefore, do not need to include cations like Mg2+ typically required for the stabilization of nanostructures made from naturally occurring nucleic acids.
Lastly, γPNA nanostructure bundling and aggregation in different solvent conditions are also dependent on the individual oligomer concentrations introduced during self-assembly. Concentrations ranging from 1 µM and higher of individual oligomers increase the propensity for bundling and aggregation and affect clear imaging of nanostructures either during TIRF or TEM imaging.
The authors have nothing to disclose.
This work was supported in part by National Science Foundation grant 1739308, NSF CAREER grant 1944130 and by the Air Force Office of Science Research grant number FA9550-18-1-0199. γPNA sequences were a generous gift from Dr. Tumul Srivastava of Trucode Gene Repair, Inc. We would like to thank Dr. Erik Winfree and Dr. Rizal Hariadi for their helpful conversations on DNA Design Toolbox MATLAB code. We would also like to thank Joseph Suhan, Mara Sullivan and the Center for Biological Imaging for their assistance in the collection of TEM data.
γPNA strands/oligomers | Trucode Gene Repair Inc. | Section 2.1 | |
UV-Vis Spectrophotometer | Agilent | Varian Cary 300 | Section 3.1.2 |
Quartz cuvettes | Starna | 29-Q-10 | Section 3.1.1 |
Thermal cycler | Bio Rad | C1000 touch | Section 4.1 |
0.2 mL PCR tubes | VWR | 53509-304 | Section 4.5 |
Anhydrous DMF | VWR | EM-DX1727-6 | Section 4.6 |
Anhydrous DMSO | VWR | EM-MX1457-6 | Section 4.6 |
Anhydrous 1,4-Dioxane | Fisher Scientific | AC615121000 | Section 4.6 |
10X Phosphate Buffered Saline (PBS) | VWR | 75800-994 | Section 3.1.1 |
Microscope slides | VWR | 89085-399 | Section 5.2 |
Glass cover slips | VWR | 48382-126 | Section 5.2 |
2% Collodion in Amyl Acetate | Sigma-Aldrich | 9817 | Section 5.2 |
Isoamyl Acetate | VWR | 200001-180 | Section 5.2 |
Biotinylated Bovine Serum Albumin (Biotin-BSA) | Sigma-Aldrich | A8549 | Section 5.3 |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153 | Section 5.4 |
Streptavidin | Sigma-Aldrich | 189730 | Section 5.5 |
Trolox | Sigma-Aldrich | 238813 | Section 5.7 |
Total Internal Reflection Fluorescence microscope | Nikon | Nikon Ti2-E | Section 5.8 |
Transmission Electron Microscope | Joel | JEM 1011 | Section 6.6 |
Tweezers | Dumont | 0203-N5AC-PO | Section 6.3 |
Uranyl Acetate | Electron Microscopy Sciences | 22400 | Section 6.1 |
Formvar, 300 mesh, Copper grids | Ted Pella Inc. | 1701-F | Section 6.2 |
Formvar-Silicon monoxide Type A, 300 mesh, Copper grids | Ted Pella Inc. | 1829 | Section 6.2 |
DNA oligomers/strands | IDT | Section 7.1 | |
Sodium Dodecyl Sulphate (SDS) | VWR | 97064-860 | Section 8.1 |