Control of Solid-Supported Intra- vs Interstrand Stille Coupling Reactions for Synthesis of DNA

With a few simple tools, structural DNA nanotechnology allows the design and assembly of an unparalleled variety of programmed molecular structures and assemblies. [1][2][3] Covalent conjugation of organic molecules to DNA imparts new functions to DNA assemblies: DNA modified with functional groups create intricate three-dimensional DNA nanostructures. 3,4 Synthetic methodology towards functionalized DNA nanostructures should amplify the impact of this field.

Covalent DNA conjugates are formed using synthetic nucleoside analogue reagents in automated DNA synthesis (a "pre-strategy"), or by chemical reaction on natural or post-synthetic DNA molecules ("post-strategy"). [5][6][7][8][9][10] The "pre-strategy" usually suffers from low yield of both multistep synthesis and coupling into DNA sequences, while the "post-strategy" is often impeded by not only difficult purification, but also low coupling yield and incompatibility to reaction conditions. 11,12 Alternatively, "on solid support" palladium-catalyzed coupling reactions functionalize nucleotides and oligonucleotides during nucleoside oligomerization, while the incipient ssDNA molecule is still attached to the solid support, [11][12][13][14][15] providing easier synthesis and purification, tolerance to harsh reaction conditions, as well as avoiding difficulties in cross-coupling of dsDNA. [16][17][18][19][20][21][22][23] (We note that some authors classify the on solid support strategy together with other post-synthetic methods. 24 ) Polymer templation by DNA sequences attracts our interest for control of dispersity and topology. 25,26 However, interstrand coupling may occur on solid support to produce mixtures with bifunctional reagents. Here, we explore interand intra-strand coupling, and find that we can control the outcome of the reaction to achieve the desired aim (Figure 1). Previously, we synthesized several monodisperse organic oligomers on DNA. We combined pre-and post-strategies to prepare nylon nucleic acids. 25,27,28 The same technology created a variety of templated linkages within and between DNA molecules. [29][30][31][32] When 2'-propargyl phosphoramidite reagents became commercially available, we constructed DNA-oligoaniline 26,33,34 and DNA-oligophenylenevinylene 26, the middle of the automated DNA synthesis. We observed on-solid support inter-strand coupling of bifunctional Figure 1. On-solid support inter-and intra-strand coupling reactions.

molecules 26 by polyacrylamide gel electrophoresis. We previously used a desymmetrization strategy to avoid interstrand coupling. 34 A recent publication did not report interstrand coupling in a similar scenario. 36 In this paper, we fabricate DNA-polyparaphenylene ladder oligomers (DNA-PPPs). Known for remarkable stiffness and strength, PPPs could provide strong reinforcement to NDA nanostructures similar to DNA-silica hybrid materials. 37 An important material in organic electronics, PPP's bipolar doping capability, stability, and other properties lead to its application in batteries and other applications. [38][39][40] PPP is difficult to synthesize by direct polymerization routes due to insolubility of even short oligomers. 41 We envisaged a sequence of automatic DNA synthesis, conjugation with exogenous reagents, and palladiumcatalyzed coupling in solid phase to access DNA-PPP conjugates. We prepared solid supported propargylmodified single-strand DNAs (ss-DNAs) with commercially available solid supports and nucleoside phosphoramidites.

A multistep synthetic sequence gave building blocks with azide groups (Figure S1), which were subsequently subjected to copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction, known as 'click reaction') [42][43][44] with previously prepared propargyl-modified ss-DNAs . MALDI-TOF characterization confirmed the complete conversion by click reaction and formation of DNA iodides on two different solid supports (Table S1).

We chose to perform aryl-aryl coupling reactions 45 on-solid support to leverage the benefits of solid-phase chemistry. 46,47 Different named reactions (Suzuki, Stille, etc.) describe reaction conditions used in Pd coupling reactions. We initially worked with organoboron reagents (Suzuki) but found the required base reagents to be incompatible with basic-cleavable CPG solid support with the standard long chain alkylamino (lcaa) linker. Suzuki conditions worked better with basic-uncleavable oligo affinity support polystyrene (OAS PS) solid support, but ultimately the organotin reagents (Stille) allowed better optimization.

We examined a DNA sequence containing a phenyl monoiodide moiety reacting with phenyltrialkyltin (Table S2). We probed solid phase coupling reaction conditions with phenyltrialkyltin, catalyzed by Pd2dba3 and AsPh3 in DMF. MALDI-TOF analysis indicated a failed coupling reaction with commonly used phenyltributyltin coupling partner. By contrast, phenyltrimethyltin gave complete conversion, consistent with the superiority of trimethyltin over its tributyl analogue in transmetalation, typically the rate-determining step in the Stille coupling reaction. [48][49][50] Besides trimethyltin species, the soluble copper(I) complex Cu(CH3CN)4PF6 also accelerated the on-solid support Stille reactions. [50][51][52] With successful coupling between monoiodide building blocks and phenyltrimethyltin, we turned our attention to the coupling reaction involving 1,4bis(trimethylstannyl)benzene (C1), which is expected to provide both intra-and inter-strand coupling products (Figure 1). The anticipated pentaphenyl species (intrastrand product, "monomer") was observed clearly as a significant product (Figure S15 To obtain intra-strand coupling, we devised a strategy to "dilute" the DNA strands on solid support by capping them with phosphoramidite without 5'-hydroxy groups and reduce the statistical proximity of the aryl iodides. Figure 5a shows the design by diluting solid supported oligodeoxynucleotides (ODNs) with a biotin additive endcap. During automated DNA synthesis, a mixture of 90%

5'-Complementary Complementary Complementary Double Helix Double Helix Double Helix Single Strand Single Strand Single Strand biotin and 10% thymidine was used instead of 100% thymidine. This approach aimed to generate a majority of terminated single-stranded DNA ODNs, which lack any reactive sites for the Stille reaction. Coupling products of S7 and C1. Lane 6 to 9: Coupling products of S8 and C1. (c) After gel purification, OD260 measurement gave relative yields of monomer vs. the sum of multimers: with 100% T, the ratio of monomer : dimer : trimer = 58% : 30% : 12%; with 90% 5'-biotin and 10% T: the ratio of multimers < 5%. ss-DNA strands: S7: 5'-TTT M1TM1 TTT XTT TTT TTT-3'; S8: 5'-TTT M1TM1 TTT TTT TTT TTT-3'; X: amidites mixtures (90% 5'-biotin and 10% T).

We hypothesized that the remaining normal ODNs, not terminated by biotin, would have greater distances between them compared to a typical solid support sample.

We conducted polyacrylamide gel electrophoresis (PAGE) analysis of the coupling reactions using these biotin-diluted ODNs. Various diluted ODNs were examined, and the results (Figure 5b) showed minimal material in the high mass range and only a few dimers compared to the reactions using normal single-stranded DNA strands. The gel purification and following OD260 measurement suggested that with the non-diluted solid support, more than 40% of the products were obtained as the dimer and trimer (higher-mass oligomers are excluded here), and less than 60% the starting strands were involved in the intrastrand coupling. By contrast, with a diluted strand, more than 95% was detected as the monomer that resulted from the intra-strand coupling. This outcome provided further confirmation that: 1) interstrand coupling indeed occurs in the on-solid support coupling reactions as the major conversion route, and 2) by increasing the distances between ODNs that possess reactive sites using the biotin dilution strategy, inter-strand coupling was significantly suppressed. These results hinted that longer coupling partners may favor inter-strand coupling over intra-strand coupling. Instead of suppressing inter-strand couplings, we could promote inter-strand crosslinking between ss-DNAs and fabricate novel materials conjugated to multiple ssDNA. To enable these inter-strand couplings, a new series of hexaarylbis(trimethylstannyl)benzenes were synthesized as coupling partners for Stille reactions. Precise mass determination using MALDI-TOF analysis for these highmass species, particularly those with large aromatic moieties, proved challenging. However, PAGE analysis 0% 20% 40% 60% 80% 100% Monomer Multimer useful in its own right. 53 Pristine DNA-PPP ladder products were freely soluble in water and owed to the high compression strength of PPP (207 MPa), 38 there's potential applications in DNA origami to improve the structural integrity of DNA nano-constructs. Further, using DNA selfassembly, the DNA-PPP would allow directional orientation of monodisperse PPP 54 -a long-standing challenge -to provide a pathway to produce better performing PPP based electronics (FET, Blue-OLED, sensors). 55,56 These achievements in intra-and inter-strand couplings present a convenient and controllable approach for DNA functionalization and the fabrication of PPP materials within DNA scaffolds.