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Biolayer interferometry (BLI) and circular dichroism (CD) spectroscopy were used to investigate the interaction between previously reported i-motif DNA (i-DNA) ligands and folded or unfolded i-DNA in acidic (pH 5.5) and near-neutral (pH 6.5) conditions. We observed that although several ligands, in particular macrocyclic bis-acridine (BisA) and pyridostatin (PDS), showed good affinities for the telomeric i-motif forming sequence, none of the ligands displayed selective interactions with the i-DNA structure nor was able to promote its formation.

i-Motifs are largely underexplored tetraplex nucleic acid structures which have been suggested to perform essential biological functions and might constitute future therapeutic targets. i-Motifs generally require acidic conditions to fold in vitro, a particularity which significantly complicates the use of native i-motif forming sequences for interaction studies with potential ligands and biological components (e.g. proteins). In this context, we report herein on the assembly of a peptide-DNA conjugate capable of folding at room temperature into a stable i-motif structure at neutral pH. To achieve the controlled assembly of the i-motif forming conjugate, we developed a new synthetic pathway of four successive orthogonal ligation reactions between bifunctional C-rich DNA strands and a tetrafunctional cyclopeptide scaffold.

G-triplex nucleic acid structures (G3) have been conjectured to form in vivo but little is known about their physiological functions. The identification of ligands capable of specific binding to G3 structures is therefore highly appealing but remains elusive. Here we report on the assembly of a DNA conjugate which folds into a stable G3 structure. The structural mimic was used to probe the interactions between a G3 ligand and first-in-class G4 ligands, revealing signification binding promiscuity.

Surface plasmon resonance (SPR) is a powerful technique to study the interactions of ligands with analytes and therefore a number of biosensor surfaces and injection methods have been developed so far. However, many experimental parameters can affect the interactions and consequently the affinity measurements. In particular, the interactions of positively charged analytes (often used for anionic nucleic acids targets) can be influenced by the sensing surfaces (e.g., negatively charged), leading to significant nonspecific interactions as well as regeneration problems. The aim of the present work is to investigate the effect of different parameters, including ionic strength, SPR biosensor (i.e., nature of the surfaces), and the injection method on the recognition of porphyrin G-quadruplex ligands. We demonstrate that the injection method does not influence the affinity whereas the ionic strength and the nature of the surface impact the recognition properties of the porphyrin for the G-quadruplex DNA. We also found that self-assembled monolayer coating surface presents many advantages in comparison with carboxymethylated dextran surface for SPR studies of G-quadruplex DNA/ligand interactions: (i) the electrostatic interaction with charged analytes is less important, (ii) its structure/composition is less sensitive to the ionic concentration and less prone to unspecific adsorption, (iii) it is easily homemade, and (iv) the cost is approximately 10 times cheaper.

G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biologically relevant DNA sequences were assembled onto addressable cyclopeptide scaffolds through sequential oxime and CuAAc reactions. The resulting conjugates are able to fold into well-defined anti-parallel DNA G-quadruplex structures, which exhibit high stability and reduced polymorphism.

A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.

Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.

Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d5SICS and analogues of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new dMMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogues with the exception of dNaM. We also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications., (© 2011 American Chemical Society)

A robust, reproducible, and scalable method for the solid-phase synthesis of 5'-triphosphates of DNA, RNA, and their chemically modified analogues using 5'-H-phosphonate intermediates is described. 5'-Triphosphates of oligonucleotides with varying lengths and sequences containing different 5'-terminal nucleotides, with and without internal sugar-backbone modifications, were efficiently prepared as crude products or further purified by HPLC.