The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed “M1.3” as a linear or cyclic scaffold and the assembly of small origami structures with just 15–24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5′-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.

M1.3 – a small scaffold for DNA origami

Eukaryotic mRNA with its 5′-cap is of central importance for the cell. Many studies involving mRNA require reliable preparation and modification of 5′-capped RNAs. Depending on the length of the desired capped RNA, chemical or enzymatic preparation – or a combination of both – can be advantageous. We review state-of-the art methods and give directions for choosing the appropriate approach. We also discuss the preparation and properties of mRNAs with non-natural caps providing novel features such as improved stability or enhanced translational efficiency.

https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-13-274.pdf

Synthetic mRNA capping

DNA has found widespread uses as a nanosized scaffold for assembly of patterned multichomophoric nanostructures. Herein we report the synthesis, self-assembly, stability, and spectroscopic studies of short alternating non-self-complementary DNA sequences 5′-(dGdA)4 and 5′-(dAdG)4 with non-charged tetraarylporphyrins covalently linked to the 5′ position of deoxyadenosine or deoxyguanosine via a phosphate or amide linker. The linker, the metal in the porphyrin coordination center, and the neighboring nucleobase have very distinct effects on the duplex formation of porphyrin–deoxyguanosine–deoxyadenosine oligodeoxynucleotides. At ionic strength between 5 mM and 40 mM, free base trispyridylphenylporphyrin appended to the 5′ termini of 5′-(dAdG)4 oligonucleotide via short non-polar amide linker served as a hydrophobic molecular cap inducing deoxyadenosine–deoxyguanosine antiparallel homoduplex. At ionic strength of ≥60 mM, the free base porphyrin functioned as a molecular ‘glue’ and induced the formation of porphyrin–DNA inter-homoduplex assemblies with characteristic tetrasignate CD Cotton effects in the porphyrin Soret band region. When the porphyrin cap was covalently attached to 5′ position of deoxyguanosine or deoxyadenosine via charged phosphate linker, no significant deoxyadenosine–deoxyguanosine hybridization was observed even at elevated ionic strengths.

/pdf/porphyrin-dna-conjugates-porphyrin-induced-adenine-guanine-3unpft7iaa.pdf

Porphyrin–DNA conjugates: porphyrin induced adenine–guanine homoduplex stabilization and interduplex assemblies

Copper‐Catalyzed Synthesis of Substituted 4‐Quinolones using Water as a Benign Reaction Media: Application for the Construction of Oxolinic Acid and BQCA

Recent studies illustrate that short oligonucleotide sequences
can be easily produced from nucleotide precursors in a
template-free non-enzymatic way under dehydrating conditions,
i.e. using essentially dry materials. Here we report that 3',5'
cyclic AMP may also serve as a substrate of the reaction, which
proceeds under moderate conditions yet with a lower efficiency
than the previously reported oligomerization of 3',5' cyclic
GMP. Optimally the oligomerization requires (i) a temperature
of 80 degrees C, (ii) a neutral to alkaline environment and
(iii) a time on the order of weeks. Differences in the yield
and required reaction conditions of the oligomerizations
utilizing 3', 5' cGMP and cAMP are discussed in terms of the
crystal structures of the compounds. Polymerization of 3',5'
cyclic nucleotides, whose paramount relevance in a prebiotic
chemistry context has been widely accepted for decades,
supports the possibility that the origin of extant genetic
materials might have followed a direct uninterrupted path since
its very beginning, starting from non-elaborately pre-activated
monomer compounds and simple reactions.

/pdf/non-enzymatic-oligomerization-of-3-5-cyclic-amp-285w29znrl.pdf

Non-Enzymatic Oligomerization of 3', 5' Cyclic AMP.

Detecting short RNA strands with high fidelity at any of the bases of their sequence, including the termini, can be challenging, since fraying, wobbling, and refolding all compete with canonical base pairing. We performed a search for 5'-substituents of oligodeoxynucleotides that increase base pairing fidelity at the terminus of duplexes with RNA target strands. From a total of over 70 caps, differing in stacking moiety and linker, a phosphodiester-linked sequence of the residues of L-prolinol, glycine, and oxolinic acid, dubbed ogOA, was identified as a 5'-cap that stabilizes any of the four canonical base pairs, with ΔT(m) values of up to +13.1 °C for an octamer. At the same time, the cap increases discrimination against any of the 12 possible terminal mismatches, including mismatches that are more stable than their perfectly matched counterparts in the control duplex, such as A:A. A probe with the cap also showed increased selectivity in the detection of two closely related microRNAs, let7c and let7a, with a ΔT(m) value of 9.2 °C. Melting curves also yielded thermodynamic data that shed light on the uniformity of molecular recognition in the sequence space of DNA:DNA and DNA:RNA duplexes. Hybridization probes with fidelity-enhancing caps should find applications in the individual and parallel detection of biologically active RNA species.

A 5′‐Cap for DNA Probes Binding RNA Target Strands

/pdf/research-paper-segmented-hybridization-probes-modulating-529xyug3us.pdf

RESEARCH PAPER Segmented hybridization probes: modulating target affinityand base pairing selectivity

Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5′-O-terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of l-prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step-wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap-bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 51:4.53.1-4.53.21. © 2012 by John Wiley & Sons, Inc.


Keywords:

oligonucleotides;
phosphoramidite;
peptides;
controlled pore glass;
hybridization probes

Synthesis of oligodeoxynucleotides with 5'-caps binding RNA targets.

Short hybridization probes bind their targets with greater base pairing fidelity, but with lower affinity than longer probes. Furthermore, their target sequence is shorter, and thus more likely not to be unique in a given genome. Long hybridization probes provide increased affinity, and their sequences are more unique, but their duplexes tolerate mismatches more readily, without a significant depression in melting point. It was reasoned that segmenting longer hybridization probes by introducing flexible, abasic linkers might lead to oligonucleotides that retain some of the sequence selectivity of short probes without losing too much of the target affinity of their unsegmented counterparts. A model study led to 1,3-propandiol-phosphates as linker residues. These spacer residues were introduced at different positions of hybridization probes 8-20 residues in length and their hybridization properties were studied in UV-melting curves with RNA or DNA target strands. Increases in base pairing selectivity (ΔΔTm of up to !7.4EC for a single mismatch) and decreased target affinities (ΔTm between !15 and !25EC) were found for the segmented probes when compared to their unsegmented counterparts, and so was a decreased selectivity for insertions at the site of the linker. Also, the increases in selectivity are not uniform in their magnitude and depend on sequence context and position. A favorable case appears to be a hybridization probe that contains two spacers, with one octamer as core segment, flanked by a heptamer and a pentamer as terminal segments.

/pdf/segmented-hybridization-probes-modulating-target-affinity-30h6l2en28.pdf

Simone Egetenmeyer

Papers

A 5′‐Cap for DNA Probes Binding RNA Target Strands

RESEARCH PAPER Segmented hybridization probes: modulating target affinityand base pairing selectivity

Synthesis of oligodeoxynucleotides with 5'-caps binding RNA targets.

Segmented hybridization probes: modulating target affinity and base pairing selectivity