Afaf H. El-Sagheer

Click chemistry with DNA

The advent of click chemistry has led to an influx of new ideas in the nucleic acids field. The copper catalysed alkyne-azide cycloaddition (CuAAC) reaction is the method of choice for DNA click chemistry due to its remarkable efficiency. It has been used to label oligonucleotides with fluorescent dyes, sugars, peptides and other reporter groups, to cyclise DNA, to synthesise DNA catenanes, to join oligonucleotides to PNA, and to produce analogues of DNA with modified nucleobases and backbones. In this critical review we describe some of the pioneering work that has been carried out in this area (78 references).

Highly sensitive DNA sensor based on upconversion nanoparticles and graphene oxide

In this work we demonstrate a DNA biosensor based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er nanoparticles and graphene oxide (GO). Monodisperse NaYF4:Yb,Er nanoparticles with a mean diameter of 29.1 ± 2.2 nm were synthesized and coated with a SiO2 shell of 11 nm, which allowed the attachment of single strands of DNA. When these DNA-functionalized NaYF4:Yb,Er@SiO2 nanoparticles were in the proximity of the GO surface, the π-π stacking interaction between the nucleobases of the DNA and the sp(2) carbons of the GO induced a FRET fluorescence quenching due to the overlap of the fluorescence emission of the NaYF4:Yb,Er@SiO2 and the absorption spectrum of GO. By contrast, in the presence of the complementary DNA strands, the hybridization leads to double-stranded DNA that does not interact with the GO surface, and thus the NaYF4:Yb,Er@SiO2 nanoparticles remain unquenched and fluorescent. The high sensitivity and specificity of this sensor introduces a new method for the detection of DNA with a detection limit of 5 pM.

Rapid and Efficient DNA Strand Cross-Linking by Click Chemistry

Click chemistry has been used to covalently cross‐link complementary DNA strands between bases to form very stable duplexes. Several alkyne‐ and azide‐modified uracil monomers were used to evaluate the effect of the linkers on the efficiency of the click reaction. All cross‐linked duplexes had much higher thermal stabilities than non‐cross‐linked ones, with increases in melting temperature of up to 30u2009°C. In some cases, the conversion was near‐quantitative, and the reaction was complete in 5 min.

Tension induces a base-paired overstretched DNA conformation

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60–70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792–794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795–799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106:18231–18236]. Here, we resolve this controversy by using optical tweezers to extend small 60–64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.

A Very Stable Cyclic DNA Miniduplex with Just Two Base Pairs

Cyclic mini-DNA duplexes, as shown in the scheme, have been synthesized in high yield on the multimicromole scale by using click chemistry. These B-like duplexes are very stable and bind to drugs, such as distamycin A and 7-aminoactinomycin D. Circular dichroism indicates that the bases are stacked, and UV melting and NMR spectroscopy showed that the interbase hydrogen bonds are very stable, even in a dimer.

Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (AT) and its photophysical characterization inside DNA. AT shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, AT shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that AT only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that AT shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, AT shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

Assessing the biocompatibility of click-linked DNA in Escherichia coli

The biocompatibility of a triazole mimic of the DNA phosphodiester linkage in Escherichia coli has been evaluated. The requirement for selective pressure on the click-containing gene was probed via a plasmid containing click DNA backbone linkages in each strand of the gene encoding the fluorescent protein mCherry. The effect of proximity of the click linkers on their biocompatibility was also probed by placing two click DNA linkers 4-bp apart at the region encoding the fluorophore of the fluorescent protein. The resulting click-containing plasmid was found to encode mCherry in E. coli at a similar level to the canonical equivalent. The ability of the cellular machinery to read through click-linked DNA was further probed by using the above click-linked plasmid to express mCherry using an in vitro transcription/translation system, and found to also be similar to that from canonical DNA. The yield and fluorescence of recombinant mCherry expressed from the click-linked plasmid was also compared to that from the canonical equivalent, and found to be the same. The biocompatibility of click DNA ligation sites at close proximity in a non-essential gene demonstrated in E. coli suggests the possibility of using click DNA ligation for the enzyme-free assembly of chemically modified genes and genomes.

Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility

DNA strands containing a triazole linkage flanked on its 3′-side by an aminoethylphenoxazine nucleobase analogue (G-clamp) have been prepared by solid-phase synthesis followed by CuAAC-mediated click oligonucleotide ligation. The stability of the doubly modified DNA duplexes and DNA–RNA hybrids is greatly increased, whereas a single base pair mismatch located at or adjacent to the modifications is strongly destabilising, making triazole G-clamp a potent mismatch/point mutation sensor. A DNA strand containing this unnatural combination was successfully amplified by PCR to produce unmodified copies of the original template, with deoxyguanosine inserted opposite to the G-clamp-triazole nucleotide analogue. This study shows for the first time that a polymerase enzyme can read through a combined backbone/nucleobase modification surprisingly well. These favourable properties suggest new applications for oligonucleotides containing the G-clamp triazole modification in biotechnology, nanotechnology, diagnostics and therapeutics.

Improved synthesis of 5-hydroxymethyl-2'-deoxycytidine phosphoramidite using a 2'-deoxyuridine to 2'-deoxycytidine conversion without temporary protecting groups.

5-Hydroxymethylcytosine has recently been characterized as the sixth base in human DNA. To enable research on this DNA modification, we report an improved method for the synthesis of 5-hydroxymethyl-2-deoxycytidine ((5-HOMe)dC) phosphoramidite for site-specific incorporation into oligonucleotides. To minimize manipulations we employed a temporary protecting group-free 2-deoxyuridine to 2-deoxycytidine conversion procedure that utilizes phase transfer catalysis. The desired (5-HOMe)dC phosphoramidite is obtained in six steps and 24% overall yield from 2-deoxyuridine.

Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Overstretching of DNA occurs at about 60–70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.