Natalya N. Degtyareva

DNA sensing by amplifying the number of near-infrared emitting, oligonucleotide-encapsulated silver clusters.

A bifunctional oligonucleotide integrates in situ synthesis of a fluorogenic silver cluster with recognition of a target DNA sequence. With the template C(3)AC(3)AC(3)GC(3)A, a complex forms with 10 silver atoms that possesses electronic transitions in the near-infrared and that is detected at nanomolar concentrations using diode laser excitation. Pendant to this cluster encoding region, the recognition component binds a target DNA strand through hybridization, and decoupling of these two regions of the composite sensor renders a modular sensor for specific oligonucleotides. A target is detected using a quencher strand that bridges the cluster template and recognition components and disturbs cluster binding, as indicated by static quenching. Competitive displacement of the quencher by the target strand restores the favored cluster environment, and our key finding is that this exchange enhances emission through a proportional increase in the number of emissive clusters. DNA detection is also accomplished in serum-containing buffers by taking advantage of the high brightness of this fluorophore and the inherently low endogenous background in the near-infrared spectral region. Cluster stability in this biological environment is enhanced by supplementing the solutions with Ag(+).

Optical sensing by transforming chromophoric silver clusters in DNA nanoreactors

Bifunctional DNA oligonucleotides serve as templates for chromophoric silver clusters and as recognition sites for target DNA strands, and communication between these two components is the basis of an oligonucleotide sensor. Few-atom silver clusters exhibit distinct electronic spectra spanning the visible and near-infrared region, and they are selectively synthesized by varying the base sequence of the DNA template. In these studies, a 16-base cluster template is adjoined with a 12-base sequence complementary to the target analyte, and hybridization induces structural changes in the composite sensor that direct the conversion between two spectrally and stoichiometrically distinct clusters. Without its complement, the sensor strand selectively harbors ~7 Ag atoms that absorb at 400 nm and fold the DNA host. Upon association of the target with its recognition site, the sensor strand opens to expose the cluster template that has the binding site for ~11 Ag atoms, and absorption at 720 nm with relatively strong emission develops in lieu of the violet absorption. Variations in the length and composition of the recognition site and the cluster template indicate that these types of dual-component sensors provide a general platform for near-infrared-based detection of oligonucleotides in challenging biological environments.

Structural studies of a trinucleotide repeat sequence using 2-aminopurine

The secondary structure of repeated trinucleotide sequences results in the development of several neurodegenerative diseases, and these studies consider the (CAG)(8) sequence that forms a stem-loop hairpin. The structural and thermodynamic properties of this hairpin are assessed using 2-aminopurine substitutions for adenine at six positions in this repeated sequence. Circular dichroism spectra and thermal denaturation experiments show that the secondary structure is not disturbed by the modifications. The local structure of the hairpin was monitored using the fluorescence intensities of 2-aminopurines, the changes in the intensity relative to the denatured state, and the sensitivity of the fluorescence to quenching by acrylamide. To establish the stem and loop characteristics in (CAG)(8), known reference points for stem, loop, and exposed base motifs were used. In the vicinity of the loop, the bases become more solvent exposed, which suggests that the instability associated with this repeated hairpin influences the global secondary structure. These results provide the basis to interpret the structures adopted by other repeated (CAG) structures.

Context Dependence of Trinucleotide Repeat Structures

Long repeated sequences of DNA and their associated secondary structure govern the development and severity of a significant class of neurological diseases. Utilizing the effect of base stacking on fluorescence quantum yield, 2-aminopurine substitutions for adenine previously demonstrated sequestered bases in the stem and exposed bases in the loop for an isolated (CAG)(8) sequence. This study evaluates (CAG)(8) that is incorporated into a duplex, as this three-way junction is a relevant model for intermediates that lead to repeat expansion during DNA replication and repair. From an energetic perspective, thermally induced denaturation indicates that the duplex arms dictate stability and that the secondary structure of the repeated sequence is disrupted. Substitutions with 2-aminopurine probe base exposure throughout this structure, and two conclusions about secondary structure are derived. First, the central region of (CAG)(8) is more solvent-exposed than single-stranded DNA, which suggests that hairpin formation in the repeated sequence is disrupted. Second, base stacking becomes compromised in the transition from the duplex to (CAG)(8), resulting in bases that are most similar to single-stranded DNA at the junction. Thus, an open (CAG)(8) loop and exposed bases in the arms indicate that the strand junction profoundly influences repeated sequences within three-way junctions.

Sequence length dictates repeated CAG folding in three-way junctions.

The etiology of a large class of inherited neurological diseases is founded on hairpin structures adopted by repeated DNA sequences, and this folding is determined by base sequence and DNA context. Using single substitutions of adenine with 2-aminopurine, we show that intrastrand folding in repeated CAG trinucleotides is also determined by the number of repeats. This isomeric analogue has a fluorescence quantum yield that varies strongly with solvent exposure, thereby distinguishing particular DNA motifs. Prior studies demonstrated that (CAG)(8) alone favors a stem-loop hairpin, yet the same sequence adopts an open loop conformation in a three-way junction. This comparison suggests that repeat folding is disrupted by base pairing in the duplex arms and by purine-purine mismatches in the repeat stem. However, these perturbations are overcome in longer CAG repeats, as demonstrated by studies of isolated and integrated forms of (CAG)(15). The oligonucleotide alone forms a symmetrically folded hairpin with looplike properties exhibited by the relatively high emission intensities from a modification in the central eighth repeat and with stemlike properties evident from the relatively low emission intensities from peripheral modifications. Significantly, these hairpin properties are retained when (CAG)(15) is integrated into a duplex. Intrastrand folding by (CAG)(15) in the three-way junction contrasts with the open loop adopted by (CAG)(8) in the analogous context. This distinction suggests that cooperative interactions in longer repeat tracts overwhelm perturbations to reassert the natural folding propensity. Given that anomalously long repeats are the genetic basis of a large class of inherited neurological diseases, studies with (CAG)-based three-way junctions suggest that their secondary structure is a key factor in the length-dependent manifestation and progression of such diseases.

Arginine-linked neomycin B dimers: synthesis, rRNA binding, and resistance enzyme activity

The nucleotides comprising the ribosomal decoding center are highly conserved, as they are important for maintaining translational fidelity. The bacterial A-site has a small base variation as compared with the human analogue, allowing aminoglycoside (AG) antibiotics to selectively bind within this region of the ribosome and negatively affect microbial protein synthesis. Here, by using a fluorescence displacement screening assay, we demonstrate that neomycin B (NEO) dimers connected by L-arginine-containing linkers of varying length and composition bind with higher affinity to model A-site RNAs compared to NEO, with IC50 values ranging from ~40-70 nM, and that a certain range of linker lengths demonstrates a clear preference for the bacterial A-site RNA over the human analogue. Furthermore, AG-modifying enzymes (AMEs), such as AG O-phosphotransferases, which are responsible for conferring antibiotic resistance in many types of infectious bacteria, demonstrate markedly reduced activity against several of the L-arginine-linked NEO dimers in vitro. The antimicrobial activity of these dimers against several bacterial strains is weaker than that of the parent NEO.

Eukaryotic Ribosomal Expansion Segments as Antimicrobial Targets

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 μM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH, and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CA in vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.

Non-B conformations of CAG repeats using 2-aminopurine.

Repetition of trinucleotide sequences is the molecular basis of ~30 hereditary neurological and neurodegenerative diseases, and alternate structures adopted by these sequences are implicated in the etiology of such diseases. Elucidating these structures is important for advancing mechanistic understanding and ultimately treatment. Studies of (CAG) repeats are motivated by their involvement in a number of these diseases, and the structures favored by (CAG)₈ are discussed in this contribution. Utilizing the strong effect of base stacking on fluorescence quantum yield, 2-aminopurine is used in place of adenine to determine the secondary structures adopted by such repeated sequences. Alone, (CAG)₈ folds into a hairpin comprised of a duplex stem and a single-stranded loop. Energetic studies indicate that the hairpin is anchored by the interactions in the stem and has a strained loop environment. As a model for intermediates that form during repeat expansion, (CAG)₈ was also incorporated into a duplex to form a three-way junction. In contrast to the isolated (CAG)₈, this integrated repeat adopts an open, unfolded loop. Enthalpy and entropy changes associated with denaturation indicate that the stability of the three-way junction is dominated by interactions in the duplex arms and that the repeated sequence tracks global unfolding. Because 2-aminopurine provides both structural and energetic information via fluorescence and also is an innocuous substitution for adenine, significant progress in elucidating the secondary structures of (CAG) repeats will be achieved.