Long-lived Ag10 6+ luminescence and a split DNA scaffold.

Molecular silver clusters emit across the visible to near-infrared, and specific chromophores can be formed using DNA strands. We study C4AC4TC3G that selectively coordinates and encapsulates Ag10 6+, and this chromophore has two distinct electronic transitions. The green emission is strong and prompt with ϕ = 18% and τ = 1.25 ns, and the near-infrared luminescence is weaker, slower with τ = 50 µs, and is partly quenched by oxygen, suggesting phosphorescence. This lifetime can be modulated by the DNA host, and we consider two derivatives of C4AC4TC3G with similar sequences but distinct structures. In one variant, thymine was excised to create an abasic gap in an otherwise intact strand. In the other, the covalent phosphate linkage was removed to split the DNA scaffold into two fragments. In relation to the contiguous strands, the broken template speeds the luminescence decay by twofold, and this difference may be due to greater DNA flexibility. These modifications suggest that a DNA can be structurally tuned to modulate metastable electronic states in its silver cluster adducts.

A single nucleobase tunes nonradiative decay in a DNA-bound silver cluster.

DNA strands are polymeric ligands that both protect and tune molecular-sized silver cluster chromophores. We studied single-stranded DNA C4AC4TC3XT4 with X = guanosine and inosine that form a green fluorescent Ag10 6+ cluster, but these two hosts are distinguished by their binding sites and the brightness of their Ag10 6+ adducts. The nucleobase subunits in these oligomers collectively coordinate this cluster, and fs time-resolved infrared spectra previously identified one point of contact between the C2-NH2 of the X = guanosine, an interaction that is precluded for inosine. Furthermore, this single nucleobase controls the cluster fluorescence as the X = guanosine complex is ∼2.5× dimmer. We discuss the electronic relaxation in these two complexes using transient absorption spectroscopy in the time window 200 fs-400 µs. Three prominent features emerged: a ground state bleach, an excited state absorption, and a stimulated emission. Stimulated emission at the earliest delay time (200 fs) suggests that the emissive state is populated promptly following photoexcitation. Concurrently, the excited state decays and the ground state recovers, and these changes are ∼2× faster for the X = guanosine compared to the X = inosine cluster, paralleling their brightness difference. In contrast to similar radiative decay rates, the nonradiative decay rate is 7× higher with the X = guanosine vs inosine strand. A minor decay channel via a dark state is discussed. The possible correlation between the nonradiative decay and selective coordination with the X = guanosine/inosine suggests that specific nucleobase subunits within a DNA strand can modulate cluster-ligand interactions and, in turn, cluster brightness.

Atomic Structure of a Fluorescent Ag8 Cluster Templated by a Multistranded DNA Scaffold.

Multinuclear silver clusters encapsulated by DNA exhibit size-tunable emission spectra and rich photophysics, but their atomic organization is poorly understood. Herein, we describe the structure of one such hybrid chromophore, a green-emitting Ag8 cluster arranged in a Big Dipper-shape bound to the oligonucleotide A2C4. Three 3' cytosine metallo-base pairs stabilize a parallel A-form-like duplex with a 5' adenine-rich pocket, which binds a metallic, trapezoidal-shaped Ag5 moiety via Ag-N bonds to endo- and exocyclic nitrogens of cytosine and adenine. The unique DNA configuration, constrained coordination environment, and templated Ag8 cluster arrangement highlight the reciprocity between the silvers and DNA in adopting this structure. These first atomic details of a DNA-encapsulated Ag cluster fluorophore illuminate many aspects of biological assembly, nanoscience, and metal cluster photophysics.

LNA Thymidine Monomer Enables Differentiation of the Four Single-Nucleotide Variants by Melting Temperature.

High-resolution melting (HRM) analysis of DNA is a closed-tube single-nucleotide polymorphism (SNP) detection method that has shown many advantages in point-of-care diagnostics and personalized medicine. While recently developed melting probes have demonstrated significantly improved discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no effort has been made to design a simple melting probe that can reliably distinguish all four SNP alleles in a single experiment. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost. Here we demonstrate that a melting probe embedded with a single locked thymidine monomer (tL) can reliably differentiate the four SNP alleles by four distinct melting temperatures (termed the "4Tm probe"). This enhanced discriminatory power comes from the decreased melting temperature of the tL·C mismatched hybrid as compared to that of the t·C mismatched hybrid, while the melting temperatures of the tL-A, tL·G and tL·T hybrids are increased or remain unchanged as compared to those of their canonical counterparts. This phenomenon is observed not only in the HRM experiments but also in the molecular dynamics simulations.

A Segregated, Partially Oxidized, and Compact Ag10 Cluster within an Encapsulating DNA Host.

Silver clusters develop within DNA strands and become optical chromophores with diverse electronic spectra and wide-ranging emission intensities. These studies consider a specific cluster that absorbs at 400 nm, has low emission, and exclusively develops with single-stranded oligonucleotides. It is also a chameleon-like chromophore that can be transformed into different highly emissive fluorophores. We describe four characteristics of this species and conclude that it is highly oxidized yet also metallic. One, the cluster size was determined via electrospray ionization mass spectrometry. A common silver mass is measured with different oligonucleotides and thereby supports a Ag10 cluster. Two, the cluster charge was determined by mass spectrometry and Ag L3-edge X-ray absorption near-edge structure spectroscopy. Respectively, the conjugate mass and the integrated white-line intensity support a partially oxidized cluster with a +6 and +6.5 charge, respectively. Three, the cluster chirality was gauged by circular dichroism spectroscopy. This chirality changes with the length and sequence of its DNA hosts, and these studies identified a dispersed binding site with ∼20 nucleobases. Four, the structure of this complex was investigated via Ag K-edge extended X-ray absorption fine structure spectroscopy. A multishell fitting analysis identified three unique scattering environments with corresponding bond lengths, coordination numbers, and Debye-Waller factors for each. Collectively, these findings support the following conclusion: a Ag10(+6) cluster develops within a 20-nucleobase DNA binding site, and this complex segregates into a compact, metal-like silver core that weakly links to an encapsulating silver-DNA shell. We consider different models that account for silver-silver coordination within the core.

Ten-atom silver cluster signaling and tempering DNA hybridization.

Silver clusters with ∼10 atoms are molecules, and specific species develop within DNA strands. These molecular metals have sparsely organized electronic states with distinctive visible and near-infrared spectra that vary with cluster size, oxidation, and shape. These small molecules also act as DNA adducts and coordinate with their DNA hosts. We investigated these characteristics using a specific cluster-DNA conjugate with the goal of developing a sensitive and selective biosensor. The silver cluster has a single violet absorption band (λ(max) = 400 nm), and its single-stranded DNA host has two domains that stabilize this cluster and hybridize with target oligonucleotides. These target analytes transform the weakly emissive violet cluster to a new chromophore with blue-green absorption (λ(max) = 490 nm) and strong green emission (λ(max) = 550 nm). Our studies consider the synthesis, cluster size, and DNA structure of the precursor violet cluster-DNA complex. This species preferentially forms with relatively low amounts of Ag(+), high concentrations of the oxidizing agent O2, and DNA strands with ≳20 nucleotides. The resulting aqueous and gaseous forms of this chromophore have 10 silvers that coalesce into a single cluster. This molecule is not only a chromophore but also an adduct that coordinates multiple nucleobases. Large-scale DNA conformational changes are manifested in a 20% smaller hydrodynamic radius and disrupted nucleobase stacking. Multidentate coordination also stabilizes the single-stranded DNA and thereby inhibits hybridization with target complements. These observations suggest that the silver cluster-DNA conjugate acts like a molecular beacon but is distinguished because the cluster chromophore not only sensitively signals target analytes but also stringently discriminates against analogous competing analytes.

Near-infrared silver cluster optically signaling oligonucleotide hybridization and assembling two DNA hosts.

Silver clusters with ~10 atoms form within DNA strands, and the conjugates are chemical sensors. The DNA host hybridizes with short oligonucleotides, and the cluster moieties optically respond to these analytes. Our studies focus on how the cluster adducts perturb the structure of their DNA hosts. Our sensor is comprised of an oligonucleotide with two components: a 5'-cluster domain that complexes silver clusters and a 3'-recognition site that hybridizes with a target oligonucleotide. The single-stranded sensor encapsulates an ~11 silver atom cluster with violet absorption at 400 nm and with minimal emission. The recognition site hybridizes with complementary oligonucleotides, and the violet cluster converts to an emissive near-infrared cluster with absorption at 730 nm. Our key finding is that the near-infrared cluster coordinates two of its hybridized hosts. The resulting tertiary structure was investigated using intermolecular and intramolecular variants of the same dimer. The intermolecular dimer assembles in concentrated (~5 µM) DNA solutions. Strand stoichiometries and orientations were chromatographically determined using thymine-modified complements that increase the overall conjugate size. The intramolecular dimer develops within a DNA scaffold that is founded on three linked duplexes. The high local cluster concentrations and relative strand arrangements again favor the antiparallel dimer for the near-infrared cluster. When the two monomeric DNA/violet cluster conjugates transform to one dimeric DNA/near-infrared conjugate, the DNA strands accumulate silver. We propose that these correlated changes in DNA structure and silver stoichiometry underlie the violet to near-infrared cluster transformation.

A silver cluster-DNA equilibrium.

DNA encapsulates silver clusters, and these hybrid nanomaterials form molecular sensors. We discuss a silver cluster-oligonucleotide sensor with four characteristics. First, a specific reporting cluster forms within a single-stranded DNA. This template uses the 5' cluster domain CCCCAACTCCTT with different 3' recognition sites for complementary oligonucleotides. The modular composite strand exclusively forms a cluster with λmax = 400 nm and with low emission. Conjugates were chromatographically purified, and their elemental analysis measured a cluster adduct with ∼11 silver atoms. Second, hybridization transforms the cluster. Size exclusion chromatography shows that the 3' recognition sites of the single-stranded conjugates hybridize with their complements. This secondary structural change both shifts cluster absorption from 400 to 490 nm and develops emission at 550 nm. Third, cluster size remains intact. Like their violet predecessors, purified blue-green clusters have ∼11 silver atoms. Cluster integrity is further supported by extracting the complement from the blue-green conjugate and reversing the spectral changes. Fourth, the cluster transformation is an equilibrium. Complementary strands generate an isosbestic point and thus directly link single-stranded hosts for the violet cluster and their hybridized analogs for the blue-green cluster. This equilibrium shifts with temperature. A van't Hoff analysis shows that longer and more stable duplexes favor the blue-green cluster. However, hybridized cluster hosts are less stable than their native DNA counterparts, and stability further degrades when short complements expose nucleobases within S1-S2. Duplex instability suggests that unpaired nucleobases coordinate the violet cluster and favor the single-stranded sensor. A balance between innate hybridization and exogenous folding highlights a distinct feature of silver clusters for sensing: they are both chromophoric reporters and ligands that modulate analyte-sensor interactions.

Silver clusters as both chromophoric reporters and DNA ligands.

Molecular silver clusters conjugated with DNA act as analyte sensors. Our studies evaluate a type of cluster-laden DNA strand whose structure and silver stoichiometry change with hybridization. The sensor strand integrates two functions: the 3' region binds target DNA strands through base recognition while the 5' sequence C(3)AC(3)AC(3)TC(3)A favors formation of a near-infrared absorbing and emitting cluster. This precursor form exclusively harbors an ∼11 silver atom cluster that absorbs at 400 nm and that condenses its single-stranded host. The 3' recognition site associates with a complementary target strand, thereby effecting a 330 nm red-shift in cluster absorption and a background-limited recovery of cluster emission at 790 nm. One factor underlying these changes is sensor unfolding and aggregation. Variations in salt and oligonucleotide concentrations control cluster development by influencing DNA association. Structural studies using fluorescence anisotropy, fluorescence correlation spectroscopy, and size exclusion chromatography show that the sensor-cluster conjugate opens and subsequently dimerizes with hybridization. A second factor contributing to the spectral and photophysical changes is cluster transformation. Empirical silver stoichiometries are preserved through hybridization, so hybridized, dimeric near-infrared conjugates host twice the amount of silver in relation to their violet absorbing predecessors. These DNA structure and net silver stoichiometry alterations provide insight into how DNA-silver conjugates recognize analytes.

Breaching the Fortress: Photochemistry of DNA-Caged Ag106.

A DNA strand can encapsulate a silver molecule to create a nanoscale, aqueous stable chromophore. A protected cluster that strongly fluoresces can also be weakly photolabile, and we describe the laser-driven photochemistry of the green fluorophore C4AC4TC3GT4/Ag106+. The embedded cluster is selectively photoexcited at 490 nm and then bleached, and we describe how the efficiency, products, and route of this photochemical reaction are controlled by the DNA cage. With irradiation at 496.5 nm, the cluster absorption progressively drops to give a photodestruction quantum yield of 1.5 (±0.2) × 10-4, ∼103× less efficient than fluorescence. A new λabs = 335 nm chromophore develops because the precursor with 4 Ag0 is converted into a group of clusters with 2 Ag0 - Ag64+, Ag75+, Ag86+, and Ag97+. The 4-7 Ag+ in this series are chemically distinct from the 2 Ag0 because they are selectively etched by iodide. This halide precipitates silver to favor only the smallest Ag64+ cluster, but the larger clusters re-develop when the precipitated Ag+ ions are replenished. DNA-bound Ag106+ decomposes because it is electronically excited and then reacts with oxygen. This two-step process may be state-specific because O2 quenches the red luminescence from Ag106+. However, the rate constant of 2.3 (±0.2) × 106 M-1 s-1 is relatively small, which suggests that the surrounding DNA matrix hinders O2 diffusion. On the basis of analogous photoproducts with methylene blue, we propose that a reactive oxygen species is produced and then oxidizes Ag106+ to leave behind a loose Ag+-DNA skeleton. These findings underscore the ability of DNA scaffolds to not only tune the spectra but also guide the reactions of their molecular silver adducts.

Interrupted DNA and Slow Silver Cluster Luminescence.

A DNA-silver cluster conjugate is a hierarchical chromophore with a partly reduced silver core embedded within the DNA nucleobases that are covalently linked by the phosphodiester backbone. Specific sites within a polymeric DNA can be targeted to spectrally tune the silver cluster. Here, the repeated (C2A)6 strand is interrupted with a thymine, and the resulting (C2A)2-T-(C2A)4 forms only Ag106+, a chromophore with both prompt (∼1 ns) green and sustained (∼102 µs) red luminescence. Thymine is an inert placeholder that can be removed, and the two fragments (C2A)2 and (C2A)4 also produce the same Ag106+ adduct. In relation to (C2A)2T(C2A)4, the (C2A)2 + (C2A)4 pair is distinguished because the red Ag106+ luminescence is ∼6× lower, relaxes ∼30% faster, and is quenched ∼2× faster with O2. These differences suggest that a specific break in the phosphodiester backbone can regulate how a contiguous vs broken scaffold wraps and better protects its cluster adduct.

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.

Tug-of-War between DNA Chelation and Silver Agglomeration in DNA-Silver Cluster Chromophores.

Supramolecular chromophores form when a DNA traps silvers that then coalesce into clusters with discrete, molecular electronic states. However, DNA strands are polymeric ligands that disperse silvers and thus curb agglomeration. We study this competition using two chromophores that share three common components: a dimeric DNA scaffold, Ag+-nucleobase base pairs, and Ag0 chromophores. The DNA host C4-A2-iC4T mimics structural elements in a DNA-cluster crystal structure using a phosphodiester backbone with combined 5' → 3' and 3' → 5' (indicated by "i") directions. The backbone directions must alternate to form the two silver clusters, and this interdependence supports a silver-linked structure. This template creates two chromophores with distinct sizes, charges, and hence spectra: (C4-A2-iC4T)2/Ag117+ with λabs/λem = 430/520 nm and (C4-A2-iC4T)2/Ag148+ with λabs/λem = 510/630 nm. The Ag+ and Ag0 constituents in these partially oxidized clusters are linked with structural elements in C4-A2-iC4T. Ag+ alone binds sparsely but strongly to form C4-A2-iC4T/3-4 Ag+ and (C4-A2-iC4T)2/7-8 Ag+ complexes, and these stoichiometries suggest that Ag+ cross-links pairs of cytosines to form a hairpin with a metallo-C4/iC4 duplex and an adenine loop. The Ag0 are chemically orthogonal because they can be oxidatively etched without disrupting the underlying Ag+-DNA matrix, and their reactivity is attributed to their valence electrons and weaker chelation by the adenines. These studies suggest that Ag+ disperses with the cytosines to create an adenine binding pocket for the Ag0 cluster chromophores.

Mapping H+ in the Nanoscale (A2C4)2-Ag8 Fluorophore.

When strands of DNA encapsulate silver clusters, supramolecular optical chromophores develop. However, how a particular structure endows a specific spectrum remains poorly understood. Here, we used neutron diffraction to map protonation in (A2C4)2-Ag8, a green-emitting fluorophore with a "Big Dipper" arrangement of silvers. The DNA host has two substructures with distinct protonation patterns. Three cytosines from each strand collectively chelate handle-like array of three silvers, and calorimetry studies suggest Ag+ cross-links. The twisted cytosines are further joined by hydrogen bonds from fully protonated amines. The adenines and their neighboring cytosine from each strand anchor a dipper-like group of five silvers via their deprotonated endo- and exocyclic nitrogens. Typically, exocyclic amines are strongly basic, so their acidification and deprotonation in (A2C4)2-Ag8 suggest that silvers perturb the electron distribution in the aromatic nucleobases. The different protonation states in (A2C4)2-Ag8 suggest that atomic level structures can pinpoint how to control and tune the electronic spectra of these nanoscale chromophores.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

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.

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(+).

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.

Time-Resolved Vibrational Fingerprints for Two Silver Cluster-DNA Fluorophores.

DNA-templated silver clusters are chromophores in which the nucleobases encode the cluster spectra and brightness. We describe the coordination environments of two nearly identical Ag106+ clusters that form with 18-nucleotide strands CCCCA CCCCT CCCX TTTT, with X = guanosine and inosine. For the first time, femtosecond time-resolved infrared (TRIR) spectroscopy with visible excitation and mid-infrared probing is used to correlate the response of nucleobase vibrational modes to electronic excitation of the metal cluster. A rich pattern of transient TRIR peaks in the 1400-1720 cm-1 range decays synchronously with the visible emission. Specific infrared signatures associated with the single guanosine/inosine along with a subset of cytidines, but not the thymidines, are observed. These fingerprints suggest that the network of bonds between a silver cluster adduct and its polydentate DNA ligands can be deciphered to rationally tune the coordination and thus spectra of molecular silver chromophores.

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.