Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.

2-Aminopurine-based quencher-free DNA tweezers with fluorescence properties well tuned by surrounding bases.

Reversible structural changes in DNA nanomachines have great potential in the field of bioanalysis. Here, we demonstrate an assembly strategy for quencher-free and tunable DNA tweezers based on 2-aminopurine (2-AP), avoiding the tedious fluorescence labelling step. The conformational state of the tweezers could be controlled by specific oligonucleotides (fuel or anti-fuel). Taking advantage of the local environmental sensitivity of 2-AP, the structural changes of the tweezers were easily tracked, and multiple cyclic switching of the tweezers between the open and closed states was achieved. In addition, the influence of oligonucleotide structure on the fluorescence properties of 2-AP was deeply explored. We figured out that the fluorescence of 2-AP was highly quenched by the base-stacking of natural bases in DNA oligonucleotides. Moreover, by comprehensively regulating the type of bases surrounding the inserted 2-AP site, a sensitive fluorescence response towards dynamic change can be obtained. This principle of quencher-free nanodevices based on 2-AP provides a convenient method for monitoring the structural changes of DNA nanomachines.

BZ Junctions and Its Application as Probe (2AP) to Detect Z-DNA Formation and Its Effector.

The left-handed Z-DNA is surrounded by right-handed canonical B-DNA, and thus the junction between B- and Z-DNA has been occurred during temporal Z-DNA formation in the genome. The base extrusion structure of the BZ junction may help detect Z-DNA formation in DNAs. Here we describe the BZ junction structural detection by using 2-aminopurine (2AP) fluorescent probe. BZ junction formation can be measured in solution by this method.

Probing RNA Structures and Interactions Using Fluorescence Lifetime Analyses.

Structural analyses of large, complex noncoding RNAs continue to lag behind their rapid discovery and functional descriptions. Site-specifically incorporated, minimally invasive fluorescent probes such as 2-aminopurine (2AP) and pyrrolo-cytosine (PyC) have provided essential complementary information about local RNA structure, conformational dynamics, and interactions. Here I describe a protocol that benchmarks and correlates local RNA conformations with their respective fluorescence lifetimes, as a general technique that confers key advantages over fluorescence intensity-based methods. The observation that fluorescence lifetimes are more sensitive to local structures than sequence contexts suggests broad utility across diverse RNA and ribonucleoprotein systems.

Base-Stacking Heterogeneity in RNA Resolved by Fluorescence-Detected Circular Dichroism Spectroscopy.

RNA plays a critical role in many biological processes, and the structures it adopts are intimately linked to those functions. Among many factors that contribute to RNA folding, van der Waals interactions between adjacent nucleobases stabilize structures in which the bases are stacked on top of one another. Here, we utilize fluorescence-detected circular dichroism spectroscopy (FDCD) to investigate base-stacking heterogeneity in RNA labeled with the fluorescent adenine analogue 2-aminopurine (2-AP). Comparison of standard (transmission-detected) CD and FDCD spectra reveals that in dinucleotides, 2-AP fluorescence is emitted almost exclusively by unstacked molecules. In a trinucleotide, some fluorescence is emitted by a population of stacked and highly quenched molecules, but more than half originates from a minor ∼10% population of unstacked molecules. The combination of FDCD and standard CD measurements reveals the prevalence of stacked and unstacked conformational subpopulations as well as their relative fluorescence quantum yields.

2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer.

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a Kd of 15.0 mM glucose, which was comparable with the 10 mM Kd previously reported using the strand displacement assay. The binding required two Na+ ions or one Mg2+ that cannot be replaced by Li+ or K+ . The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher Kd values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8.

All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3'-5' exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA.

Bacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host's restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

A widespread pathway for substitution of adenine by diaminopurine in phage genomes.

DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. Diaminopurine (Z) is an exception because it completely replaces adenine and forms three hydrogen bonds with thymine in cyanophage S-2L genomic DNA. However, the biosynthesis, prevalence, and importance of Z genomes remain unexplored. Here, we report a multienzyme system that supports Z-genome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables Z-DNA production on a large scale for a diverse range of applications.

A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes.

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology.

Noncanonical DNA polymerization by aminoadenine-based siphoviruses.

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.

Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution.

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance.

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide "shoulders" of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5-7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5'- and 3'-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.

A simple and affordable kinetic assay of nucleic acids with SYBR Gold gel staining.

Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.

Incorporation of Pseudo-complementary Bases 2,6-Diaminopurine and 2-Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization.

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.

Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution.

2-aminopurine (2AP) is a responsive fluorescent base analogue that is used widely as a probe of the local molecular environment in DNA. The ability of 2AP to report changes in local conformation and base-stacking interactions arises from the efficient quenching of its fluorescence by the natural DNA bases. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of the collisional quenching of 2AP by the natural bases, in different buffer solutions, showed that dynamic quenching efficiency depends on the identity of the natural base, but disagreed on the relative quenching efficiencies of the bases. We report a comprehensive investigation of inter-base quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies. Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a charge-transfer mechanism. Time-resolved measurements also provide insight into static quenching, revealing formation of 2AP-NMP ground-state complexes in which 2AP displays a very short fluorescence lifetime, comparable to that seen in oligonucleotides. In these complexes, the dependence of the rate of quenching on the partner base also supports a charge-transfer mechanism.

Nucleobase derivatives induce in-source decay of oligonucleotides as new matrix-assisted laser desorption/ionization matrices.

RATIONALE: For quality control of oligonucleotide therapeutics, accurate and efficient structural characterization using mass spectrometry techniques, such as liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), is essential. In MALDI MS analysis, matrix selection is critical and a new matrix could enable more efficient and rapid structural analysis. METHODS: We hypothesized that nucleobase derivatives could act as matrices more efficiently than the currently used matrices for oligonucleotides because of structural similarity, which leads to close contact with the analyte. To evaluate their suitability as matrices, 16 nucleobase derivatives were selected and tested as matrix candidates for oligonucleotide analysis. RESULTS: Six of the 16 nucleobase derivatives acted as matrices for oligonucleotides. Particularly, 6-thioguanine (TG) performed well and induced clear in-source decay fragmentation. When TG or 2-amino-6-chloropurine was used as the matrix, oligonucleotides were ionized, and mainly the w and d fragment ions were observed. CONCLUSIONS: Herein we demonstrate that a 10-mer RNA or DNA sequence can be successfully characterized using TG as matrix and suggest the possibility of using nucleobase derivatives as novel matrices in oligonucleotide sequencing.

A quencher-free 2-aminopurine modified hairpin aptasensor for ultrasensitive detection of Ochratoxin A.

A sensitive, efficient and quencher-free fluorescence aptasensor to detect Ochratoxin A (OTA) based on aptamer, 2-aminopurine (2AP) labeled Oligonucleotide sequence, as well as exonuclease I (Exo I) activity was developed. In which the aptamer specific to OTA was modified into a hairpin structure, and 8 bases at the 3' ends are exposed (H); also, 2AP is embedded in the oligonucleotide complementary to the 8 bases (2AP-probe).The detection principle based on 2AP-probe could be bonded to its complementary sequence and quenches the fluorescence of 2AP; The aptamer has a stronger affinity for the target than its complementary sequence; Exo I can dissociate single-stranded DNA and has little effect on double-stranded DNA as well as folded DNA. In the absence of OTA, the fluorescence of 2AP is quenched due to the complementary pairing of H and 2AP-probe; in the presence of OTA, H selective binding target is detached from 2AP-probe, and the fluorescence of 2AP is slightly restored. Moreover, when the Exo I is added to the detection system, 2AP-probe is dissociated by the Exo I to release the free 2AP, and the fluorescence of the system is further enhanced thereby realizing the detection of OTA. The detection limit of the aptasensor was low as 0.03 nM with a linear range of 0.5-100 nM. Moreover, the aptasensor has good selectivity and practicability and also has good potential in realizing the detection of toxic and harmful substances in food complex matrices.

Common Secondary and Tertiary Structural Features of Aptamer-Ligand Interaction Shared by RNA Aptamers with Different Primary Sequences.

Aptamer selection can yield many oligonucleotides with different sequences and affinities for the target molecule. Here, we have combined computational and experimental approaches to understand if aptamers with different sequences but the same molecular target share structural and dynamical features. NEO1A, with a known NMR-solved structure, displays a flexible loop that interacts differently with individual aminoglycosides, its ligand affinities and specificities are responsive to ionic strength, and it possesses an adenosine in the loop that is critical for high-affinity ligand binding. NEO2A was obtained from the same selection and, although they are only 43% identical in overall sequence, NEO1A and NEO2A share similar loop sequences. Experimental analysis by 1D NMR and 2-aminopurine reporters combined with molecular dynamics modeling revealed similar structural and dynamical characteristics in both aptamers. These results are consistent with the hypothesis that the target ligand drives aptamer structure and also selects relevant dynamical characteristics for high-affinity aptamer-ligand interaction. Furthermore, they suggest that it might be possible to "migrate" structural and dynamical features between aptamer group members with different primary sequences but with the same target ligand.

Interstrand DNA Cross-Links Derived from Reaction of a 2-Aminopurine Residue with an Abasic Site.

Efficient methods for the site-specific installation of structurally defined interstrand cross-links in duplex DNA may be useful in a wide variety of fields. The work described here developed a high-yield synthesis of chemically stable interstrand cross-links resulting from a reductive amination reaction between an abasic site and the noncanonical nucleobase 2-aminopurine in duplex DNA. Results from footprinting, liquid chromatography-mass spectrometry, and stability studies support the formation of an N2-alkylamine attachment between the 2-aminopurine residue and the Ap site. The reaction performs best when the 2-aminopurine residue on the opposing strand is offset 1 nt to the 5'-side of the abasic site. The cross-link confers substantial resistance to thermal denaturation (melting). The cross-linking reaction is fast (complete in 4 h), employs only commercially available reagents, and can be used to generate cross-linked duplexes in sufficient quantities for biophysical, structural, and DNA repair studies.