Fluorescence energy transfer monitored competitive equilibria of nucleic acids: applications in thermodynamics and screening

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins.     

Fluorescence energy transfer monitored competitive equilibria of nucleic acids: Applications in thermodynamics and screening

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 214–223, 2002     

Pyrophosphate-condensing activity linked to nucleic acid synthesis.

In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.     

Computational investigation of proton transfer,pKa shifts and pH‐optimum of protein–DNA and protein–RNA complexes

Protein–nucleic acid interactions play a crucial role in many biological processes. This work investigates the changes of pKa values and protonation states of ionizable groups (including nucleic acid bases) that may occur at protein–nucleic acid binding. Taking advantage of the recently developed pKa calculation tool DelphiPka, we utilize the large protein–nucleic acid interaction database (NPIDB database) to model pKa shifts caused by binding. It has been found that the protein's interfacial basic residues experience favorable electrostatic interactions while the protein acidic residues undergo proton uptake to reduce the energy cost upon the binding. This is in contrast with observations made for protein–protein complexes. In terms of DNA/RNA, both base groups and phosphate groups of nucleotides are found to participate in binding. Some DNA/RNA bases undergo pKa shifts at complex formation, with the binding process tending to suppress charged states of nucleic acid bases. In addition, a weak correlation is found between the pH‐optimum of protein–DNA/RNA binding free energy and the pH‐optimum of protein folding free energy. Overall, the pH‐dependence of protein–nucleic acid binding is not predicted to be as significant as that of protein–protein association. Proteins 2017; 85:282–295. © 2016 Wiley Periodicals, Inc.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase gamma in vitro is not impaired

Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4-13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp 'common deletion' were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase gamma-containing mitochondrial fraction was used.     

A sequential expression system for high-throughput functional genomic analysis

A method employing sequential rounds of cell-free protein synthesis (CFPS) was developed to identify gene products influencing the complex metabolic systems that result in protein accumulation and folding in vitro. The first round of CFPS creates an array of cell extracts individually enriched with a single gene product expressed in-parallel from linear DNA expression templates (ETs). The cell extract is engineered to enhance template stability and to provide reaction conditions conducive for general protein activation. Following first-round expression, linear templates are selectively degraded and a plasmid template for a reporter enzyme is added to initiate a subsequent round of protein expression. Reporter concentration and activity identify first-round gene products that affect amino acid and nucleic acid stability, energy supply, protein expression, stability, and activation. This sequential CFPS system provides a unique format for the functional genomic identification of broadly diverse metabolic activities.     

Isolation of polyadenylic acid-containing RNA using Ca(II)

Purine ribonucleotide homopolymers are insoluble in dilute Ca(II)- or Mg(II)-containing solutions whereas nucleic acid heteropolymers are soluble. The differences in solubility serve as a basis of a new method for separating polyadenylic acid-containing RNA from other nucleic acids. In the presence of as little as 10 mM Ca(II) polyadenylic acid-containing RNA of nucleic acid mixtures selectively coprecipitates with a small amount of added synthetic polyadenylic acid.     

Molecular Complexes of acridine orange and nucleosides

Summary The addition of various nucleosides to the aqueous AO solution brings about the red shift of the absorption band of AO monomer and the enhancement of the AO fluorescence emission. These phenomena are attributable to the formation of a kind of molecular complex between AO monomer and nucleoside.The absorption and fluorescence characteristics and their thermal behaviours enable us to determine the association constant and the binding energy. The binding energy of AO with purines is larger than that with pyrimidines, and the association constant between AO and the deoxyribonucleoside is larger than that between AO and the ribonucleoside. For the molecular complexes dealt with, the face-to-face arrangement of AO and nucleoside, linking of AO with sugar by hydrogen bridge, may be more preferential than the side-by-side arrangement with the direct linkage between AO and nucleic acid base by hydrogen bonding. The Van der Waals-London interactions may be one of the essential factors for the binding in these molecular complexes.The association constant and the binding energy for AO-DNA and -RNA systems were also determined. The magnitude of these quantities seems to reflect the difference in the structure of these nucleic acids. The rather open structure of RNA compared with DNA is in favour of affinity with AO and gives the larger association constant than that for AO-DNA. The binding energy is somewhat larger for AO-DNA complex than for AO-RNA complex, probably due to the structural difference of the base arrangement; the stacked base pairs for DNA and the stacked bases for RNA.This may explain the selective degradation [12] of guanine photo-sensitized by dyes either in the free state or when incorporated to DNA or RNA, as discussed elsewhere.     

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.     

Ned Seeman and the prediction of amino acid-basepair motifs mediating protein-nucleic acid recognition

Fifty years ago, the first atomic-resolution structure of a nucleic acid double helix, the mini-duplex (ApU)₂, revealed details of basepair geometry, stacking, sugar conformation, and backbone torsion angles, thereby superseding earlier models based on x-ray fiber diffraction, including the original DNA double helix proposed by Watson and Crick. Just 3 years later, in 1976, Ned Seeman, John Rosenberg, and Alex Rich leapt from their structures of mini-duplexes and H-bonding motifs between bases in small-molecule structures and transfer RNA to predicting how proteins could sequence specifically recognize double helix nucleic acids. They proposed interactions between amino acid side chains and nucleobases mediated by two hydrogen bonds in the major or minor grooves. One of these, the arginine-guanine pair, emerged as the most favored amino acid-base interaction in experimental structures of protein-nucleic acid complexes determined since 1986. In this brief review we revisit the pioneering work by Seeman et al. and discuss the importance of the arginine-guanine pairing motif.     

Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA

The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.     

Nonspecific prion protein-nucleic Acid interactions lead to different aggregates and cytotoxic species

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP-DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP-D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP-nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP-nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.     

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids

The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide.     

Ab initio conformational analysis of nucleic acid components: intrinsic energetic contributions to nucleic acid structure and dynamics

In recent years, the use of high-level ab initio calculations has allowed for the intrinsic conformational properties of nucleic acid building blocks to be revisited. This has provided new insights into the intrinsic conformational energetics of these compounds and its relationship to nucleic acids structure and dynamics. In this article we review recent developments and present new results. New data include comparison of various levels of theory on conformational properties of nucleic acid building blocks, calculations on the abasic sugar, known to occur in vivo in DNA, on the TA conformation of DNA observed in the complex with the TATA box binding protein, and on inosine. Tests of the Hartree-Fock (HF), second-order M?ller-Plesset (MP2), and Density Functional Theory/Becke3, Lee, Yang and Par (DFT/B3LYP) levels of theory show the overall shape of backbone torsional energy profiles (for gamma, epsilon, and chi) to be similar for the different levels, though some systematic differences are identified between the MP2 and DFT/B3LYP profiles. The east pseudorotation energy barrier in deoxyribonucleosides is also sensitive to the level of theory, with the HF and DFT/B3LYP east barriers being significantly lower (approximately 2.5 kcal/mol) than the MP2 counterpart (approximately 4.0 kcal/mol). Additional calculations at various levels of theory suggest that the east barrier in deoxyribonucleosides is between 3.0 and 4.0 kcal/mol. In the abasic sugar, the west pseudorotation energy barrier is found to be slightly lower than the east barrier and the south pucker is favored more than in standard nucleosides. Results on the TA conformation suggest that, at the nucleoside level, this conformation is significantly destabilized relative to the global energy minimum, or relative to the A- and B-DNA conformations. Deoxyribocytosine would destabilize the TA conformation more than other bases relative to the A-DNA conformation, but not relative to the B-DNA conformation.     

Strand Invasion Based Amplification (SIBA?): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.