| 1. |
- Nordén, Bengt, 1945, et al.
(author)
-
Thermodynamics of PNA Interactions
- 2005
-
In: Peptide Nucleic Acids. Protocols and Applications. - 9780954523244 ; , s. 77-105
-
Book chapter (other academic/artistic)
|
|
| 2. |
- Ratilainen, Tommi, 1970, et al.
(author)
-
A simple model for gene targeting
- 2001
-
In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 81:5, s. 2876-2885
-
Journal article (peer-reviewed)abstract
- Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial DeltaH(o) associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell.
|
|
| 3. |
- Ratilainen, Tommi, 1970, et al.
(author)
-
Hybridization of peptide nucleic acid
- 1998
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 37:35, s. 12331-12342
-
Journal article (peer-reviewed)abstract
- The thermodynamics of hybridization and the conformations of decameric mixed purine-pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 degrees C increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (Delta T-m = -12 degrees C) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA.
|
|
| 4. |
- Ratilainen, Tommi, 1970
(author)
-
Thermodynamics of Peptide Nucleic Acid Interactions with DNA
- 2000
-
Doctoral thesis (other academic/artistic)abstract
- Peptide Nucleic Acid (PNA) is a DNA mimic with the natural nucleobases attached to a charge-neutral pseudopeptide backbone. It has remarkably strong and sequence-specific binding affinity to natural nucleic acids (DNA and RNA), mainly attributed to the lack of charge in the PNA backbone. The ionic effects on the thermal stability of 10 and 15 base pairs long PNA-DNA duplexes have been studied. At low ionic strength ( 1 M), increased salt concentration decreased the stability of the three kinds of duplexes in a similar fashion. By measuring the fluorescence energy transfer (FET) efficiency from fluorescein to rhodamine labels attached to opposite ends of 10-mer PNA-PNA, PNA-DNA, and DNA-DNA duplexes, the estimated end-to-end distances were all found to be consistent with relatively extended duplex conformations. While quite useful for structural characterization, due to label-duplex interactions, FET was found to be less suitable than absorption hyperchromicity for thermodynamic analysis. A systematic study of the thermodynamics of PNA-DNA duplexes established a length- and sequence-averaged binding constant of 14 M-1bp-1 at 25°C, while the stability of single mismatches in mixed sequence 9-12-mers was found to vary significantly and depend on both stability and nature of the surrounding base pairs. On average, one mismatch turned out to cost as much as the gain of two perfect base pairs. Further analysis of the thermodynamic parameters revealed that strong enthalpy-entropy compensation is important for PNA-DNA interactions. Based on the thermodynamic data, the hybridization properties of PNA versus a hypothetical genome target was modeled using a simple statistical approach. Though simple, the model gives general insight into sequence-specific binding, and should be applicable to a variety of situations, ranging from gene-targeting to biotechnical applications such as gene-diagnostics. In addition, FET was also used for kinetic investigation of interactions between structured, naturally occurring regulatory RNA pairs. The overall kinetic rate constants for complex formation of wild-type and mutated pairs were obtained, as well as a preliminary characterization of the early interaction event using the fluorescent nucleobase analog 2-aminopurine.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Ratilainen, Tommi, 1970, et al.
(author)
-
Thermodynamics of sequence-specific binding of PNA to DNA
- 2000
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 39:26, s. 7781-7791
-
Journal article (peer-reviewed)abstract
- For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (Delta G degrees) was determined to be -6.5 +/- 0.3 kT mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M-1 bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T-m) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kT mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M-1 per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.
|
|
| 8. |
- Tomac, S., et al.
(author)
-
Ionic effects on the stability and conformation of peptide nucleic acid complexes
- 1996
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 118:24, s. 5544-5552
-
Journal article (peer-reviewed)abstract
- Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA-DNA duplex and the corresponding DNA-DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson-Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA-DNA, PNA-PNA, and DNA-DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3') and parallel (N/5') PNA-DNA duplexes whereas they stabilize the DNA-DNA duplex. Studies on the effect of monovalent salt such as NaCl on T-m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T-m of the N/3' PNA-DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA-PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3' PNA-DNA compared to the DNA-DNA duplex (Delta Delta G similar to-7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA-DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T-m, were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3' PNA-DNA and PNA-PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.
|
|
