Biomedical subjects

Elzbieta Kierzek

Publications and source records attributed to Elzbieta Kierzek.

11 recordsLinked to original sources

PubMed2006

Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides.

Results from optical melting studies of Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson-Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.

Base Pairing↗

PubMed2006

Exploring the energy landscape of a small RNA hairpin.

The energy landscape of a small RNA tetraloop hairpin is explored by temperature jump kinetics and base-substitution. The folding kinetics are single-exponential near the folding transition midpoint T(m). An additional fast phase appears below the midpoint, and an additional slow phase appears above the midpoint. Stem mutation affects the high-temperature phase, while loop mutation affects the low-temperature phase. An adjusted 2-D lattice model reproduces the temperature-dependent phases, although it oversimplifies the structural interpretation. A four-state free energy landscape model is generated based on the lattice model. This model explains the thermodynamics and multiphase kinetics over the full temperature range of the experiments. An analysis of three variants shows that one of the intermediate RNA structures is a stacking-related trap affected by stem but not loop modification, while the other is an early intermediate that forms some stem and loop structure. Even a very fast-folding 8-mer RNA with an ideal tetraloop sequence has a rugged energy landscape, ideal for testing analytical and computational models.

Kinetics↗

PubMed2006

Facilitating RNA structure prediction with microarrays.

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

Base Pairing↗

PubMed2005

The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2'-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3' terminal U LNA and 5' terminal LNAs are less stabilizing than interior and other 3' terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2'-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2'-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2'-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2'-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37 degrees C are presented for 2'-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.

Base Pair Mismatch↗

PubMed2005

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure.

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.

Models, Molecular↗

PubMed2004

Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate.

Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.

Base Pairing↗

PubMed2003

The synthesis of oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modification of precursor oligomers.

The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm and the contribution of these hypermodified nucleosides to codon-anticodon interactions, as well as translation, are significant, although not fully understood. Herein we described a new chemical synthesis method of the oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N6-alkyladenosines, the precursor oligoribonucleotide carrying 6-methylthiopurine riboside residue was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N6-alkyladenosines the precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. Among the modified oligoribonucleotides of different length and secondary structures, there were several containing naturally occurring modified nucleosides such as: N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), and 2-methylthio-N6-methyladenosine (ms2m6A), as well as several unnaturally modified adenosine derivatives.

Adenosine↗

PubMed2003

The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines.

The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N6-alkyladenosines and 2-methylthio-N6-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A(Mod) base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3'-terminal unpaired nucleotide. Beside naturally modified adenosines such as N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A) and 2-methylthio-N6-methyladenosine (ms2m6A), we studied several artificial modifications to evaluate the steric and electronic effects of N6-alkyl substituents. Moreover, some N6-alkyladenosines and 2-methylthio-N6-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A(Mod) base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.

Adenosine↗

PubMed2003

Restricting the conformational heterogeneity of RNA by specific incorporation of 8-bromoguanosine.

In an effort to reduce the conformational heterogeneity of RNA, the modified nucleobase 8-bromoguanosine (8BrG) was introduced into oligonucleotides having the hairpin tetraloop motif YNMG (Y = U or C and M = C or A). Purine nucleobases with bromine at position eight are known to preferentially adopt the syn conformation as nucleosides. The hairpin tetraloop motif YNMG was chosen as a model system because it has a syn guanosine at position four of the loop that is essential for thermodynamic stability. Thermodynamic and structural characterization of modified oligonucleotides with the hairpin sequences UUCG, CGCG, and CGAG by UV-melting and NMR spectroscopy revealed that 8BrG substitution has a small effect upon the hairpin conformation, while the duplex conformation is strongly destabilized (DeltaDeltaG degrees 37 approximately +4.7 kcal mol-1), thus inhibiting dimerization. These results support a model in which 8BrG substitution shifts the hairpin-duplex equilibrium constant toward the hairpin conformation by destabilizing the duplex. This methodology should be useful for limiting conformational heterogeneity in large RNAs, with potential applications in structural biology and enzymology.

Guanosine↗

PubMed2002

The influence of various modified nucleotides placed as 3'-dangling end on thermal stability of RNA duplexes.

The ribonucleic acids (RNA) form highly folded structures, which behind the helical fragments contain several secondary and tertiary structural motives. All of them have an influence on thermodynamic stability of the RNA. The 5'- and 3'-dangling ends are one of those structural motives, which effect stability of the adjacent helixes. In this paper, we described the influence of 14 different modified nucleotides, placed as 3'-dangling ends, on thermal stability of the RNA duplexes. Collected data demonstrate that: (i) 5-substituents of the uridine have an impact on the 3'-dangling end effect and the largest changes were observed for 5-chloro, bromo and methyl substituents; (ii) position of the methyl group within the uracil residue affect the thermal stability of the duplex; (iii) increasing a size of the heterocycle base placed as the 3'-terminal unpaired nucleotide enhances stabilization of duplexes.

DNA↗

PubMed2002

The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides.

The presence of various modifications within oligomers changes their thermodynamic stability. To get more systematic data, we measured effects of 5- and 6-substituted uridine on thermal stability of (AUCU(Mod.)AGAU)2 and (AUCUAGAU(Mod.))2. Collected results lead to the following conclusions: (i) 5-halogenated and 5-alkylated substituents of the uridine affect thermal stability of the RNA duplexes differently. Moreover, the 5-fluorouridine changes stability of the RNA duplexes opposite to remaining 5-halogenouridines; (ii) for oligomers containing 5-chloro, 5-bromo or 5-iodouridine stronger hydrogen bond formed between oxygen-4 of the 5-halogenated uracil and 6-amino group of the adenine is presumably responsible for stabilizing effect; (iii) placing of A-U(5R) base pairs closer to the end of the duplex enhance thermal stability relatively to oligomer with central position of this base pair; (iv) the effects of 5-substituents are additive, particularly for substituents which stabilize RNA duplexes; (v) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine present at internal position of A-U(Mod.) inhibit duplexes formation; (vi) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine placed as terminal base pairs stabilize the duplexes mostly via 3'-dangling end effect.

Hot Temperature↗