Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
1.
RNA ; 23(2): 217-228, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27872162

RESUMO

Fifty-three RNA duplexes containing two single nucleotide bulge loops were optically melted in 1 M NaCl in order to determine the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each duplex. Because of the large number of possible combinations and lack of sequence effects observed previously, we limited our initial investigation to adenosine bulges, the most common naturally occurring bulge. For example, the following duplexes were investigated: 5'GGCAXYAGGC/3'CCG YX CCG, 5'GGCAXY GCC/3'CCG YXACGG, and 5'GGC XYAGCC/3'CCGAYX CGG. The identity of XY (where XY are Watson-Crick base pairs) and the total number of base pairs in the terminal and central stems were varied. As observed for duplexes with a single bulge loop, the effect of the two bulge loops on duplex stability is primarily influenced by non-nearest neighbor interactions. In particular, the stability of the stems influences the destabilization of the duplex by the inserted bulge loops. The model proposed to predict the influence of multiple bulge loops on duplex stability suggests that the destabilization of each bulge is related to the stability of the adjacent stems. A database of RNA secondary structures was examined to determine the naturally occurring abundance of duplexes containing multiple bulge loops. Of the 2000 examples found in the database, over 65% of the two bulge loops occur within 3 base pairs of each other. A database of RNA three-dimensional structures was examined to determine the structure of duplexes containing two single nucleotide bulge loops. The structures of the bulge loops are described.


Assuntos
Sequências Repetidas Invertidas , RNA de Cadeia Dupla/química , RNA Ribossômico 23S/química , Pareamento de Bases , Bases de Dados de Ácidos Nucleicos , Modelos Moleculares , Conformação de Ácido Nucleico , Estabilidade de RNA , Termodinâmica
2.
RNA ; 21(5): 975-84, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25805856

RESUMO

Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5' or 3' position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5' or 3' side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.


Assuntos
2-Aminopurina/química , Corantes Fluorescentes/química , Sequências Repetidas Invertidas/genética , Conformação de Ácido Nucleico , RNA/química , Pareamento de Bases , Sequência de Bases , Estabilidade de RNA , Termodinâmica
3.
RNA ; 20(6): 825-34, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24742935

RESUMO

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson-Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson-Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson-Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3' or 5' of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.


Assuntos
Nucleotídeos/química , Nucleotídeos/genética , Estabilidade de RNA/genética , RNA/química , RNA/genética , Pareamento de Bases/genética , Sequências Repetidas Invertidas/genética , Modelos Teóricos , Conformação de Ácido Nucleico , Termodinâmica
4.
Biochemistry ; 52(5): 765-72, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23356429

RESUMO

The rates of duplex formation for two octamers of DNA (5' d-CACGGCTC/5' d-GAGCCGTG and 5' d-CACAGCAC/5' d-GTGCTGTG), the homologous RNA, and both sets of hybrids in 1 M NaCl buffer have been measured using stopped-flow spectroscopy. In addition, the thermodynamic parameters, ΔH° and ΔS°, have been determined for the same sequences under the same buffer conditions using optical melting techniques. These data reveal a linear free energy relationship between the free energy of activation for denaturation and the change in free energy for formation of the duplexes. This relationship indicates that these duplex formation reactions occur through a common unstructured transition state that is more similar to the single strands in solution than to the ensuing duplex. In addition, these data confirm that the greater stability of RNA duplexes relative to that of homologous DNA and hybrid duplexes is controlled by the denaturation rate and not the duplex formation rate.


Assuntos
DNA/química , RNA/química , Sequência de Bases , Cinética , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Estabilidade de RNA , Termodinâmica
5.
Biochemistry ; 51(16): 3508-22, 2012 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-22490167

RESUMO

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5'GG/3'UU motif were added to the single representation in the previous database. This revises the ΔG°(37) for the 5'GG/3'UU motif from an unfavorable 0.5 kcal/mol to a favorable -0.2 kcal/mol. Similarly, the ΔG°(37) for the 5'UG/3'GU motif changes from 0.3 to -0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°(37), ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.


Assuntos
Pareamento de Bases , RNA/química , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Termodinâmica
6.
RNA ; 18(4): 807-14, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22345128

RESUMO

Forty-six RNA hairpins containing combinations of 3' or 5' bulge loops and a 3' or 5' fluorescein label were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each hairpin were determined. The bulge loops were of the group I variety, in which the identity of the bulge is known, and the group II variety, in which the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The fluorescein label at either the 3' end or 5' end of the hairpin did not significantly influence the stability of the hairpin. As observed with bulge loops inserted into a duplex motif, the insertion of a bulge loop into the stem of a hairpin loop was destabilizing. The model developed to predict the influence of bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability. Specifically, the influence of the bulge is related to the stability of the hairpin stem distal from the hairpin loop.


Assuntos
Nucleotídeos/química , RNA/química , Termodinâmica
7.
RNA ; 17(1): 108-19, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21088109

RESUMO

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.


Assuntos
Estabilidade de RNA , RNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/genética , Termodinâmica
8.
Nucleic Acids Res ; 37(1): 70-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19015151

RESUMO

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2'-fluoro/2'-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.


Assuntos
MicroRNAs/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Regulação da Expressão Gênica , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/análise , MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/química , Ácidos Nucleicos Peptídicos/química
9.
Nucleic Acids Res ; 36(17): 5652-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18765476

RESUMO

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3' single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3' dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3' single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3' double-nucleotide overhang. Thermodynamic parameters for the 5' single terminal overhangs adjacent to wobble closing base pairs are also presented.


Assuntos
RNA de Cadeia Dupla/química , Termodinâmica , Pareamento de Bases , MicroRNAs/química , MicroRNAs/classificação , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Nucleotídeos/química , Filogenia , RNA Interferente Pequeno/química
10.
Biochemistry ; 46(51): 15123-35, 2007 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-18047298

RESUMO

Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.


Assuntos
Nucleotídeos/química , Estabilidade de RNA , RNA/química , RNA/genética , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleotídeos/genética , Termodinâmica
11.
Nucleic Acids Res ; 34(11): 3338-44, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16820533

RESUMO

Thermodynamic parameters are reported for duplex formation of 48 self-complementary RNA duplexes containing Watson-Crick terminal base pairs (GC, AU and UA) with all 16 possible 3' double-nucleotide overhangs; mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest-neighbor analysis, the addition of a second dangling nucleotide to a single 3' dangling nucleotide increases stability of duplex formation up to 0.8 kcal/mol in a sequence dependent manner. Results from this study in conjunction with data from a previous study [A. S. O'Toole, S. Miller and M. J. Serra (2005) RNA, 11, 512.] allows for the development of a refined nearest-neighbor model to predict the influence of 3' double-nucleotide overhangs on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against five oligomers with various core duplex sequences. Phylogenetic analysis of naturally occurring miRNAs was performed to support our results. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent upon the identity of the 3' double-nucleotide overhang. Thermodynamic parameters for 3' single terminal overhangs adjacent to a UA pair are also presented.


Assuntos
MicroRNAs/química , RNA de Cadeia Dupla/química , Termodinâmica , Adenina/química , Pareamento de Bases , MicroRNAs/classificação , Filogenia , RNA Interferente Pequeno/química , Ribonucleotídeos/química , Uracila/química
12.
Biochemistry ; 45(5): 1400-7, 2006 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-16445282

RESUMO

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the types GCGXUAAUYCGC and GGUXUAAUYACC with Watson-Crick loop closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.1 to 5.1 kcal/mol. These results agree with the model previously developed [Vecenie, C. J., and Serra, M. J. (2004) Biochemistry 43, 11813] to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on the 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Thermodynamic parameters are also reported for hairpin formation in 1 M NaCl by RNA sequence of the types GACGXUAAUYUGUC and GGUXUAAUYGCC with GU base pair closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.6 to 5.3 kcal/mol. These results allow the development of a model for predicting the stability of hairpin loops closed by GU base pairs. DeltaG degrees (37L(n) (kcal/mol) = DeltaG degrees (37i(n) - 0.8 (if the first mismatch is GA) - 0.8 (if the first mismatch is GG and the loop is closed on the 5' side by a purine). Note that for these hairpins, the stability of the loops does not depend on DeltaG degrees (37MM). For hairpin loops closed by GU base pairs, the DeltaG degrees (37i(n) values, when n = 4, 5, 6, 7, and 8, are 4.9, 5.0, 4.6, 5.0, and 4.8 kcal/mol, respectively. The model gives good agreement when tested against six naturally occurring hairpin sequences. Thermodynamic values for terminal mismatches adjacent to GC, GU, and UG base pairs are also reported.


Assuntos
Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Estabilidade de RNA , RNA/química , Composição de Bases , Pareamento de Bases , Sequência de Bases , Modelos Químicos , Modelos Moleculares , Desnaturação de Ácido Nucleico , RNA/síntese química , RNA/isolamento & purificação , Cloreto de Sódio/química , Termodinâmica
13.
RNA ; 11(4): 512-6, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15769878

RESUMO

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.


Assuntos
Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , RNA Interferente Pequeno/química , Modelos Moleculares , Oligorribonucleotídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Termodinâmica , Temperatura de Transição
14.
Biochemistry ; 43(37): 11813-7, 2004 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-15362866

RESUMO

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the type GCAXUAAUYUGC, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates that the free energy of loop formation at 37 degrees C varies from 3.2 to 5.0 kcal/mol. These results combined with the model previously developed [Dale et al. (2000) RNA 6, 608] allow improvements in the model to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n)) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Hairpins with GG first mismatches were found to vary in stability depending upon the orientation of the closing base pair (5' or 3' purine relative to the loop). The model gives good agreement when tested against four naturally occurring hairpin sequences.


Assuntos
Adenina/metabolismo , Pareamento de Bases , Conformação de Ácido Nucleico , RNA/química , Uracila/metabolismo , Sequência de Bases , Desnaturação de Ácido Nucleico , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , RNA/genética , Estabilidade de RNA , Termodinâmica
15.
Nucleic Acids Res ; 32(5): 1824-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15037659

RESUMO

Optical melting was used to determine the stabilities of three series of RNA oligomers containing tandem XU base pairs, GGCXUGCC (5'XU3'), GGCUXGCC (5'UX3') and GGCXXGGC/CCGUUCCG (5'XX3'), where X is either A, G or I (inosine). The helices containing tandem AU base pairs were the most stable in the first two series (5'XU3' and 5'UX3'), with an average melting temperature approximately 11 degrees C higher than the helices with tandem 5'GU3' base pairs and 25 degrees C higher than the helices with tandem 5'IU3' base pairs. For the third series (5'XX3'), the helix containing tandem GG is the most stable, with an average melting temperature approximately 2 degrees C higher than the helix with tandem AA base pairs and approximately 24 degrees C higher than the helix with tandem II base pairs. The thermodynamic stability of the oligomers with tandem IU base pairs was also investigated as a function of magnesium ion concentration. As with normal A-U or G-U tandem duplexes, the data could best be interpreted as non-specific binding of magnesium ions to the inosine-containing RNA oligonucleotides.


Assuntos
Inosina/química , RNA/química , Uracila/química , Pareamento de Bases , Magnésio/química , Conformação de Ácido Nucleico , Estabilidade de RNA , Termodinâmica
16.
Biochemistry ; 41(33): 10406-17, 2002 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-12173927

RESUMO

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.


Assuntos
Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Nucleotídeos/química , RNA Ribossômico/química , Termodinâmica , Adenosina/química , Pareamento de Bases , Haloarcula marismortui , Modelos Químicos , Nucleotídeos de Purina/química , Nucleotídeos de Pirimidina/química , Thermus thermophilus
17.
RNA ; 8(3): 307-23, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12003491

RESUMO

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.


Assuntos
Magnésio/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA/metabolismo , Pareamento de Bases , Temperatura Alta , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Termodinâmica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA