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The importance of loop length on the stability of i-motif structures.

Gurung SP, Schwarz C, Hall JP, Cardin CJ, Brazier JA - Chem. Commun. (Camb.) (2015)

Bottom Line: Using UV and srCD spectroscopy it is found that loop length within the i-motif structure is important for both thermal and pH stability, but in contrast to previous statements, it is the shorter loops that exhibit the highest stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Reading, Whiteknights, Reading, Berks RG6 6AD, UK.

ABSTRACT
Using UV and srCD spectroscopy it is found that loop length within the i-motif structure is important for both thermal and pH stability, but in contrast to previous statements, it is the shorter loops that exhibit the highest stability.

Show MeSH
(a) Protonated-cytosine: cytosine base pair. (b) Schematic representation of an i-motif structure, where N denotes any DNA heterocyclic base.
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fig1: (a) Protonated-cytosine: cytosine base pair. (b) Schematic representation of an i-motif structure, where N denotes any DNA heterocyclic base.

Mentions: The i-motif is a quadruplex structure that is formed by cytosine-rich sequences of DNA (Fig. 1a) and has been found to form in several sequences complementary to G-quadruplexes.1–3 The role of the i-motif in modulating gene expression has recently been elucidated, with the contribution of the loop regions within the structure key to the recognition and binding of transcriptional proteins.4,5 The i-motif is not only of interest for its biological role in gene expression, especially due to its presence in the promoter regions of several genes associated with cancer, but also for its use as a switch within functional nanodevices.6 The majority of studies using an i-motif based nanoswitch have used the human telomeric sequence (CCCTAA)3CCC or a subtle variant. It is known from research on biologically derived sequences, that i-motifs formed by different sequences exhibit different thermal and pH stability.3 Recent research focussed on the design of a pH sensor based on i-motif structures has shown that changes in the sequence composition can change the pH sensitivity in a controlled way.7 It has been reported in the literature that i-motif forming sequences containing longer loops (N = 5–8, Fig. 1b) are thermally more stable than those with short loops, with the length of the loop often suggested to be the contributing factor for this stability.8 This is in contrast to research carried out on the loop length of G-quadruplex forming sequences, which showed that longer loops were less stable.9 Recently we published results that showed unusually high stability for a sequence containing short loops (N = 1–4, Fig. 1b) which questioned the need for long loops to impart stability.3 To probe this question further, we report here a systematic study of loop length versus both, thermal and pH stability. A series of i-motif forming sequences (Table 1), containing an increasing number of thymine bases within the loop region, with the general sequence CCCT(3-8)CCCT(3-8)CCCT(3-8)CCC, were assessed for their thermal (Fig. 2a) and pH stability (Fig. 2b) using both UV and synchrotron CD spectroscopy. By confining the loop bases to thymine, the possibility of interaction between the bases was limited to thymine: thymine and were consistent throughout the systematic series of oligonucleotides. The results of the thermal stability experiments show that C3T333 has the highest stability and C3T888 has the lowest stability. The other sequences in the series follow a predominately linear trend with respect to thermal stability with a decreasing order of C3T555, C3T666 and C3T777. The only exception is C3T444 which has the same thermal stability as C3T333. The results clearly show that the stability of the i-motif structure decreases as the length of the loop increases, a finding which is in contrast with statements made in the literature. One rationale for this observation, is as the loop length increases, the structure gains more flexibility, and therefore stabilising interactions become weaker. This decrease in stability is also present in the results from the pH titration experiments, where the transitional pH (TpH, the pH at which half of the i-motif is unfolded) decreases as loop length increases. Once again C3T333 shows the highest TpH, with C3T888 demonstrating the lowest value. Other i-motif forming sequences show a systematic decrease between these two values, with an order of C3T444, C3T555, C3T666 and C3T777. The pH sensitivity was also confirmed by srCD,‡


The importance of loop length on the stability of i-motif structures.

Gurung SP, Schwarz C, Hall JP, Cardin CJ, Brazier JA - Chem. Commun. (Camb.) (2015)

(a) Protonated-cytosine: cytosine base pair. (b) Schematic representation of an i-motif structure, where N denotes any DNA heterocyclic base.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384421&req=5

fig1: (a) Protonated-cytosine: cytosine base pair. (b) Schematic representation of an i-motif structure, where N denotes any DNA heterocyclic base.
Mentions: The i-motif is a quadruplex structure that is formed by cytosine-rich sequences of DNA (Fig. 1a) and has been found to form in several sequences complementary to G-quadruplexes.1–3 The role of the i-motif in modulating gene expression has recently been elucidated, with the contribution of the loop regions within the structure key to the recognition and binding of transcriptional proteins.4,5 The i-motif is not only of interest for its biological role in gene expression, especially due to its presence in the promoter regions of several genes associated with cancer, but also for its use as a switch within functional nanodevices.6 The majority of studies using an i-motif based nanoswitch have used the human telomeric sequence (CCCTAA)3CCC or a subtle variant. It is known from research on biologically derived sequences, that i-motifs formed by different sequences exhibit different thermal and pH stability.3 Recent research focussed on the design of a pH sensor based on i-motif structures has shown that changes in the sequence composition can change the pH sensitivity in a controlled way.7 It has been reported in the literature that i-motif forming sequences containing longer loops (N = 5–8, Fig. 1b) are thermally more stable than those with short loops, with the length of the loop often suggested to be the contributing factor for this stability.8 This is in contrast to research carried out on the loop length of G-quadruplex forming sequences, which showed that longer loops were less stable.9 Recently we published results that showed unusually high stability for a sequence containing short loops (N = 1–4, Fig. 1b) which questioned the need for long loops to impart stability.3 To probe this question further, we report here a systematic study of loop length versus both, thermal and pH stability. A series of i-motif forming sequences (Table 1), containing an increasing number of thymine bases within the loop region, with the general sequence CCCT(3-8)CCCT(3-8)CCCT(3-8)CCC, were assessed for their thermal (Fig. 2a) and pH stability (Fig. 2b) using both UV and synchrotron CD spectroscopy. By confining the loop bases to thymine, the possibility of interaction between the bases was limited to thymine: thymine and were consistent throughout the systematic series of oligonucleotides. The results of the thermal stability experiments show that C3T333 has the highest stability and C3T888 has the lowest stability. The other sequences in the series follow a predominately linear trend with respect to thermal stability with a decreasing order of C3T555, C3T666 and C3T777. The only exception is C3T444 which has the same thermal stability as C3T333. The results clearly show that the stability of the i-motif structure decreases as the length of the loop increases, a finding which is in contrast with statements made in the literature. One rationale for this observation, is as the loop length increases, the structure gains more flexibility, and therefore stabilising interactions become weaker. This decrease in stability is also present in the results from the pH titration experiments, where the transitional pH (TpH, the pH at which half of the i-motif is unfolded) decreases as loop length increases. Once again C3T333 shows the highest TpH, with C3T888 demonstrating the lowest value. Other i-motif forming sequences show a systematic decrease between these two values, with an order of C3T444, C3T555, C3T666 and C3T777. The pH sensitivity was also confirmed by srCD,‡

Bottom Line: Using UV and srCD spectroscopy it is found that loop length within the i-motif structure is important for both thermal and pH stability, but in contrast to previous statements, it is the shorter loops that exhibit the highest stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Reading, Whiteknights, Reading, Berks RG6 6AD, UK.

ABSTRACT
Using UV and srCD spectroscopy it is found that loop length within the i-motif structure is important for both thermal and pH stability, but in contrast to previous statements, it is the shorter loops that exhibit the highest stability.

Show MeSH