Nucleic Acids Res. 2017 Feb 28;45(4):2099-2111.

Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA-a study of duplex, hairpins and the aptamer for preQ1 as models.

Choi YJ1, Gibala KS1, Ayele T1, Deventer KV1, Resendiz MJE1.

1 Department of Chemistry, University of Colorado Denver, Science Building 1151 Arapahoe St, Denver, CO 80204, USA.

Abstract

A better understanding of the effects that oxidative lesions have on RNA is of importance to understand their role in the development/progression of disease. 8-oxo-7,8-dihydroguanine was incorporated into RNA to understand its structural and functional impact on RNA:RNA and RNA:DNA duplexes, hairpins and pseudoknots. One to three modifications were incorporated into dodecamers of RNA [AAGA GGG AUGAC] resulting in thermal destabilization (Δ T m – 10°C per lesion). Hairpins with tetraloops c-UUCG*-g* ( ), a-ACCG-g* ( ), c-UUG*G*-g* ( ) and c-ACG*G*-g* ( ) were modified and used to determine thermal stabilities, concluding that: (i) modifying the stem leads to destabilization unless adenosine is the opposing basepair of 8-oxoGua; (ii) modification at the loop is position- and sequence-dependent and varies from slight stabilization to large destabilization, in some cases leading to formation of other secondary structures (hairpin→duplex). Functional effects were established using the aptamer for preQ 1 as model. Modification at G5 disrupted the stem P1 and inhibited recognition of the target molecule 7-methylamino-7-deazaguanine (preQ 1 ). Modifying G11 results in increased thermal stability, albeit with a K d 4-fold larger than its canonical analog. These studies show the capability of 8-oxoG to affect structure and function of RNA, resulting in distinct outcomes as a function of number and position of the lesion.

PMID: 28426093

 

Supplement:

RNA damage resulting from oxidative stress is an area of research that has attracted interest in recent years (1).  This phenomenon has been associated with various diseases (2), and certain focus has been placed on those involving neurodegeneration.  It is likely that oxidative lesions affect the interactions between RNA and other cellular molecules, thus unexpectedly affecting biochemical pathways associated with these strands.(3)  Reactions between reactive oxygen species (ROS) and RNA can lead to a variety of oxidative lesions, of which 8-oxo-7,8-dihydropurines [8-oxoguanine (8-oxoGua) and 8-oxoadenine (8-oxoAde)] represent among the most abundant, and studied (4).  These lesions are known to have adverse effects when present in DNA and are therefore potentially toxic if generated within strands of RNA.  Both oxidative lesions have interesting structural features that result in conformational changes that affect the overall function and structure of the modified strand.  Where, the preferred syn-conformation (rotation around the N9-C1’ glycosidic bond), induces a pattern change in their base pairing interactions that result in 8-oxoG:A and 8-oxoA:G basepair mismatches (Figure 1).  This conformational change has been reported as the reason for variances in structure that lead to functional changes in protein binding, basepair mismatching, and translational stalling among other important biological processes.

 

Figure 1.  Basepair mismatches between 8-oxo-7,8-dihydropurines and their canonical pairs.

 

In our group, we are interested in structure-function relationships arising from these oxidatively generated modifications and have developed synthetic methodology to obtain the phosphoramidites (necessary precursors in solid-phase synthesis (5)) of 8-oxoG and 8-oxoA for their incorporation into oligonucleotides of RNA.  This report focuses on establishing structural and functional implications on duplexes, hairpins and pseudoknots of RNA containing 8-oxoG to address the potential implications that this lesion may have if generated within cellular environments.  These structural motifs are ubiquitously found in nature and are therefore important in regards to the effects that the lesions may have on thermal stability, structure, and function.  The methodology relies on the use of circular dichroism, electrophoretic analyses, and isothermal titration calorimetry as tools.

These studies showed that the structural impact is position dependent and may stabilize or destabilize RNA structure as a function of number and position of the lesion, in some cases, leading to unexpected structural transformations (hairpin→duplex).  Thus, potentially posing a threat in the downstream pathway of cellular RNAs.  Dodecamers of RNA were used as models and their sequence was varied to explore the effects on: 1) duplexes containing 1-3 modifications; 2) tetraloop and hexaloop hairpins containing one modification at the stem or loop; and 3) a pseudoknot containing one lesion at key positions.  Circular dichroism was used to determine thermal denaturation transitions (Tm), where, disappearance of a band with negative ellipticity at 210 nm along with a hyperdichroic shift on the band at 270 nm is characteristic of denaturation of an A-form double strand to their corresponding coil structures (Figure 2).  It was shown that following the changes of either band results in the same Tm values, an observation that we had corroborated with other systems at different wavelengths.(6)

 

Figure 2.  Examples illustrating the differences between a duplex and a coil RNA (A), and the disappearance in the dichroic bands of a duplex upon heating.

 

The structural results are highlighted on Figure 3 (select samples were chosen), and displayed destabilization on duplexes containing 8-oxoG, reflected in drops of ca. 10° C per lesion.  A value that is in close agreement with that observed for duplexes containing 8-oxoA.(7)  The results obtained upon positioning the 8-oxoG lesion within hairpins was sequence and position dependent and can be summarized as follows: 1) 8-oxoG within the stem resulted in destabilization by undergoing a transformation to a hexaloop with a shorter stem; 2) 8-oxoG within the loop stabilized or destabilized the hairpin depending on the sequence context, or steered the equilibrium towards the formation of a duplex (given the pseudo-palindromic character when an A is present on the loop).

 

 

Figure 3.  Destabilization of duplex samples is more pronounced as the number of modifications increases (left).  Placement of a modification at the closing basepair of the stem destabilizes the hairpin significantly, while modification at the loop results in destabilization (right, top) or stabilization (right, middle).  Modification at the loop also resulted in formation of the hairpin to their corresponding duplex (right, bottom).

 

In order to assess the functional outcomes arising from the presence of this lesion, we resorted to the use of the aptamer for preQ1 (7-aminomethyl-7-deazaguanine).(8)  This aptamer is known to undergo a hairpin → pseudoknot transformation in the presence of the small molecule.  Positions G5 and G11 were substituted with the corresponding 8-oxoG oxidative modification and their stability and binding capabilities were explored using circular dichroism and isothermal titration calorimetry respectively (Figure 5).  As expected, given its position within the stem, substitution at G5 resulted in a drop in the thermal denaturation transition and loss of recognition for preQ1.  However, substitution at G11 resulted in enhanced stability (higher Tm value than its canonical analogue) along with a 5-fold increase in the KD value, obtained via ITC.

 

 

Figure 4.  Modification of the pseudoknot with 8-oxoG at G5 or G11 resulted in destabilization or stabilization of structure (left).  Recognition of preQ1 upon substitution with 8-oxoG is summarized on the right.

 

Overall, we found that the lesion 8-oxo-7,8-dihydroguanine can have a destabilizing or stabilizing effect on structure of RNA.  This in turn may affect its function, as illustrated with the recognition patterns observed on the aptamer system.  As more discoveries are made in this field, it will be important to have a good understanding of the structural and functional impacts that this and other, oxidatively generated, lesion(s) have on various processes involving RNA.  This will shed light on its potential role on the development/progression of disease and other cellular mechanisms.

 

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