Model systems for understanding DNA base pairing
Introduction
The information that is stored and transferred in a cell is carried in the structure and sequence of nucleic acid bases. These heterocycles must be distinguished from one another by the cellular machinery, and this recognition and discrimination is carried out both by nucleobases themselves, and by proteins as well. For successful cellular function, the genetic information as a base sequence must be recognized accurately during many processes, including DNA replication, transcription, DNA repair, recombination, translation, and RNA interference. All of these processes involve the pairing of one base with another in a selective way. Errors in this pairing can lead to medically serious outcomes such as cancer and drug resistance; but at the same time, such errors also make the process of evolution, and thus life on earth, possible.
A central and basic question is what chemically defines the nucleobases as different from one another, and how does the answer vary in the different biological processes mentioned above. The four bases adenine, guanine, thymine, and cytosine vary from one another in their size, shape, polarity, local electrostatics, and functional groups (Figure 1). In principle, all of these differences can be used in the identification of a base, and yet which of these are most important is often not clear. We know from many solution studies that the bases have some ability to recognize each other in nonpolar solvents by complementary hydrogen bonding, and of course we also know that short molecules of DNA and RNA can self-assemble into double helices in water. However, studies have also shown that simple base–base recognition is not strong or selective enough on its own to account for the selectivities observed in many biological processes, which suggests that proteins act to enhance the specificity and efficiency of nucleobase recognition.
Understanding the mechanisms of accurate genetic pairing matters in both basic and practical ways. Since this transfer of information is central to many diseases, a full understanding of its origin and propagation in living systems will require a better grasp of these recognition processes. In addition, design of therapeutic agents aimed at disrupting or enhancing this recognition also can benefit from such knowledge. Finally, the development of improved tools for postgenomic analysis can result from better understanding of the recognition of genetic sequences as well.
This brief review describes recent work in which non-natural nucleobases are used as tools to study base recognition and pairing mechanisms. It covers only very recent work, mainly carried out in the past two to three years, because multiple reviews have addressed related topics recently. In addition, because of the large increase in work in this field lately, it focuses mainly on studies aimed at basic science, and only briefly notes some designed systems, which have been already reviewed in this journal.
Section snippets
Testing electrostatic effects in pairing
Electrostatic effects can be important in DNA since the structure is highly polar. This is of course tempered by the fact that the solvent is water, which is not only highly polar itself (providing a high dielectric that suppresses electrostatic attractions) but also competes directly for electrostatic interactions such as with hydrogen-bonding groups. One of the main electrostatic interactions to be studied recently is base–base hydrogen bonding, and the question of the role of Watson–Crick
Probes for minor groove effects
Studies in several laboratories have shown that polar interactions in the minor groove of DNA are exceedingly important in pairing stability and structure. All natural DNA bases have minor groove hydrogen-bond acceptors in similar positions, and these are typically well solvated. McLaughlin has developed a number of base analogs lacking minor groove hydrogen-bond acceptors, which have shown a strong destabilizing effect on DNA [19]. Minor groove carbonyl groups of thymine have also been
Testing shape and size effects
The canonical pairs adopt very similar geometries, occupying space that overlaps to a large extent (Figure 2). Since both correctly and incorrectly matched base pairs in DNA are hydrogen-bonded, it is clear that the nonstandard geometry of mismatches plays a substantial role in pairing selectivity. An important question is how much do steric effects alone (in the absence of hydrogen bonds) affect pairing selectivity in the double helix? And secondly, it is clear that pairing selectivity in DNA
Probes for recognition of damaged bases
DNA analogs have also been recently implemented as probes of damage in DNA. Sturla has demonstrated selective and stable base pairing between the biologically relevant damage adduct O6-benzyldeoxyguanosine and a designed diaminonaphthyl-derived nucleoside [34]. In addition, two laboratories have constructed analogs of oxidized purines as probes for how such damage is recognized by repair and polymerase enzymes. Hamm et al. used 8-haloguanines to measure the steric effect of the 8-substituent on
Structural studies
Structural studies of nucleobase analogs in DNA or RNA yield the benefit of helping to understand how the helix adjusts to changes in structure or interactions. Manoharan, Frank-Kamenetskii, and Egli (as part of their siRNA studies) published the X-ray crystal structure of an RNA–RNA duplex containing a non-H-bonded pair between adenine and difluorotoluene. The majority of the RNA and local base pair structure was unaffected by this pair, although small local adjustments to pair geometry were
Alternative principles for base pairing
Some of the insights gained from studies of pairing have led to the development and testing of other, non-natural principles for design of new base pairs. For example, Sekine recently tested the interesting idea of using ‘halogen bonds’ to help pairs associate [41]. Saito examined the use of Schiff base linkage to connect opposing bases [42]. Purine–purine pairs were examined by Battersby et al. [43]. A number of laboratories have recently described metal-bridged base pairs; in this light,
Conclusions
There are multiple points in the transmission of cellular genetic information where accurate base pairing is crucial, and the growing complement of DNA base analogs developed by researchers (Figure 4) will be essential in evaluating how this recognition occurs. It is no doubt the case that both steric and electrostatic effects have important influences on base pairing, but the relative importance of these factors will vary depending on the context, whether it is DNA or RNA. In addition, it is
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
This work was supported by the U.S. National Institutes of Health (GM072705 and GM63587).
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