Strength Of Base Pairing Research Articles

Influence of 5-Halogenation on the Base-Pairing Energies of Protonated Cytidine Nucleoside Analogue Base Pairs: Implications for the Stabilities of Synthetic i-Motif Structures for DNA Nanotechnology Applications.

DNA nanotechnology has been employed to develop devices based on i-motif structures. The protonated cytosine-cytosine base pairs that stabilize i-motif conformations are favored under slightly acidic conditions. This unique property has enabled development of the first DNA molecular motor driven by pH changes. The ability to alter the stability and pH transition range of such DNA molecular motors is desirable. Understanding how i-motif structures are influenced by modifications, and which modifications enhance stability and/or affect the pH characteristics, are therefore of great interest. Here, the influence of 5-halogenation of the cytosine nucleobases on the base pairing of protonated cytidine nucleoside analogue base pairs is examined using complementary threshold collision-induced dissociation techniques and computational methods. The nucleoside analogues examined here include the 5-halogenated forms of the canonical DNA and RNA cytidine nucleosides. Comparisons among these systems and to the analogous canonical base pairs previously examined enable the influence of 5-halogenation and the 2'-hydroxy substituent on the base pairing to be elucidated. 5-Halogenation of the cytosine nucleobases is found to enhance the strength of base pairing of DNA base pairs and generally weakens the base pairing for RNA base pairs. Trends in the strength of base pairing indicate that both inductive and polarizability effects influence the strength of base pairing. Overall, the present results suggest that 5-halogenation, and in particular, 5-fluorination and 5-iodination, provide effective means of stabilizing DNA i-motif conformations for applications in nanotechnology, whereas only 5-iodination is effective for stabilizing RNA i-motif conformations but the enhancement in stability is less significant.

Acid‐Stable Nucleobase Protection for a Strongly Pairing Pyridone C‐Nucleoside Suitable for Solid‐Phase Synthesis of Oligonucleotides

AbstractOligonucleotides are indispensable tools in diagnostics, therapeutic applications and molecular biology. The low base pairing strength of thymine with adenine complicates their use. EthynylpyridoneC‐nucleosides are analogs of thymidine that pair more strongly and with improved base selectivity, and sequences containing these analogs show improved target affinity and selectivity, but their routine use is hampered by diminished yields of solid‐phase syntheses with the known building blocks. A partial loss of base protecting groups during the acidic deblocking step of chain extension cycles was identified as the cause of lower yields. Here we report the synthesis of an improved phosphoramidite building block featuring a pivaloyloxymethyl (POM) base protecting group. This building block gives oligonucleotides containing the strongly pairing ethynylmethylpyridoneC‐nucleoside in high yield and purity via solid‐phase synthesis.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson–Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.

Bias at the third nucleotide of codon pairs in virus and host genomes

Genomes of different sizes and complexity can be compared using common features. Most genomes contain open reading frames, and most genomes use the same genetic code. Redundancy in the genetic code means that different biases in the third nucleotide position of a codon exist in different genomes. However, the nucleotide composition of viruses can be quite different from host nucleotide composition making it difficult to assess the relevance of these biases. Here we show that grouping codons of a codon-pair according to the GC content of the first two nucleotide positions of each codon reveals patterns in nucleotide usage at the third position of the 1st codon. Differences between the observed and expected biases occur predominantly when the first two nucleotides of the 2nd codon are both S (strong, G or C) or both W (weak, A or T), not a mixture of strong and weak. The data indicates that some codon pairs are preferred because of the strength of the interactions between the codon and anticodon, the adjacent tRNAs and the ribosome. Using base-pairing strength and third position bias facilitates the comparison of genomes of different size and nucleotide composition and reveals patterns not previously described.

Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide—substrate RNA pairs.

A Search for Structural Alternatives of RNA

This account describes a search for potentially primordial informational oligomers; the work is the direct outcome of the research program that was initiated by the Eschenmoser group —at ETH Zürich about 20 years ago and was continued at The Scripps Research Institute since 1996— in order to understand the chemical basis on which nucleic acids were chosen by nature as the molecular foundation of its genetic apparatus. The investigation began with the study of the base-pairing properties of structural alternatives of nucleic acids —constructed from different sugars (hexo- & pentopyranoses and tetrofuranose) retaining the canonical nucleobases and phosphates. The outcome from these studies led to the conclusion that Watson-Crick type base-pairing is not unique to RNA/DNA, and that it can be compatible with a wide variety of backbone edifice. This provided the motivation to map the landscape of potentially primordial informational oligomer systems that may contain backbones, recognition elements and linker groups structurally quite different from those known so far. The oligomer systems chosen for study are, conceptually, deemed to be (a) potentially primordial (based on the nature of the starting materials and reaction conditions considered to be prebiotically realistic) and (b) informational (based on their ability to adopt a repetitive conformation such that the information encoded by the recognition elements can be transmitted intermolecularly). Though such studies suggest the possibility of finding informational systems that could lay claim as functional ancestors of RNA —they are more likely to generate results that provide the opportunity to assess the structural and functional uniqueness of nature’s choice. The experimental investigation described here deals with the base-pairing properties of oligomer systems derived from 2,4-disubsituted -triazines, -5-aminopyrimdines and -6-carboxy pyrimidines as recognition elements that are tagged to oligo-dipeptide backbones via different linker groups. The results from the inter- and intra-system cross-pairing studies reveal that there is, on first approximation, a direct correlation between the magnitude of the difference in DpKa of the recognition elements and their base-pairing strength —smaller the Dpka between the base-pairing partners, weaker is the base-pairing strength (in aqueous medium at near neutral pH). These results exemplify the inherent singularity of the canonical nucleobases— their ability to remain un-ionized under physiological conditions based on their constitution – emphasizing the relationship between their physicochemical properties and their functional competence in connection with their role in informational base-pairing.

A strongly pairing fifth base: oligonucleotides with a C-nucleoside replacing thymidine

There are five canonical bases in DNA and RNA. Each base has its particular molecular recognition properties and base pairing strength. Thymine and uracil form only two hydrogen bonds when pairing with adenine, and duplexes rich in A:T base pairs are more labile than duplexes rich in C and G, making some sequences difficult to detect via hybridization in a genomic context. Here we report the synthesis of an ethynylmethylpyridone C-nucleoside, abbreviated ‘W’, that presents a similar recognition surface as thymidine in the major groove but pairs with A about as strongly as C pairs with G. A phosphoramidite building block was synthesized that allows for incorporation of W residues via automated synthesis in high yield. Melting point increases over duplexes containing T:A pairs of up to 17.5°C, or up to 5.8°C per residue were measured for oligonucleotides containing W. Further, the new base shows excellent fidelity, with a single mismatched G opposite W causing a melting point depression of up to 20.5°C. The strongly pairing replacement for thymidine is only slightly larger than its natural counterpart and performs well in different sequence contexts. It can be used to target weakly pairing A-rich sequences in biological studies.

Molecular Mechanism for Folding Cooperativity of Functional RNAs in Living Organisms.

A diverse set of organisms has adapted to live under extreme conditions. The molecular origin of the stability is unclear, however. It is not known whether the adaptation of functional RNAs, which have intricate tertiary structures, arises from strengthening of tertiary or secondary structure. Herein we evaluate effects of sequence changes on the thermostability of tRNAphe using experimental and computational approaches. To separate out effects of secondary and tertiary structure on thermostability, we modify base pairing strength in the acceptor stem, which does not participate in tertiary structure. In dilute solution conditions, strengthening secondary structure leads to non-two-state thermal denaturation curves and has small effects on thermostability, or the temperature at which tertiary structure and function are lost. In contrast, under cellular conditions with crowding and Mg2+-chelated amino acids, where two-state cooperative unfolding is maintained, strengthening secondary structure enhances thermostability. Investigation of stabilities of each tRNA stem across 44 organisms with a range of optimal growing temperatures revealed that organisms that grow in warmer environments have more stable stems. We also used Shannon entropies to identify positions of higher and lower information content, or sequence conservation, in tRNAphe and found that secondary structures have modest information content allowing them to drive thermal adaptation, while tertiary structures have maximal information content hindering them from participating in thermal adaptation. Base-paired regions with no tertiary structure and modest information content thus offer a facile evolutionary route to enhancing the thermostability of functional RNA by the simple molecular rules of base pairing.

The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects.

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules.

Analysis of stacking overlap in nucleic acid structures: algorithm and application.

RNA contains different secondary structural motifs like pseudo-helices, hairpin loops, internal loops, etc. in addition to anti-parallel double helices and random coils. The secondary structures are mainly stabilized by base-pairing and stacking interactions between the planar aromatic bases. The hydrogen bonding strength and geometries of base pairs are characterized by six intra-base pair parameters. Similarly, stacking can be represented by six local doublet parameters. These dinucleotide step parameters can describe the quality of stacking between Watson-Crick base pairs very effectively. However, it is quite difficult to understand the stacking pattern for dinucleotides consisting of non canonical base pairs from these parameters. Stacking interaction is a manifestation of the interaction between two aromatic bases or base pairs and thus can be estimated best by the overlap area between the planar aromatic moieties. We have calculated base pair overlap between two consecutive base pairs as the buried van der Waals surface between them. In general, overlap values show normal distribution for the Watson-Crick base pairs in most double helices within a range from 45 to 50Å(2) irrespective of base sequence. The dinucleotide steps with non-canonical base pairs also are seen to have high overlap value, although their twist and few other parameters are rather unusual. We have analyzed hairpin loops of different length, bulges within double helical structures and pseudo-continuous helices using our algorithm. The overlap area analyses indicate good stacking between few looped out bases especially in GNRA tetraloop, which was difficult to quantitatively characterise from analysis of the base pair or dinucleotide step parameters. This parameter is also seen to be capable to distinguish pseudo-continuous helices from kinked helix junctions.

Allele-Selective Inhibition of Expression of Huntingtin and Ataxin-3 by RNA Duplexes Containing Unlocked Nucleic Acid Substitutions

Unlocked nucleic acid (UNA) is an acyclic analogue of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base pairing, creating opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of expression of trinucleotide repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3). We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent, and selectivities of >40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action.

PKa Shifting in Double-Stranded RNA Is Highly Dependent upon Nearest Neighbors and Bulge Positioning

Shifting of pKa's in RNA is important for many biological processes; however, the driving forces responsible for shifting are not well understood. Herein, we determine how structural environments surrounding protonated bases affect pKa shifting in double-stranded RNA (dsRNA). Using (31)P NMR, we determined the pKa of the adenine in an A(+)·C base pair in various sequence and structural environments. We found a significant dependence of pKa on the base pairing strength of nearest neighbors and the location of a nearby bulge. Increasing nearest neighbor base pairing strength shifted the pKa of the adenine in an A(+)·C base pair higher by an additional 1.6 pKa units, from 6.5 to 8.1, which is well above neutrality. The addition of a bulge two base pairs away from a protonated A(+)·C base pair shifted the pKa by only ~0.5 units less than a perfectly base paired hairpin; however, positioning the bulge just one base pair away from the A(+)·C base pair prohibited formation of the protonated base pair as well as several flanking base pairs. Comparison of data collected at 25 °C and 100 mM KCl to biological temperature and Mg(2+) concentration revealed only slight pKa changes, suggesting that similar sequence contexts in biological systems have the potential to be protonated at biological pH. We present a general model to aid in the determination of the roles protonated bases may play in various dsRNA-mediated processes including ADAR editing, miRNA processing, programmed ribosomal frameshifting, and general acid-base catalysis in ribozymes.

Pick one, but be quick: 5′ splice sites and the problems of too many choices

Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5' Splice sites (5'ss) are the critical elements at the 5' end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5'ss in the human transcriptome. Most 5'ss are recognized by base-pairing with the 5' end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5'ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5'ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5'ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5'ss might lead to selection of a single 5'ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5'ss selection.

Does base-pairing strength play a role in microRNA repression?

MicroRNAs (miRNAs) are short, single-stranded RNAs that silence gene expression by either degrading mRNA or repressing translation. Each miRNA regulates a specific set of mRNA "targets" by binding to complementary sequences in their 3' untranslated region. In this study, we examined the importance of the base-pairing strength of the miRNA-target duplex to repression. We hypothesized that if base-pairing strength affects the functionality of miRNA repression, organisms with higher body temperature or that live at higher temperatures will have miRNAs with higher G/C content so that the miRNA-target complex will remain stable. In the nine model organisms examined, we found a significant correlation between the average G/C content of miRNAs and physiological temperature, supporting our hypothesis. Next, for each organism examined, we compared the average G/C content of miRNAs that are conserved among distant organisms and that of miRNAs that are evolutionarily recent. We found that the average G/C content of ancient miRNAs is lower than recent miRNAs in homeotherms, whereas the trend was inversed in poikilotherms, suggesting that G/C content is associated with temperature, thus further supporting our hypothesis. In the organisms examined, the average G/C content of miRNA "seed" sequences was higher than that of mature miRNAs, which was higher than pre-miRNA loops, suggesting an association between the degree of functionality of the sequence and its average G/C content. Our analyses show a possible association between the base-pairing strength of miRNA-targets and the temperature of an organism, suggesting that base-pairing strength plays a role in repression by miRNAs.

Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modified RNA

Various chemical modifications are currently being evaluated for improving the efficacy of short interfering RNA (siRNA) duplexes as antisense agents for gene silencing in vivo. Among the 2′-ribose modifications assessed to date, 2′deoxy-2′-fluoro-RNA (2′-F-RNA) has unique properties for RNA interference (RNAi) applications. Thus, 2′-F-modified nucleotides are well tolerated in the guide (antisense) and passenger (sense) siRNA strands and the corresponding duplexes lack immunostimulatory effects, enhance nuclease resistance and display improved efficacy in vitro and in vivo compared with unmodified siRNAs. To identify potential origins of the distinct behaviors of RNA and 2′-F-RNA we carried out thermodynamic and X-ray crystallographic analyses of fully and partially 2′-F-modified RNAs. Surprisingly, we found that the increased pairing affinity of 2′-F-RNA relative to RNA is not, as commonly assumed, the result of a favorable entropic contribution (‘conformational preorganization’), but instead primarily based on enthalpy. Crystal structures at high resolution and osmotic stress demonstrate that the 2′-F-RNA duplex is less hydrated than the RNA duplex. The enthalpy-driven, higher stability of the former hints at the possibility that the 2′-substituent, in addition to its important function in sculpting RNA conformation, plays an underappreciated role in modulating Watson–Crick base pairing strength and potentially π–π stacking interactions.

Structural and thermodynamic properties of a linearly perturbed matrix model for RNA folding

The structural and thermodynamic properties of a matrix model of homo-RNA folding with linear external interaction are studied. The interaction distinguishes paired bases of the homo-RNA chain from the unpaired bases hence dividing the possible RNA structures given by the linear model into two structural regimes. The genus distribution functions show that the total number of structures for any given length of the chain are reduced for the simple linear interaction considered. The partition function of the model exhibits a scaling relation with the matrix model in which the base pairing strength parameter is re-scaled (G. Vernizzi, H. Orland, A. Zee, Phys. Rev. Lett. 94, 168103 (2005)). The thermodynamics of the model are computed for i) largely secondary structures, (with tertiary structures suppressed by a factor 10(-4)) and ii) secondary plus tertiary structures. A structural change for large even lengths is observed in the free energy and specific heat. This change with largely secondary structures appears much before (with respect to length of the chain) than when all the structures (secondary and pseudoknots) are considered. The appearance of different structures which dominate the ensemble with varying temperatures is also found as a function of the interaction parameter for different types of structures (given by different numbers of pairings).

Overexpression of adapted U1snRNA in patients' cells to correct a 5′ splice site mutation in propionic acidemia

Splicing defects account for 16% of the mutant alleles in the PCCA and PCCB genes, encoding both subunits of the propionyl-CoA carboxylase (PCC) enzyme, defective in propionic acidemia, one of the most frequent organic acidemias causing variable neurological impairment. Most of the splicing mutations identified affect the conserved 3′ splice (3′ ss) or 5′ splice (5′ ss) sites, the latter predictably through lowering the strength of base pairing with U1snRNA. Among the 5′ ss mutations we have focused on the c.1209 + 3A > G (IVS13 + 3A > G) mutation in the PCCA gene, identified in four patients (three homozygous and one heterozygous) of common geographical origin and causing exon 13 skipping. To study the potential of splicing modulation to restore PCC function, we analyzed the effect of transient transfections in patients' cells with modified U1snRNA adapted to compensate the mutant change and other mismatches at different positions of the 5′ ss. Using this strategy normal transcript could be efficiently recovered with the concomitant disappearance of the aberrant exon skipping transcript, as observed after standard RT-PCR and sequence analysis or using fluorescent primers and semiquantitative RT-PCR. Different efficiencies with up to 100% exon inclusion were observed depending on the transfection conditions and specifically on the adapted U1snRNA used, confirming previously reported dependencies between nucleotides at the 5′ ss for its correct recognition by the spliceosome. The reversal of the splicing defect did not result in a significant increase in enzyme activity, suggesting other factors must be taken into account for the application of overexpression of splice factors such as U1 as therapeutic strategy for splice defects.

Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

The 2008 Benjamin Franklin Medal in Chemistry is presented to Albert Eschenmoser, Dr. sc.nat

Abstract The Franklin Institute, Philadelphia, Pennsylvania, awards the 2008 Benjamin Franklin Medal in Chemistry to Professor Albert Eschenmoser for his seminal efforts in the chemical etiology of nucleic acid structure, which through systematic exploration employing chemical synthesis of potentially natural structural alternatives to DNA, demonstrated that Watson–Crick base pairing is not unique to the ribofuranosyl system, and that DNA’s structure represents an optimization of several factors rather than a maximization of base-pairing strength. In his early scientific career, Professor Albert Eschenmoser established himself as one of the premier synthetic organic chemists in the world, with the monumental achievement of two total syntheses of Vitamin B12, the first in collaboration with Professor Robert B. Woodward of Harvard University. However, since the 1980s, Professor Eschenmoser has concentrated his research interest in establishing the area of chemical etiology of nucleic acid structures to understand better the structural and functional uniqueness of the molecular basis of nature’s genetic information carrier—DNA and RNA. This systematic investigation of potential natural nucleic acid alternatives has demonstrated experimentally that Watson–Crick base pairing is not a unique property of DNA and RNA. Moreover, his research on homo-DNA revealed that the helicality of the famous double-stranded DNA is a direct outcome of the 5-membered ring nature of the deoxyribofuranose structure unit, while the study on p-RNA demonstrated that nature did not choose her genetic system by the standard of maximal base pairing strength, but instead optimization. Of equal significance, his recent design and synthesis of TNA (α-threofuranosyl nucleic acid), found to possess extraordinary base-paring properties, led to the hypothesis suggesting TNA as a possible precursor to the “RNA World”. Finally, his research on the correlation between the differences in the pKa of nucleic bases and their base-pairing strength led to a novel principal for the selection of nucleic base alternatives with proper bonding strength.

Site-Specific Dynamics in TAT Triplex DNA As Revealed by Time-Domain Fluorescence of 2-Aminopurine

Triple helices of DNA are finding increasing level of applications in several areas, including antigene therapy and gene regulation. We have probed site-specific dynamic aspects of TAT triple helices of DNA by using steady-state and time-domain fluorescence of 2-aminopurine (2-AP), a fluorescent analog of adenine. TAT triplexes were formed from repeats of adenine and thymine with 2-AP incorporated at various locations in the polyadenine strand. We find an overall decrease in the level of near-neighbor base-stacking interaction in the TAT triplex when compared to AT duplex as reported by fluorescence decay kinetics of 2-AP. More strikingly, we have observed a stark asymmetry in both the level of base stacking and motional dynamics of the bases in the two ends of TAT triplexes, namely, the 5' end having a higher level of base stacking and segmental dynamics when compared to the 3' end. The possible implications of this asymmetry, which reflects the asymmetry in the strength of Hoogstein base-pairing with the 3' end having stronger Hoogstein pairing when compared to the 5' end, is discussed.