Next Article in Journal
Ivabradine: An Intelligent Drug for the Treatment of Ischemic Heart Disease
Previous Article in Journal
Synthesis and Modifications of Phosphinic Dipeptide Analogues
Previous Article in Special Issue
Spatial Control of DNA Reaction Networks by DNA Sequence
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 17
Issue 11
10.3390/molecules171113569
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids

by
Marcel Hollenstein
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
Molecules 2012, 17(11), 13569-13591; https://doi.org/10.3390/molecules171113569
Submission received: 21 September 2012 / Revised: 7 November 2012 / Accepted: 9 November 2012 / Published: 15 November 2012
(This article belongs to the Special Issue DNA-Directed Chemistry)
Browse Figures
Versions Notes

Abstract

:
Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.

1. Introduction

The importance of the role played by (2'-deoxy)ribonucleoside triphosphates [(d)NTPs] in numerous biological processes needs not to be further underscored. For instance, natural (d)NTPs serve as the fundamental building blocks in the polymerase-mediated synthesis of nucleic acids, both in vitro and in vivo, while ATP acts as the universal unit of molecular currency. Unsurprisingly, modified triphosphates have been the target of numerous synthetic campaigns since they represent the final and active form of DNA polymerase and reverse transcriptase inhibitors [1]. Moreover, since the advent of the polymerase chain reaction (PCR), numerous engineered (thermostable) DNA polymerases have been crafted [2]. Unlike their wild-type counterparts, these DNA polymerase variants possess a much broader substrate tolerance allowing for the polymerization of modified dNTPs and thus, for the generation of modified oligonucleotides and functional nucleic acids for a wide-ranging palette of applications [3].
This review article will focus on the recent progress made in nucleoside triphosphate synthesis and will highlight a few of the most prominent applications, including the use of modified dNTPs both as probes for unraveling the mechanism of polymerases, and in systematic evolution of ligands by exponential enrichment (SELEX) and related combinatorial methods of in vitro selection for the development of functional nucleic acids (i.e., ribozymes, DNAzymes, and aptamers) [4,5,6].

2. Synthesis of Modified dNTPs

Even though a generally applicable and high-yielding method for the generation of dNTPs remains elusive, and despite the tedious purification that these reactive species must undergo, recent advances have certainly facilitated access to modified nucleoside triphosphates [7].

2.1. Yoshikawa Protocol

One of the first, and still one of the most popular methods for the synthesis of nucleoside triphosphates is the Yoshikawa method (Scheme 1) [8,9]. This procedure involves the selective 5'-monophosphorylation of an unprotected nucleoside precursor 1 with the electrophilic phosphorous oxychloride (POCl3), yielding the highly reactive phosphorodichlorate intermediate 2. This intermediate is then subsequently reacted in situ with pyrophosphate to yield the cyclic triphosphate 3, which in turn is hydrolyzed to the desired compound 4.
Molecules 17 13569 g007 550
Scheme 1. Yoshikawa method for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).
Scheme 1. Yoshikawa method for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).
Molecules 17 13569 g007
The advantages of this methodology emanate from its simplicity. Indeed, no protecting group is required and the use of trialkylphosphate solvents mainly directs the phosphorylation to the 5'-regioisomer [7,10,11]. Consequently, a vast array of dNTPs modified with functionalities such as organic polymers [12], diamondoid-like structures [13], amino acid and amino acid-like residues [14,15,16,17,18,19,20,21], modified dNTPs with an sp3-hybridized carbon connecting the nucleobase and the linker arm [22,23,24], perfluorinated side-chains [25], unnatural bases [26,27,28,29], boronic acids [30], 2'-methylseleno triphosphates [31,32], and dual modified 4'-C-aminomethyl-2'-O-methylthymidine [33] have been reported. However, the use of a strong electrophilic phosphorous reagent is not compatible with all nucleosides [34], and modern analytical techniques have revealed the formation of a quantity of undesirable by-products [35].

2.2. Ludwig-Eckstein Method

The “one-pot, three-steps” method developed by Ludwig and Eckstein in the late 80s, is still amongst the most reliable and popular procedures for the synthesis of modified triphosphates [36]. Briefly, the suitably 3'-O-protected (and 2'-O in the case of NTPs) modified nucleoside precursor 5 (see Scheme 2) is reacted with salicyl phosphorochlorite, which is active enough to specifically react with the free 5'-hydroxyl group to yield the activated phosphite intermediate 6. The bifunctional phosphite 6 then undergoes two nucleophilic substitution reactions triggered by tris(tetra-n-butylammonium) hydrogen pyrophosphate, which leads to the displacement of salicylic acid and the formation of the cyclic intermediate 7[36]. Finally, the iodine-mediated oxidation of derivative 7 yields the modified (d)NTP 8 via a cyclic nucleoside triphosphate.
Molecules 17 13569 g008 550
Scheme 2. Ludwig-Eckstein synthetic approach (B = modified or natural nucleobase; R = H, OH, OAc or modification).
Scheme 2. Ludwig-Eckstein synthetic approach (B = modified or natural nucleobase; R = H, OH, OAc or modification).
Molecules 17 13569 g008
The Ludwig-Eckstein protocol has the advantage of reducing the amount of undesired by-products (e.g., regioisomers, mono-, di-, and oligo-phosphates) that are generated in the Yoshikawa methodology, which in turn drastically simplifies the ensuing HPLC purification [37]. In addition, the reaction can easily be followed by 31P-NMR and thus, despite being a one-flask protocol, the formation of all the intermediates can be monitored [36,38,39]. The only disadvantage of this protocol is the slightly longer synthetic route, especially when compared to the Yoshikawa method, since the free nucleoside needs first to be tritylated, then 3'-O-acetylated, before the DMTr protecting group can be unmasked to yield the precursor 5. Despite this slight drawback, an impressive palette of modified dNTPs has been generated by application of the Ludwig-Eckstein protocol. Indeed, dNTPs adorned with amino acid-like side chains [38,40,41], α-L-threofuranosyl nucleoside triphosphates (tNTPs) [42,43], locked nucleic acid NTPs [44,45,46,47] and unnatural bases [48,49,50] have been generated. More recently, five modified deoxyuridine triphosphate derivatives (see Figure 1) bearing side-chains capable of organocatalysis (i.e., bearing proline, urea, and sulfonamide groups) were synthesized and shown to be fully compatible with in vitro selection protocols since these dNTPs acted as substrates for polymerases in both primer extension reactions and in PCR [51].
Molecules 17 13569 g001 550
Figure 1. Chemical structures of the proline-containing analogues dUtPTP (9); dUcPTP (10); and dUFPTP (11); the urea modified dUBpuTP (12); and the sulfonamide functionalized dUBsTP (13) [51].
Figure 1. Chemical structures of the proline-containing analogues dUtPTP (9); dUcPTP (10); and dUFPTP (11); the urea modified dUBpuTP (12); and the sulfonamide functionalized dUBsTP (13) [51].
Molecules 17 13569 g001

2.3. The Borch Approach and Other Strategies

In order to circumvent the drawbacks associated with other methods, i.e., low yields, formation of side products, and incompatibility of functional groups with the strong electrophilic phosphorous reagents, an alternative strategy that employs a highly reactive zwitterionic intermediate has been developed (Scheme 3) [52]. Indeed, the O-benzyl-protected phosphoramidate ester 14 is activated by removal of the protecting group (usually by means of catalytic hydrogenation), which then leads to the formation of the reactive pyrrolidinium phosphoramidate zwitterion 16. Intermediate 16 is prone to react in situ with a nucleophile in general, and pyrophosphate in particular. This approach was successfully applied in the synthesis of farnesyltransferase inhibitors [53], and phosphoramidate prodrugs [54]. However, this methodology has not been used for the development of modified dNTPs as yet, probably because of the rather sinuous synthetic route leading to intermediate 14, an issue that has recently been addressed by the direct activation of more readily accessible H-phosphonate nucleosides [55].
Molecules 17 13569 g009 550
Scheme 3. The Borch approach for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).
Scheme 3. The Borch approach for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).
Molecules 17 13569 g009
In a completely different approach, halogenated nucleoside triphosphates are used in direct aqueous cross-coupling reactions for the synthesis of modified dNTPs (Scheme 4) [56]. This convenient strategy, which completely bypasses the traditional multi-step procedures, has successfully been applied for the generation of dNTPs bearing a vast array of functional groups ranging from functional tags [57,58,59,60,61,62,63,64,65,66] and bile acids [67] to amino acids [68,69]. Finally, amide bond formation reactions were extensively used to connect dNTPs equipped with amine residues to side-chains bearing carboxylic acid groups, so as to yield triphosphates adorned with amino acid-like functionalities [14] or metal complexes [70,71].
Molecules 17 13569 g010 550
Scheme 4. Synthesis of modified dNTPs via direct aqueous Sonogashira (compound 19a) or Suzuki (compound 19b) coupling reactions (R1 = H, OH, or modification; R2 = functional group) [56].
Scheme 4. Synthesis of modified dNTPs via direct aqueous Sonogashira (compound 19a) or Suzuki (compound 19b) coupling reactions (R1 = H, OH, or modification; R2 = functional group) [56].
Molecules 17 13569 g010

3. Applications of Modified dNTPs

The enzymatic polymerization of chemically altered dNTPs represents a milder and alternative way for the synthesis of oligonucleotides, especially when compared to more traditional methods for the generation of modified nucleic acids such as solid-phase synthesis using phosphoramidite building blocks or post synthetic approaches [72,73]. Moreover, since modified dNTPs have been engaged in a motley array of applications such as the generation of modified oligonucleotides by the bias of the Nicking Enzyme Amplification Reaction (NEAR) [74,75], only a few will be considered in this section and the interested reader is directed to other excellent reviews dealing with other facets of modified dNTPs [3,56,72,76,77,78,79].

3.1. As Probes for Polymerases and Substrate Acceptance

Numerous modified dNTPs have been shown to be accepted as substrates and incorporated by DNA polymerases. In this context, the modifications are usually anchored at the C5 positions of pyrimidines and at the C7 of 7-deazapurines by means of rather rigid alkyne- or alkene-based linker arms. The polymerases seem to be quite tolerant to the nature of the chemical alteration appended, since both small substituents [25,80], as well as bulky groups [81], do not reduce the acceptance of the nucleosides. On the other hand, minute alterations in the structure of the linker arm, for instance substituting an E- for a Z-alkene, can have drastic and deleterious effects on the substance abilities of the dNTPs [19,82]. This clearly demonstrates that the prediction of the acceptance of dNTPs by polymerases is hazy and the underlying mechanisms are still not well understood, even though both factors are intrinsic for the rational design of modified triphosphates and the engineering of new polymerases with extended substrate tolerance [2,83]. In order to investigate the mechanisms that dictate DNA polymerase substrate selectivity and acceptance, suitably modified dNTPs were employed [84].
In this context, non-hydrolysable dNTP substrate analogues in which one or all the bridging oxygen atoms of the triphosphate residue are replaced by methylene or other alkyl units, have been the target of numerous synthetic campaigns starting in the early 60s when 5'-adenylyl methylenediphosphonate (AMP-PCP) 20 (Figure 2a) was first synthesized [85]. Substitution of the β,γ-oxygen atom for a methylene linkage in 21 had only a limited impact on the leaving group efficiency of the corresponding pyrophosphate in RNA polymerase mediated reactions [86]. Moreover, a crystal structure of the DNA polymerase (pol) β with (β,γ)-CH2 dGTP revealed that the triphosphate region of both the modified analogue and the native dGTP could be superimposed with no significant deviation, suggesting that the active site was not perturbed by such a modification [87]. However, single-turnover kinetic assays (Figure 2b) revealed that despite the lack of structural disturbance, the nature of the substitution had a profound impact on the pol β-mediated nucleotidyl transfer efficiency and mechanism [87]. Indeed, replacement of the β,γ-bridging oxygen by CF2, CHF, CH2, and CCl2 units (X in Figure 2b) revealed a strong correlation between the rate constants for the slowest nucleotide insertion step (kpol) and the pKa values of the corresponding bisphosphonates or pyrophosphate (when X = O), suggesting that the altered leaving groups had an impact on a chemical step rather than on the conformation of the enzyme. More in particular, the lower pKas corresponding to the more electronegative bridges (X = CF2 and O), could stabilize the build-up of negative charge at the α,β-bridging oxygen during bond breaking and thus induce higher kpol values. These findings further underscored that halogenated, especially fluorinated, bridging methylene residues were better surrogates than the original (β,γ)-CH2 analogues [88,89]. Recently, individual β,γ-CXY dNTP diastereomers could be synthesized and the absolute configuration at the chiral carbon was confirmed by X-ray crystallographic analysis of the complexes formed by the dNTP analogues and DNA pol β [90]. These analogues, especially the mono-fluorinated derivatives, can be useful probes for further investigating the mechanism of DNA polymerases [90,91]. Finally, (α,β)(β,γ)-bisCF2 substituted dNTP analogues were synthesized (21 in Figure 2a) [92]. Unsurprisingly, these derivatives efficiently blocked DNA pol β in single-turnover gap-filling assays, proving their non-hydrolysable nature. More importantly, an X-ray structure of the complex formed by (α,β)(β,γ)-bisCF2 dTTP (21) and DNA pol β showed minimal distortion from the structure with the native dTTP [92].
DNA replication is an intricate and complex process, during which the polymerases switch from an “open” and catalytically inept form to a “closed” conformation that allows for the polymerization of the incoming triphosphate on the nascent chain [93,94]. Moreover, the incoming dNTP forms a Watson-Crick base pair with the templating nucleotide in the ternary complex formed by the DNA polymerase with the primer-template duplex. The Watson-Crick base pair is often complemented by hydrogen bonds and/or stacking interactions between the dNTP and some amino acid residues of the polymerase. It is thus of crucial importance to investigate the effect caused by base modification on the acceptance of dNTPs by DNA polymerases.
Molecules 17 13569 g002 550
Figure 2. (a) Chemical structures of AMP-PCP 20 and (α,β)(β,γ)-bisCF2 dTTP 21; (b) Schematic representation of the single-turnover kinetic assay using the modified dGTP and DNA polymerase β (X = CH2, CHF, CF2, CCl2, or O; M = C or T) [87].
Figure 2. (a) Chemical structures of AMP-PCP 20 and (α,β)(β,γ)-bisCF2 dTTP 21; (b) Schematic representation of the single-turnover kinetic assay using the modified dGTP and DNA polymerase β (X = CH2, CHF, CF2, CCl2, or O; M = C or T) [87].
Molecules 17 13569 g002
In this context, two nucleoside triphosphates embellished with a nitroxide modification (dTspinTP 22 in Figure 3a) [95] and a flexible dendron (dTdendTP 23 in Figure 3a) [12] were first engaged in single nucleotide incorporation assays [96]. Both modified dNTPs are readily incorporated by the large fragment of Thermus aquaticus DNA polymerase (KlenTaq), albeit with a 2500- and 137-fold reduction in efficiency compared to the natural dTTP, respectively. The crystal structures of the modified dNTPs with KlenTaq bound to a primer-template duplex were obtained and both dTspinTP 22 and dTdendTP 23 caused only minor disturbances in the overall structure when compared to that of an unmodified dTTP. However, certain distinctions were apparent: in the structure of dTspinTP 22 (Figure 3b), Arg660 was in a different orientation in order to encompass the nitroxide residue, while in the structure with dTdendTP 23, Arg660 interacts both with the phosphate-backbone and the amide moiety of the rigid propargylamide linker arm [96]. It was surmised that these subtle differences do account for the variation in the acceptance efficiency of both dNTPs.
Finally, in a recent report by Marx et al., the polymerase acceptance of a series of aminopentinyl-modified nucleoside triphosphates was gauged at by incorporation assays and X-ray crystal structure analysis [97]. The polymerase KlenTaq presented a higher tolerance for the C7-modified 7-deazapurine analogues since they were incorporated with similar efficiencies compared to their natural unmodified counterparts, while the C5-derivatized pyrimidines caused a drop in the catalytic competence of the polymerase. The rather small flexible side-chains caused only minor disturbances in the X-ray structures of all the modified dNTPs trapped in the active site of KlenTaq when compared to that of a natural dTTP. Moreover, these modifications induced only a minor displacement of Arg660, especially when compared to the bulkier dNTPs 22 and 23, which certainly explains their higher acceptance by the DNA polymerase [96,97].
Molecules 17 13569 g003 550
Figure 3. (a) Chemical structures of dTspinTP 22 and dTdendTP 23; (b) Close-up view of the X-ray structure of the DNA polymerase KlenTaq with the modified triphosphate dTspinTP 22, picture taken from reference [96].
Figure 3. (a) Chemical structures of dTspinTP 22 and dTdendTP 23; (b) Close-up view of the X-ray structure of the DNA polymerase KlenTaq with the modified triphosphate dTspinTP 22, picture taken from reference [96].
Molecules 17 13569 g003

3.2. Modified dNTPs and SELEX

While Nature is reluctant to leave DNA in a single-stranded form and rather compels it to the well-known double-helical structure, this constraint does not apply to chemists. This realization, in conjunction with the advent of the polymerase chain reaction, prompted Szostak et al., Joyce et al., and Gold et al. to develop a combinatorial methodology for the parallel screening of large populations of nucleic acid sequences, coined SELEX (systematic evolution of ligands by exponential enrichment) [4,5,98,99]. Application of this chemical variant of Darwinian evolution allowed for the generation of nucleic acids (aptamers) capable of selectively and tightly binding to specific targets [100] and of catalytic RNAs (ribozymes) and DNAs (DNAzymes) [101,102], all of which present an enormous potential for in vivo applications [103,104]. However, the use of natural nucleic acids imposes certain drastic restriction on the applicability of aptamers and nucleic acid enzymes gained through these in vitro selection experiments. Indeed, wild-type based DNA and RNA aptamers have quite a limited tolerance to nucleases and might endure chemical degradation [105]. Furthermore, the narrow chemical arsenal presented by nucleic acids, especially when compared to the wealth of functional groups endemic to proteins, restricts both the catalytic efficiencies and the range of reactions covered by DNAzymes (Dz) and ribozymes. In addition, natural nucleic acid enzymes often have to rely on external cofactors such as divalent metal cations (M2+) to achieve reasonable catalytic activities [106]. Consequently, the paucity of functional groups and the insignificant nuclease-resistance of wild-type nucleic acids, prompted the development of SELEX and related methods of in vitro selection using modified triphosphates [3,105]. Furthermore, the in vitro selection protocol involving modified dNTPs for the generation of DNA aptamers or DNAzymes is less cumbersome and time consuming than the RNA equivalent since the transcription and reverse transcription steps can be omitted. Thus, this part of the review will essentially deal with in vitro selections of DNA molecules.

3.2.1. Selections of Modified DNAzymes

Since the discovery of the first Pb2+-dependent RNA-cleaving DNAzyme [107], numerous deoxyribozymes have been identified, including the very potent Dz10-23 and 8-17 [108]. However, it was soon recognized that in order to alleviate the strong M2+-dependence of DNAzymes that is often not compatible with in vivo applications and in view of replenishing natural DNAs with functionalities capable of promoting catalysis, modified dNTPs had to be used in selection experiments. In this context, Dz16.2-11, a Zn2+-dependent RNA-cleaving DNAzyme, was isolated by in vitro selection using a C5-imidazole-functionalized dUTP that was used in lieu of its natural counterpart (Figure 4) [109]. In its minimal composition, Dz16.2-11 requires the presence of three essential imidazole modifications for optimal catalytic activity (kcat > 1 min−1). These imidazole moieties are probably chelating the aminophilic Zn2+ and thus promoting bond cleavage via a mechanism reminiscent of protein enzymes such as carboxypeptidase A [109]. The rather short Dz16.2-11 presents a high catalytic efficiency (kcat/KM ~ 108 M−1min−1) under simulated physiological conditions (10 μM Zn2+, 1 mM Mg2+, 150 mM Na+, pH 7.5, at 37 °C).
Molecules 17 13569 g004 550
Figure 4. Sequence and hypothetical 2D structure of Dz16.2-11 (bold-face U’s indicate the position of the modified nucleoside; the arrow shows the cleavage site within the RNA substrate) [109].
Figure 4. Sequence and hypothetical 2D structure of Dz16.2-11 (bold-face U’s indicate the position of the modified nucleoside; the arrow shows the cleavage site within the RNA substrate) [109].
Molecules 17 13569 g004
Other in vitro selection efforts focused on the generation of M2+-independent ribophosphodiester-cleaving DNAzymes using a combination of dNTPs equipped with amino acid-like residues to compensate for the absence of M2+-cofactors [110,111,112,113,114]. Most notably, dAimTP 24 (Figure 5a) [115] along with the commercially available allylamino-dUTP (dUaaTP) were used conjunctly in an in vitro selection experiment that culminated with the identification of the RNA-cleaving DNAzyme 925-11 [110]. Indeed, the cationic amine and the imidazole residues act in synergy through a general acid and base mechanism and convey robust catalytic activity to the self-cleaving Dz925-11c (c for cis) in the absence of M2+ (kobs ~ 0.2–0.3 min−1). Furthermore, Dz925-11c could successfully be converted into a small 31 nucleotide trans-cleaving species, Dz925-11t, which presented an appreciable catalytic efficiency (kcat/KM ~ 5105 M−1min−1) under multiple turnover, again in the absence of M2+ [116,117,118]. In a similar attempt to mimic the active site of RNase A, Sidorov et al. simultaneously applied a C7-dATP analogue embellished with a cationic amine, in combination with a dUTP nucleotide bearing an imidazole function anchored at position C5 of the nucleobase in an in vitro selection [111]. The resulting DNAzyme employs the two protein-like residues for the M2+-independent and sequence-specific cleavage of a 12nt-long all-RNA substrate. While the first-order rate constants remain modest (kobs ~ 0.07 min−1) when compared to protein enzymes, the selected DNAzyme is ~50-fold more proficient at promoting the cleavage of a ribophosphodiester linkage than unmodified DNAzymes in the absence of M2+ and other cofactors [111,119].
Molecules 17 13569 g005 550
Figure 5. (a) Chemical structures of dAimTP 24, dCaaTP 25, dUgaTP 26, and the phenol-modified dUTP 27; (b) Sequence and hypothetical 2D structure of Dz9-86 (bold-face A’s, U’s, and C’s indicate the position of the modified nucleosides) [112].
Figure 5. (a) Chemical structures of dAimTP 24, dCaaTP 25, dUgaTP 26, and the phenol-modified dUTP 27; (b) Sequence and hypothetical 2D structure of Dz9-86 (bold-face A’s, U’s, and C’s indicate the position of the modified nucleosides) [112].
Molecules 17 13569 g005
All the aforementioned RNA-cleaving modified DNAzymes induce impressive rate enhancements when compared to the uncatalyzed scission of ribophosphodiester linkages [120], but they are still inferior catalysts than their protein counterparts. Consequently, it was surmised that increasing the chemical space that can be explored during in vitro selections could possibly improve the catalytic efficiency of DNAzymes. By the same token, the selected DNAzymes were deemed to be sequentially different from known nucleic acid enzymes, thus avoiding the so-called “tyranny of the small motif” effect, which is known to have poisoned numerous selections [6,121]. Consequently, in order to expand the chemical landscape available for exploration during a selection experiment, a third modified dNTP was included in the process. Indeed, dUgaTP 26 (Figure 5a) was equipped with a guanidinium functionality that mimics the amino acid arginine and was thus expected to help stabilizing secondary and tertiary structures through its cationic nature [112]. This modified deoxyuridine derivative was used in a selection experiment for the generation of M2+-independent RNA-cleaving DNAzymes along with the analogues dAimTP 24 and dCaaTP 25 (Figure 5a) bearing imidazole and cationic amine residues, respectively. The resulting highly functionalized DNAzyme, Dz9-86 (Figure 5b), catalyzes the cleavage of a single embedded ribo(cytosine)phosphodiester bond with a rate constant for self-cleavage that was comparable to that of Dz925-11c (kobs = 0.13 min−1) [112]. However, unlike Dz925-11, the catalytic activity of Dz9-86 increased with the temperature before reaching an apparent maximum at 37 °C. This temperature dependence along with the indifference of the catalytic rates on variation of the ionic strength was attributed to the additional stabilisation of the secondary and tertiary structures conveyed by the extra guandinium residues. Finally, even though Dz9-86 was selected for the cleavage of a single embedded RNA linkage, cleavage of 12 nt long RNA and 2'OMe substrates was also achieved, albeit with a ~100-fold drop in catalytic efficiency. While Dz9-86 represents a significant improvement compared to other modified DNAzymes, especially in terms of the specific catalytic M2+-independent scission of RNA linkages, this catalytic nucleic acid still presented some shortcomings, including rather low rate constants especially with all-RNA substrates and absence of a trans-cleaver that is necessary for practical applications [113].
In an effort to cover both wider chemical and sequence space and at the same time avoiding the tyranny of the small motif, the very same modified dNTPs as used in the selection for Dz9-86 (i.e., dAimTP 24, dCaaTP 25, and dUgaTP 26) were applied in combination with a larger randomized domain (N40 rather than N20) in a selection experiment. The resulting Dz10-66c displayed improved kinetics for self-cleavage in the absence of M2+-cofactors (kobs = 0.5 min−1) when compared to both Dz9-86 and Dz925-11c and was more effective at higher temperatures [113]. Moreover, Dz10-66c displayed a robust catalytic activity in a minimal buffer (kobs = 0.1 min−1 in 1 mM EDTA, 5 mM NaHPO4 pH 7.4), conditions that are usually incompatible with nucleic acid mediated catalysis. These favorable assets were partially attributed to the stabilizing effect induced by the guanidinium groups. Finally, Dz10-66c could be converted to a trans-cleaving species Dz10-66t by means of a slight modification of the primer extension reaction protocol. Dz10-66t showed catalytic efficiencies under multiple turnover conditions (kcat/KM ~ 6105 M−1min−1) that compared favorably with certain unmodified DNAzymes, but were still much inferior to that of Dz16.2-11 (Figure 4) or Dz10-23 when assayed under their optimum working conditions (i.e., 10 μM Zn2+ and 2–100 mM Mg2+, respectively).
As mentioned in Section 3.1, numerous factors govern the efficiency of polymerizability of modified dNTPs, including the nature and the positioning of the functional groups and/or the connecting side chains [19]. These factors seem also to strongly affect the extent of the catalytic enhancement that emanate from DNAzymes that were generated through in vitro selections with modified dNTPs. Indeed, the polymerase acceptance of dAimTP 24 remains virtually unaltered when the linker arm connecting the imidazole moiety to the nucleobase was shortened in size by one methylene unit, while a similar increase in size had a deleterious effect on the polymerizability [38]. In order to assess the effect of the size of the linker arm on the catalytic proficiency of DNAzymes, an in vitro selection was carried out [114]. More specifically, this selection experiment encompassed the simultaneous use of the modified analogues dCaaTP 25 and dUgaTP 26 in conjunction with dAimmTP, a close mimic of dAimTP 24 where the linker arm is shrunk by one CH2-unit, and eventually led to the isolation of Dz20-49 [114]. A marked depletion in terms of catalytic efficiency for the M2+-independent cleavage of a single embedded RNA linkage was observed for Dz20-49 (kobs = 310−3 min−1) when compared to Dz10-66c which utilizes the slightly longer ethylamino-linker arm to support the imidazole residues. It is quite baffling that such a minute change in the chemical structure and composition could have such a drastic impact on the catalytic efficiency of a DNAzyme, and more generally on the outcome of an in vitro selection experiment.
The inclusion of the dUTP analogue 27 equipped with a side-chain mimicking the amino-acid tyrosine (Figure 5a) in an in vitro selection protocol using a similar construct as had been used in the case of Dz925-11, allowed for the isolation of Dz11-17PheO [122]. This DNAzyme self-cleaved an embedded ribo(cytosine)phosphodiester linkage with an appreciable rate-constant (kobs > 0.2 min−1) when supported by the presence of either Ca2+, Zn2+, Mg2+ or Mn2+.
Finally, an RNA amide synthetase [123] and a Diels-Alder ribozyme [124] were isolated by in vitro selection making use of a 5-imidazole modified UTP analogue and a UTP equipped with a pyridiylmethyl unit, respectively. The Diels-Alder ribozyme presented a strong requirement for the presence of Cu2+, while this transition metal only enhanced the catalytic efficiency of the RNA amide synthetase by changing the affinity of the RNA for its substrate. Both ribozymes induce significant rate enhancements when compared to the uncatalyzed reactions, and certainly help to broaden the catalytic repertoire of nucleic acid based enzymes.

3.2.2. Aptamer Selections

Since the advent of SELEX [4,5,98], a flourishing number of aptamers have appeared, propelling these nucleic acids into a leading class of molecular biosensors for a wide ranging diversity of analytes [125,126]. Besides their role as biosensors, aptamers serve in many other applications such as drug development, therapy, target validation, and functional characterization [100,127]. Despite this large success, aptamers consisting of wild-type DNA or RNA are subjected to nuclease degradation which is highly detrimental for numerous practical applications. Furthermore, the rather functionality deprived nucleic acids offer, besides intricate binding motifs, few chemical handles for the interaction with specific residues on the intended targets. Therefore, the chemical modification of aptamers for improving their nuclease-resistance and/or their binding affinities is an important and necessary improvement. Initially, aptamers were modified post-SELEX by capping the 3'- or 5'-termini or by introducing point-mutations (e.g., 2'-fluoronucleotides or LNAs) at various locations on a trial-and-error basis [127]. Nevertheless, as seen in Section 3.2.1 for DNAzymes, the inclusion and alteration of chemical functionalities in the sequence of an aptamer can result in a dramatic loss of binding affinity. Consequently, modified dNTPs and NTPs have advanced as convenient vectors for the elaboration of chemically altered aptamers. In this context, 2'-modified nucleoside triphosphates and thiophosphate analogues have found extensive usage due to their acceptance by RNA and DNA polymerases [79]. Due to the wealth of aptamers that have been crafted using the aforementioned modifications, this section will focus on only a few recent examples involving different NTPs and dNTPs and the interested reader is directed to other excellent reviews covering 2'-modified and thiophosphate nucleotide-based aptamers [79,100,105,127,128].
The first example of an aptamer in vitro selection using a modified dNTP was reported in 1999 [129]. Indeed, Benner et al. used a dUTP analogue carrying a cationic amine (connected to the nucleobase via a propynyl-linker arm) to select for ATP, ADP, and AMP binding aptamers. The resulting sequences bore few similarities to the motifs that were observed in the selection with wild-type triphosphates [130,131]. Unexpectedly, both the modified and the natural aptamers appeared to form bimolecular complexes with ATP and this with rather similar affinity constants (~110−5 M2), which was attributed to the influence of an external stimulus on the selection stringency, namely the effective concentration of the adenosine derivative bound to the solid support (~3 mM). Moreover, the sequences emanating from the selection with the modified dNTP were much shorter than those stemming from the selection with the natural triphosphates (25 vs. 69 nucleotides, respectively), a shrinking effect that has been observed in the selection of modified DNAzymes, albeit to a lesser extent [112,132]. Finally, substituting the amino-modified dU residues for their natural counterparts results only in a two-fold loss of binding affinity, suggesting that the chemical alterations only had a modest impact on the overall properties of the aptamers [129].
An elegant landmark example was reported by Sawai et al. where a deoxyuridine triphosphate analogue adorned with a cationic amine connected via a hexamethylene linker arm to the C5 of the nucleobase (Figure 6a) [133] was used in an in vitro selection for thalidomide-binding aptamers [134]. After 15 rounds of selection, 44 individual clones were isolated. The most proficient binder, DNA aptamer T5 (Figure 6b), displayed a dissociation constant (Kd) of 113 μM for thalidomide. After dividing T5 into three distinct domains, it could be shown that the T5-1 region (square in Figure 6b) was mainly responsible for binding to the target compound since the Kd of a truncated version of T5 with the exact sequence composition of T5-1 was similar to that of the entire aptamer. Surprisingly, even though T5 was selected with a racemic mixture of thalidomide, this aptamer showed high binding to the (R)-form of thalidomide and displayed no affinity to the (S)-enantiomer [134]. Finally, it was shown that the aptamer T5 crucially depended on the presence of the modifications since the corresponding natural DNA sequence lost all binding-propensity.
Molecules 17 13569 g006 550
Figure 6. (a) Chemical structure of the amine-modified dUTP; (b) Sequence and hypothetical 2D structure of aptamer T5 (bold-face U’s indicate the position of the modified nucleosides) [134]; (c) Chemical structure of the carboxamide-modified dUTPs [40].
Figure 6. (a) Chemical structure of the amine-modified dUTP; (b) Sequence and hypothetical 2D structure of aptamer T5 (bold-face U’s indicate the position of the modified nucleosides) [134]; (c) Chemical structure of the carboxamide-modified dUTPs [40].
Molecules 17 13569 g006
Various carboxamide-modified dUTP analogues were recently synthesized (Figure 6c) and shown to be good substrates for D. Vent and KOD XL DNA polymerases in primer extension reactions but were quite reluctant to amplification under PCR conditions [40]. In order to gauge at the effect of a side-chain on the outcome of an in vitro selection, these modified dNTPs were engaged in selections for aptamers binding to either the challenging human tumor necrosis factor receptor super family member 9 (TNFRSF9) or the tumor-associated calcium signal transducer 2 (TACSTD2) as a positive control [40]. These specific protein targets were chosen because no DNA aptamer (natural or modified) for TNFRSF9 has ever been selected despite various attempts, and TACSTD2 has a rather strong affinity (Kd < 100 nM) to random DNA pools [40]. After 8 rounds of selection, strong binding to TNFRSF9 was observed in the selections with dUTP 29 and 32, while that with triphosphate 30 led to only poorly active aptamers (i.e., with Kd > 100 nM). In the case of the control experiment, all the selections (TTP, 29, 30, and 32) yielded aptamers with strong binding affinities to TACSTD2 (Kd ≤ 9 nM). Individual molecules were cloned from the population of the 8th round of the selection for TNFRSF9-binders using dUTP 32 and one single clone was further characterized. This specific aptamer, DNA clone 1684-40, showed a similarly high binding affinity for the target protein (Kd~5 nM) than that of the entire population of the 8th generation. Finally, an enzymatic synthesis of DNA clone 1684-40 performed using TTP, dUTP 29, and triphosphate 31 resulted in a total ablation of the binding affinity of the aptamers to TNFRSF9 and thus further highlighting the need for the modification and revealing that minute alterations in the chemical structures can have drastic consequences.
It is noteworthy mentioning that not all the selections with modified dNTPs have such positive outcomes as the examples outlined above. Indeed, the in vitro selection of aptamers against human thrombin using a triphosphate analogue adorned with a pentynyl-side chain [135] resulted in modified DNA molecules that had only moderate binding affinities (Kd~0.4–1.0 mM) when compared to aptamers resulting from a selection with natural dNTPs (Kd~25–200 nM) [136].

4. Conclusions

The synthesis of modified analogues of nucleoside triphosphates still remains a rather knotty task, mainly because of the rather intricate and extensive purification step that is required. Nonetheless, recent progress certainly greatly facilitates access to these interesting and promising derivatives. Analogues where the bridging oxygen atoms of the triphosphate unit have been replaced by substituted methylene linkages have vastly improved the knowledge on the kinetics and the mechanisms underlying the uptake of dNTPs by polymerases. Furthermore, the X-ray crystal structure of base-modified dNTPs trapped within the active site of DNA polymerases provided insight on the criteria governing polymerase uptake of triphosphates. Surprisingly, most of these modified dNTPs caused only minor disturbances in the overall crystal structures when compared to that of unmodified triphosphates, suggesting that subtle interactions are important for a good substrate acceptance.
dNTPs equipped with functionalities anchored on the nucleobases have been used in selection experiments for the generation of DNAzymes with enhanced catalytic properties. In this context, triphosphates embellished with amino-acid-like side chains have proven to be particularly proficient for the development of M2+-independent RNA-cleaving DNAzymes. These modified DNAzymes represent a significant improvement over nucleic acid enzymes obtained with natural DNA since they do not depend on M2+ or any other type of cofactors and can thus exert their catalytic activity in media that are not suitable for unmodified DNAzymes. Hopefully, the use of modified dNTPs in selection experiments will result in the generation of DNAzymes that present higher nuclease resistance, better catalytic activities (especially in media with low M2+ concentrations), and/or cellular delivery. In this context, LNA-modified DNAzymes have already shown promising properties [137,138,139]. Modified dNTPs have also extensively been employed in selection experiments for the generation of aptamers with improved binding affinities and/or nuclease resistance. In a landmark experiment, a DNA aptamer bound enantioselectively to the (R)-form of thalidomide, highlighting the usefulness of supplementing DNA with additional functionalities.
Finally, modified dNTPs are advancing as important building blocks for the generation of functionalized nucleic acids, especially in the context of in vitro selection experiments. However, numerous parameters including the size, the nature and the location of the modification on the nucleoside can dramatically change the outcome of a selection experiment and determine the level of polymerase acceptance of the modified dNTPs. Certainly, more chemical examples are needed to better understand all these underlying factors so as to improve the polymerase uptake and the efficiency of functionalized nucleic acids.

Acknowledgments

This work was supported by grants from the Swiss National Science Foundation (Grants n° PZ00P2_126430/1 and PZ00P2_144595). I would like to gratefully acknowledge C. Leumann (University of Bern) for providing me the lab space and equipment and for his constant support. I would like to thank A. Calabretta and C. Smith for helpful discussions and critical reading of the manuscript.

References

  1. Lewis, W.; Day, B.J.; Copeland, W.C. Mitochondrial Toxicity of NRTI Antiviral Drugs: An Integrated Cellular Perspective. Nat. Rev. Drug Discov. 2003, 2, 812–822. [Google Scholar] [CrossRef]
  2. Kranaster, R.; Marx, A. Engineered DNA Polymerases in Biotechnology. ChemBioChem 2010, 11, 2077–2084. [Google Scholar] [CrossRef]
  3. Kuwahara, M.; Sugimoto, N. Molecular Evolution of Functional Nucleic Acids with Chemical Modifications. Molecules 2010, 15, 5423–5444. [Google Scholar] [CrossRef]
  4. Ellington, A.D.; Szostak, J.D. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818–822. [Google Scholar] [CrossRef]
  5. Tuerk, C.; Gold, L. Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase. Science 1990, 249, 505–510. [Google Scholar]
  6. Joyce, G.F. Directed Evolution of Nucleic Acid Enzymes. Annu. Rev. Biochem. 2004, 73, 791–836. [Google Scholar] [CrossRef]
  7. Burgess, K.; Cook, D. Syntheses of Nucleoside Triphosphates. Chem. Rev. 2000, 100, 2047–2059. [Google Scholar] [CrossRef]
  8. Yoshikawa, M.; Kato, T.; Takenishi, T. A Novel Method for Phosphorylation of Nucleosides to 5'-Nucleotides. Tetrahedron Lett. 1967, 5065–5068. [Google Scholar]
  9. Yoshikawa, M.; Kato, T.; Takenishi, T. Studies of phosphorylation III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 1969, 42, 3505–3508. [Google Scholar] [CrossRef]
  10. Cramer, F.; Schaller, H. Zur Chemie der energiereichen Phosphate, XIII. Phosphorylierungsreaktionen mit Diestern der Imidazolyl-phosphonsäure und Monoestern der Diimidazolylphosphinsäure. Chem. Ber. 1961, 94, 1634–1640. [Google Scholar] [CrossRef]
  11. Gillerman, I.; Fischer, B. An Improved One-Pot Synthesis of Nucleoside 5'-Triphosphate analogues. Nucleosides Nucleotides Nucleic Acids 2010, 29, 245–256. [Google Scholar] [CrossRef]
  12. Baccaro, A.; Marx, A. Enzymatic Synthesis of Organic-Polymer-Grafted DNA. Chem. Eur. J. 2010, 16, 218–226. [Google Scholar] [CrossRef]
  13. Wang, Y.; Tkachenko, B.A.; Schreiner, P.R.; Marx, A. Diamondoid-modified DNA. Org. Biomol. Chem. 2011, 9, 7482–7490. [Google Scholar] [CrossRef]
  14. Sakthivel, K.; Barbas, C.F. Expanding the Potential of DNA for Binding and Catalysis: Highly Functionalized dUTP Derivatives That Are Substrates for Thermostable DNA Polymerases. Angew. Chem. Int. Ed. 1998, 37, 2872–2875. [Google Scholar] [CrossRef]
  15. Thum, O.; Jäger, S.; Famulok, M. Functionalized DNA: A New Replicable Biopolymer. Angew. Chem. Int. Ed. 2001, 40, 3990–3993. [Google Scholar] [CrossRef]
  16. Jäger, S.; Famulok, M. Generation and Enzymatic Amplification of High-Density Functionalized DNA Double Strands. Angew. Chem. Int. Ed. 2004, 43, 3337–3340. [Google Scholar] [CrossRef]
  17. Jäger, S.; Rasched, G.; Kornreich-Leshem, H.; Engeser, M.; Thum, O.; Famulok, M. A versatile toolbox for variable DNA functionalization at high density. J. Am. Chem. Soc. 2005, 127, 15071–15082. [Google Scholar]
  18. Vaish, N.K.; Fraley, A.W.; Szostak, J.W.; McLaughlin, L.W. Expanding the Structural and Functional Diversity of RNA: Analog Uridine Triphosphates as Candidates for in vitro Selection of Nucleic Acids. Nucleic Acids Res. 2000, 28, 3316–3322. [Google Scholar] [CrossRef]
  19. Lee, S.E.; Sidorov, A.V.; Gourlain, T.; Mignet, N.; Thorpe, S.J.; Brazier, J.A.; Dickman, M.J.; Hornby, D.P.; Grasby, J.A.; Williams, D.M. Enhancing the catalytic repertoire of nucleic acids: A systematic study of linker length and rigidity. Nucleic Acids Res. 2001, 29, 1565–1573. [Google Scholar]
  20. Roychowdhury, A.; Illangkoon, H.; Hendrickson, C.L.; Benner, S.A. 2'-Deoxycytidines Carrying Amino and Thiol Functionality: Synthesis and Incorporation by Vent (Exo−) Polymerase. Org. Lett. 2004, 6, 489–492. [Google Scholar] [CrossRef]
  21. Obayashi, T.; Masud, M.M.; Ozaki, A.N.; Ozaki, H.; Kuwahara, M.; Sawai, H. Enzymatic Synthesis of Labeled DNA by PCR Using New Fluorescent Thymidine Nucleotide Analogue and Superthermophilic KOD Dash DNA Polymerase. Bioorg. Med. Chem. Lett. 2002, 12, 1167–1170. [Google Scholar] [CrossRef]
  22. Sawai, H.; Ozaki, A.N.; Mine, M.; Ozaki, H. Synthesis of New Modified DNAs by Hyperthermophilic DNA Polymerase: Substrate and Template Specificity of Functionalized Thymidine Analogues Bearing an sp3-Hybridized Carbon at the C5 α-Position for Several DNA Polymerases. Bioconjug. Chem. 2002, 13, 309–316. [Google Scholar] [CrossRef]
  23. Kuwahara, M.; Nagashima, J.-I.; Hasegawa, M.; Tamura, T.; Kitagata, R.; Hanawa, K.; Hososhima, S.-I.; Kasamatsu, T.; Ozaki, H.; Sawai, H. Systematic characterization of 2'-deoxynucleoside-5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA. Nucleic Acids Res. 2006, 34, 5383–5394. [Google Scholar]
  24. Kuwahara, M.; Hanawa, K.; Ohsawa, K.; Kitagata, R.; Ozaki, H.; Sawai, H. Direct PCR amplification of various modified DNAs having amino acids: Convenient preparation of DNA libraries with high-potential activities for in vitro selection. Bioorg. Med. Chem. 2006, 14, 2518–2526. [Google Scholar] [CrossRef]
  25. Holzberger, B.; Marx, A. Enzymatic synthesis of perfluoroalkylated DNA. Bioorg. Med. Chem. 2009, 17, 3653–3658. [Google Scholar] [CrossRef]
  26. Hirao, I.; Harada, Y.; Kimoto, M.; Mitsui, T.; Fujiwara, T.; Yokoyama, S. A Two-Unnatural-Base-Pair System toward the Expansion of the Genetic Code. J. Am. Chem. Soc. 2004, 126, 13298–13305. [Google Scholar] [CrossRef]
  27. Kimoto, M.; Mitsui, T.; Yokoyama, S.; Hirao, I. A Unique Fluorescent Base Analogue for the Expansion of the Genetic Alphabet. J. Am. Chem. Soc. 2010, 132, 4988–4989. [Google Scholar] [CrossRef]
  28. Seo, Y.J.; Malyshev, D.A.; Lavergne, T.; Ordoukhanian, P.; Romesberg, F.E. Site-Specific Labeling of DNA and RNA Using an Efficiently Replicated and Transcribed Class of Unnatural Base Pairs. J. Am. Chem. Soc. 2011, 133, 19878–19888. [Google Scholar]
  29. Matray, T.J.; Kool, E.T. Aspecific partner for abasic damage in DNA. Nature 1999, 309, 704–708. [Google Scholar]
  30. Cheng, Y.; Dai, C.; Peng, H.; Zheng, S.; Jin, S.; Wang, B. Design, Synthesis, and Polymerase-Catalyzed Incorporation of Click-Modified Boronic Acid-TTP Analogues. Chem. Asian J. 2011, 6, 2747–2752. [Google Scholar] [CrossRef]
  31. Santner, T.; Siegmund, V.; Marx, A.; Micura, R. The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates. Bioorg. Med. Chem. 2012, 20, 2416–2418. [Google Scholar] [CrossRef]
  32. Siegmund, V.; Santner, T.; Micura, R.; Marx, A. Enzymatic synthesis of 2'-methylseleno-modified RNA. Chem. Sci. 2011, 2, 2224–2231. [Google Scholar] [CrossRef]
  33. Nawale, G.N.; Gore, K.R.; Höbartner, C.; Pradeepkumar, P.I. Incorporation of 4'-C-aminomethyl-2'-O-methylthymidine into DNA by thermophilic DNA polymerases. Chem. Commun. 2012, 48, 9619–9621. [Google Scholar] [CrossRef]
  34. Wu, W.; Bergstrom, D.E.; Davisson, V.J. A Combination Chemical and Enzymatic Approach for the Preparation of Azole Carboxamide Nucleoside Triphosphate. J. Org. Chem. 2003, 68, 3860–3865. [Google Scholar] [CrossRef]
  35. Borsenberger, V.; Kukwikila, M.; Howorka, S. Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates. Org. Biomol. Chem. 2009, 7, 3826–3835. [Google Scholar] [CrossRef]
  36. Ludwig, J.; Eckstein, F. Rapid and efficient synthesis of nucleoside 5'-0-(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 1989, 54, 631–635. [Google Scholar] [CrossRef]
  37. Caton-Williams, J.; Lin, L.; Smith, M.; Huang, Z. Convenient synthesis of nucleoside 50-triphosphates for RNA transcription. Chem. Commun. 2011, 47, 8142–8144. [Google Scholar]
  38. Lam, C.; Hipolito, C.; Perrin, D.M. Synthesis and Enzymatic Incorporation of Modified Deoxyadenosine Triphosphates. Eur. J. Org. Chem. 2008, 2008, 4915–4923. [Google Scholar]
  39. Caton-Williams, J.; Smith, M.; Carrasco, N.; Huang, Z. Protection-Free One-Pot Synthesis of 2'-Deoxynucleoside 5'-Triphosphates and DNA Polymerization. Org. Lett. 2011, 13, 4156–4159. [Google Scholar] [CrossRef]
  40. Vaught, J.D.; Bock, C.; Carter, J.; Fitzwater, T.; Otis, M.; Schneider, D.; Rolando, J.; Waugh, S.; Wilcox, S.K.; Eaton, B.E. Expanding the Chemistry of DNA for in Vitro Selection. J. Am. Chem. Soc. 2010, 132, 4141–4151. [Google Scholar]
  41. Schoetzau, T.; Langner, J.; Moyroud, E.; Roehl, I.; Vonhoff, S.; Klussmann, S. Aminomodified Nucleobases: Functionalized Nucleoside Triphosphates Applicable for SELEX. Bioconjug. Chem. 2003, 14, 919–926. [Google Scholar] [CrossRef]
  42. Zou, K.; Horhota, A.; Yu, B.; Szostak, J.W.; McLaughlin, L.W. Synthesis of α-L-Threofuranosyl Nucleoside Triphosphates (tNTPs). Org. Lett. 2005, 7, 1485–1487. [Google Scholar] [CrossRef]
  43. Horhota, A.; Zou, K.; Ichida, J.K.; Yu, B.; McLaughlin, L.W.; Szostak, J.W.; Chaput, J.C. Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase. J. Am. Chem. Soc. 2005, 127, 7427–7434. [Google Scholar]
  44. Veedu, R.N.; Burri, H.V.; Kumar, P.; Sharma, P.K.; Hrdlicka, P.J.; Vester, B.; Wengel, J. Polymerase-directed synthesis of C5-ethynyl locked nucleic acids. Bioorg. Med. Chem. Lett. 2010, 20, 6565–6568. [Google Scholar] [Green Version]
  45. Veedu, R.N.; Vester, B.; Wengel, J. Enzymatic Incorporation of LNA Nucleotides into DNA Strands. ChemBioChem 2007, 8, 490–492. [Google Scholar] [CrossRef] [Green Version]
  46. Veedu, R.N.; Vester, B.; Wengel, J. Polymerase directed incorporation studies of LNA-G nucleoside 5'-triphosphate and primer extension involving all four LNA nucleotides. New J. Chem. 2010, 34, 877–879. [Google Scholar] [CrossRef]
  47. Johannsen, M.W.; Veedu, R.N.; Madsen, A.S.; Wengel, J. Enzymatic polymerisation involving 2'-amino-LNA nucleotides. Bioorg. Med. Chem. Lett. 2012, 22, 3522–3526. [Google Scholar] [CrossRef]
  48. Hirao, I.; Mitsui, T.; Kimoto, M.; Yokoyama, S. An Efficient Unnatural Base Pair for PCR Amplification. J. Am. Chem. Soc. 2007, 129, 15549–15555. [Google Scholar] [CrossRef]
  49. Yang, Z.; Sismour, A.M.; Sheng, P.; Puskar, N.L.; Benner, S.A. Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 2007, 35, 4238–4249. [Google Scholar] [CrossRef]
  50. Hollenstein, M.; Wojciechowski, F.; Leumann, C.J. Polymerase incorporation of pyrene-nucleoside triphosphates. Bioorg. Med. Chem. Lett. 2012, 22, 4428–4430. [Google Scholar] [CrossRef]
  51. Hollenstein, M. Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfamide groups and their polymerase incorporation into DNA. Chem. Eur. J. 2012, 18, 13320–13330. [Google Scholar] [CrossRef]
  52. Wu, W.; Freel Meyers, C.L.; Borch, R.F. A Novel Method for the Preparation of Nucleoside Triphosphates from Activated Nucleoside Phosphoramidates. Org. Lett. 2004, 6, 2257–2260. [Google Scholar] [CrossRef]
  53. Clark, M.K.; Scott, S.A.; Wojtkowiak, J.; Chirco, R.; Mathieu, P.; Reiners, J.J., Jr.; Mattingly, R.R.; Borch, R.F.; Gibbs, R.A. Synthesis, Biochemical, and Cellular Evaluation of Farnesyl Monophosphate Prodrugs as Farnesyltransferase Inhibitors. J. Med. Chem. 2007, 50, 3274–3282. [Google Scholar] [CrossRef]
  54. Wu, W.; Sigmond, J.; Peters, G.J.; Borch, R.F. Synthesis and Biological Activity of a Gemcitabine Phosphoramidate Prodrug. J. Med. Chem. 2007, 50, 3743–3746. [Google Scholar] [CrossRef]
  55. Sun, Q.; Edathil, J.P.; Wu, R.; Smidansky, E.D.; Cameron, C.E.; Peterson, B.R. One-Pot Synthesis of Nucleoside 5'-Triphosphates from Nucleoside 5'-H-Phosphonates. Org. Lett. 2008, 10, 1703–1706. [Google Scholar] [CrossRef]
  56. Hocek, M.; Fojta, M. Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids. Org. Biomol. Chem. 2008, 6, 2233–2241. [Google Scholar] [CrossRef]
  57. Brázdilová, P.; Vrábel, M.; Pohl, R.; Pivoňková, H.; Havran, L.; Hocek, M.; Fojta, M. Ferrocenylethynyl Derivatives of Nucleoside Triphosphates: Synthesis, Incorporation, Electrochemistry, and Bioanalytical Applications. Chem. Eur. J. 2007, 13, 9527–9533. [Google Scholar] [CrossRef]
  58. Vrábel, M.; Horáková, P.; Pivoňková, H.; Kalachova, L.; Černocká, H.; Cahová, H.; Pohl, R.; Šebest, P.; Havran, L.; Hocek, M.; Fojta, M. Base-modified DNA labeled by [Ru(bpy)3]2+ and [Os(bpy)3]2+ complexes: Construction by polymerase incorporation of modified nucleoside triphosphates, electrochemical and luminescent properties, and applications. Chem. Eur. J. 2009, 15, 1144–1154. [Google Scholar] [CrossRef]
  59. Cahová, H.; Havran, L.; Brázdilová, P.; Pivoňková, H.; Pohl, R.; Fojta, M.; Hocek, M. Aminophenyl- and Nitrophenyl-Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection. Angew. Chem. Int. Ed. 2008, 47, 2059–2062. [Google Scholar]
  60. Macíčková-Cahová, H.; Pohl, R.; Horáková, P.; Havran, L.; Špaček, J.; Fojta, M.; Hocek, M. Alkylsulfanylphenyl Derivatives of Cytosine and 7-Deazaadenine Nucleosides, Nucleotides and Nucleoside Triphosphates: Synthesis, Polymerase Incorporation to DNA and Electrochemical Study. Chem. Eur. J. 2011, 17, 5833–5841. [Google Scholar]
  61. Balintová, J.; Pohl, R.; Horáková, P.; Vidláková, P.; Havran, L.; Fojta, M.; Hocek, M. Anthraquinone as a Redox Label for DNA: Synthesis, Enzymatic Incorporation, and Electrochemistry of Anthraquinone-Modified Nucleosides, Nucleotides, and DNA. Chem. Eur. J. 2011, 17, 14063–14073. [Google Scholar] [CrossRef]
  62. Raindlová, V.; Pohl, R.; Šanda, M.; Hocek, M. Direct Polymerase Synthesis of Reactive Aldehyde-Functionalized DNA and Its Conjugation and Staining with Hydrazines. Angew. Chem. Int. Ed. 2010, 49, 1064–1066. [Google Scholar]
  63. Thoresen, L.H.; Jiao, G.-S.; Haaland, W.C.; Metzker, M.L.; Burgess, K. Rigid, Conjugated, Fluoresceinated Thymidine Triphosphates: Syntheses and Polymerase Mediated Incorporation into DNA Analogues. Chem. Eur. J. 2003, 9, 4603–4610. [Google Scholar] [CrossRef]
  64. Riedl, J.; Pohl, R.; Rulíšek, L.; Hocek, M. Synthesis and Photophysical Properties of Biaryl-Substituted Nucleos(t)ides. Polymerase Synthesis of DNA Probes Bearing Solvatochromic and pH-Sensitive Dual Fluorescent and 19F-NMR Labels. J. Org. Chem. 2012, 77, 1026–1044. [Google Scholar] [CrossRef]
  65. Riedl, J.; Pohl, R.; Ernsting, N.P.; Orság, P.; Fojta, M.; Hocek, M. Labelling of nucleosides and oligonucleotides by solvatochromic 4-aminophthalimide fluorophore for studying DNA-protein interactions. Chem. Sci. 2012, 3, 2797–2806. [Google Scholar] [CrossRef]
  66. Raindlová, V.; Pohl, R.; Klepetářová, B.; Havran, L.; Šimková, E.; Horáková, P.; Pivoňková, H.; Fojta, M.; Hocek, M. Synthesis of Hydrazone-Modified Nucleotides and Their Polymerase Incorporation onto DNA for Redox Labeling. ChemPlusChem 2012, 77, 652–662. [Google Scholar] [CrossRef]
  67. Ikonen, S.; Macíčková-Cahová, H.; Pohl, R.; Šanda, M.; Hocek, M. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA. Org. Biomol. Chem. 2010, 8, 1194–1201. [Google Scholar] [CrossRef]
  68. Čapek, P.; Cahová, H.; Pohl, R.; Hocek, M.; Gloeckner, C.; Marx, A. An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross-Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR. Chem. Eur. J. 2007, 13, 6196–6203. [Google Scholar] [CrossRef]
  69. Raindlová, V.; Pohl, R.; Hocek, M. Synthesis of Aldehyde-Linked Nucleotides and DNA and Their Bioconjugations with Lysine and Peptides through Reductive Amination. Chem. Eur. J. 2012, 18, 4080–4087. [Google Scholar] [CrossRef]
  70. Alpha-Bazin, B.; bazin, H.; Guillemer, S.; Sauvaigo, S.; Mathis, G. Europium Cryptate Labeled Deoxyuridine-Triphosphate Analog: Synthesis and Enzymatic Incorporation. Nucleos. Nucleot. 2000, 19, 1463–1474. [Google Scholar] [CrossRef]
  71. Weizman, H.; Tor, Y. Redox-Active Metal-Containing Nucleotides: Synthesis, Tunability, and Enzymatic Incorporation into DNA. J. Am. Chem. Soc. 2002, 124, 1568–1569. [Google Scholar] [CrossRef]
  72. Weisbrod, S.H.; Marx, A. Novel strategies for the site-specific covalent labelling of nucleic acids. Chem. Commun. 2008, 5675–5685. [Google Scholar] [CrossRef]
  73. Wicke, L.; Engels, J.W. Postsynthetic on Column RNA Labeling via Stille Coupling. Bioconjug. Chem. 2012, 23, 627–642. [Google Scholar] [CrossRef]
  74. Ménová, P.; Hocek, M. Preparation of short cytosine-modified oligonucleotides by nicking enzyme amplification reaction. Chem. Commun. 2012, 48, 6921–6923. [Google Scholar]
  75. Van Ness, J.; van Ness, L.K.; Galas, D.J. Isothermal reactions for the amplification of oligonucleotides. Proc. Natl. Acad. Sci. USA 2003, 100, 4504–4509. [Google Scholar] [CrossRef]
  76. Gramlich, P.M.E.; Wirges, C.T.; Manetto, A.; Carell, T. Postsynthetic DNA Modification through the Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction. Angew. Chem. Int. Ed. 2008, 47, 8350–8358. [Google Scholar] [CrossRef]
  77. El-Sagheer, A.H.; Brown, T. Click chemistry with DNA. Chem. Soc. Rev. 2010, 39, 1388–1405. [Google Scholar] [CrossRef]
  78. Hocek, M.; Fojta, M. Nucleobase modification as redox DNA labelling for electrochemical detection. Chem. Soc. Rev. 2011, 40, 5802–5814. [Google Scholar] [CrossRef]
  79. Lauridsen, L.H.; Rothnagel, J.A.; Veedu, R.N. Enzymatic Recognition of 2'-Modified Ribonucleoside 5'-Triphosphates: Towards the Evolution of Versatile Aptamers. ChemBioChem 2012, 13, 19–25. [Google Scholar] [CrossRef]
  80. Gierlich, J.; Gutsmiedl, K.; Gramlich, P.M.E.; Schmidt, A.; Burley, G.A.; Carell, T. Synthesis of Highly Modified DNA by aCombina tion of PCR with Alkyne-Bearing Triphosphates and Click Chemistry. Chem. Eur. J. 2007, 13, 9486–9494. [Google Scholar]
  81. Baccaro, A.; Steck, A.-L.; Marx, A. Barcoded Nucleotides. Angew. Chem. Int. Ed. 2012, 51, 254–257. [Google Scholar] [CrossRef]
  82. Gourlain, T.; Sidorov, A.V.; Mignet, N.; Thorpe, S.J.; Lee, S.E.; Grasby, J.A.; Williams, D.M. Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR. Nucleic Acids Res. 2001, 29, 1898–1905. [Google Scholar] [CrossRef]
  83. Loakes, D.; Holliger, P. Polymerase engineering: Towards the encoded synthesis of unnatural biopolymers. Chem. Commun. 2009, 4619–4631. [Google Scholar] [CrossRef]
  84. Betz, K.; Streckenbach, F.; Schnur, A.; Exner, T.; Welte, W.; Diedrichs, K.; Marx, A. Structures of DNA Polymerases Caught Processing Size-Augmented Nucleotide Probes. Angew. Chem. Int. Ed. 2010, 49, 5181–5184. [Google Scholar] [CrossRef]
  85. Myers, T.C.; Nakamura, K.; Flesher, J.W. Phosphonic Acid Analogs of Nucleoside Phosphates. I. The Synthesis of 5'-Adenylyl Methylenediphosphonate, a Phosphonic Acid Analog of ATP. J. Am. Chem. Soc. 1963, 85, 3292–3295. [Google Scholar] [CrossRef]
  86. Herdewijn, P.; Marlière, P. Redesigning the leaving group in nucleic acid polymerization. FEBS Lett. 2012, 586, 2049–2056. [Google Scholar] [CrossRef]
  87. Sucato, C.A.; Upton, T.G.; Kashemirov, B.A.; Batra, V.K.; Martínek, V.; Xiang, Y.; Beard, W.A.; Pedersen, L.C.; Wilson, S.H.; McKenna, C.E.; et al. Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β. Biochemistry 2007, 46, 461–471. [Google Scholar]
  88. Mohamady, S.; Jakeman, D.L. An Improved Method for the Synthesis of Nucleoside Triphosphate Analogues. J. Org. Chem. 2005, 70, 10588–10591. [Google Scholar] [CrossRef]
  89. Batra, V.K.; Pedersen, L.C.; Beard, W.A.; Wilson, S.H.; Kashemirov, B.A.; Upton, T.G.; Goodman, M.F.; McKenna, C.E. Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding of the Fluoromethylene Analogues. J. Am. Chem. Soc. 2010, 132, 7617–7625. [Google Scholar]
  90. Wu, Y.; Zakharova, V.M.; Kashemirov, B.A.; Goodman, M.F.; Batra, V.K.; Wilson, S.H.; McKenna, C.E. β,γ-CHF- and β,γ-CHCl-dGTP Diastereomers: Synthesis, Discrete 31P-NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases. J. Am. Chem. Soc. 2012, 134, 8734–8737. [Google Scholar]
  91. Chamberlain, B.T.; Upton, T.G.; Kashemirov, B.A.; McKenna, C.E. α-Azido Bisphosphonates: Synthesis and Nucleotide Analogues. J. Org. Chem. 2011, 76, 5132–5136. [Google Scholar] [CrossRef]
  92. Prakash, G.K.S.; Zibinsky, M.; Upton, T.G.; Kashemirov, B.A.; McKenna, C.E.; Oertell, K.; Goodman, M.F.; Batra, V.K.; Pedersen, L.C.; Beard, W.A.; et al. Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid. Proc. Natl. Acad. Sci. USA 2010, 107, 15693–15698. [Google Scholar]
  93. Berman, A.J.; Kamtekar, S.; Goodman, J.L.; Lázaro, J.M.; de Vega, M.; Blanco, L.; Salas, M.; Steitz, T.A. Structures of phi29 DNA polymerase complexed with substrate: The mechanism of translocation in B-family polymerases. EMBO J. 2007, 26, 3494–3505. [Google Scholar] [CrossRef] [Green Version]
  94. Betz, K.; Malyshev, D.A.; Lavergne, T.; Welte, W.; Diedrichs, K.; Dwyer, T.J.; Ordoukhanian, P.; Romesberg, F.E.; Marx, A. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry. Nat. Chem. Biol. 2012, 8, 612–614. [Google Scholar] [CrossRef]
  95. Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Enzymatic Synthesis of Multiple Spin-Labeled DNA. Angew. Chem. Int. Ed. 2008, 47, 6782–6785. [Google Scholar]
  96. Obeid, S.; Baccaro, A.; Welte, W.; Diedrichs, K.; Marx, A. Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 2010, 107, 21327–21331. [Google Scholar]
  97. Bergen, K.; Steck, A.-L.; Strütt, S.; Baccaro, A.; Welte, W.; Diedrichs, K.; Marx, A. Structures of KlenTaq DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates. J. Am. Chem. Soc. 2012, 134, 11840–11843. [Google Scholar]
  98. Robertson, D.L.; Joyce, G.F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 1990, 344, 467–468. [Google Scholar] [CrossRef]
  99. Joyce, G.F. Forty Years of In Vitro Evolution. Angew. Chem. Int. Ed. 2007, 46, 6420–6436. [Google Scholar] [CrossRef]
  100. Mayer, G. The Chemical Biology of Aptamers. Angew. Chem. Int. Ed. 2009, 48, 2672–2689. [Google Scholar] [CrossRef]
  101. Breaker, R.R. Catalytic DNA: In training and seeking employment. Nat. Biotechnol. 1999, 17, 422–423. [Google Scholar] [CrossRef]
  102. Fiammengo, R.; Jäschke, A. Nucleic acid enzymes. Curr. Opin. Biotechnol. 2005, 16, 614–621. [Google Scholar]
  103. Dass, C.R. Deoxyribozymes: Cleaving a path to clinical trials. Trends Pharmacol. Sci. 2004, 25, 395–397. [Google Scholar] [CrossRef]
  104. Dass, C.R.; Choong, P.F.M.; Khachigian, L.M. DNAzyme technology and cancer therapy: Cleave and let die. Mol. Cancer Ther. 2008, 7, 243–251. [Google Scholar]
  105. Keefe, A.D.; Cload, S.T. SELEX with modified nucleotides. Curr. Opin. Chem. Biol. 2008, 12, 448–456. [Google Scholar] [CrossRef]
  106. Hollenstein, M. Expanding the Catalytic Repertoire of DNAzymes by Modified Nucleosides. Chimia 2011, 65, 770–775. [Google Scholar] [CrossRef]
  107. Breaker, R.R.; Joyce, G.F. A DNA enzyme that cleaves RNA. Chem. Biol. 1994, 1, 223–229. [Google Scholar] [CrossRef]
  108. Santoro, S.W.; Joyce, G.F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 1997, 94, 4262–4266. [Google Scholar] [CrossRef]
  109. Santoro, S.W.; Joyce, G.F.; Sakthivel, K.; Gramatikova, S.; Barbas, C.F. RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 2000, 122, 2433–2439. [Google Scholar] [CrossRef]
  110. Perrin, D.M.; Garestier, T.; Hélène, C. Bridging the gap between proteins and nucleic acids: A metal-independent RNAseA mimic with two protein-like functionalities. J. Am. Chem. Soc. 2001, 123, 1556–1563. [Google Scholar] [CrossRef]
  111. Sidorov, A.V.; Grasby, J.A.; Williams, D.M. Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities. Nucleic Acids Res. 2004, 32, 1591–1601. [Google Scholar] [CrossRef]
  112. Hollenstein, M.; Hipolito, C.J.; Lam, C.H.; Perrin, D.M. A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+). Nucleic Acids Res. 2009, 37, 1638–1649. [Google Scholar] [CrossRef]
  113. Hollenstein, M.; Hipolito, C.J.; Lam, C.H.; Perrin, D.M. A DNAzyme with Three Protein-Like Functional Groups: Enhancing Catalytic Efficiency of M2+-Independent RNA Cleavage. ChemBioChem 2009, 10, 1988–1992. [Google Scholar] [CrossRef]
  114. Hipolito, C.J.; Hollenstein, M.; Lam, C.H.; Perrin, D.M. Protein-inspired modified DNAzymes: Dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes. Org. Biomol. Chem. 2011, 9, 2266–2273. [Google Scholar] [CrossRef]
  115. Perrin, D.M.; Garestier, T.; Hélène, C. Expanding the catalytic repertoire of nucleic acid catalysts: Simultaneous incorporation of two modified deoxyribonucleoside triphosphates bearing ammonium and imidazolyl functionalities. Nucleos. Nucleot. 1999, 18, 377–391. [Google Scholar] [CrossRef]
  116. Lermer, L.; Roupioz, Y.; Ting, R.; Perrin, D.M. Toward an RNaseA mimic: A DNAzyme with imidazoles and cationic amines. J. Am. Chem. Soc. 2002, 124, 9960–9961. [Google Scholar] [CrossRef]
  117. Ting, R.; Thomas, J.M.; Lermer, L.; Perrin, D.M. Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: A putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation. Nucleic Acids Res. 2004, 32, 6660–6672. [Google Scholar] [CrossRef]
  118. Ting, R.; Thomas, J.M.; Perrin, D.M. Kinetic characterization of a cis- and trans-acting M2+-independent DNAzyme that depends on synthetic RNaseA-like functionality—Burst-phase kinetics from the coalescence of two active DNAzyme folds. Can. J. Chem. 2007, 85, 313–329. [Google Scholar] [CrossRef]
  119. Geyer, C.R.; Sen, D. Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. Chem. Biol. 1997, 4, 579–593. [Google Scholar] [CrossRef]
  120. Li, Y.; Breaker, R.R. Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2'-Hydroxyl Group. J. Am. Chem. Soc. 1999, 121, 5364–5372. [Google Scholar] [CrossRef]
  121. Schlosser, K.; Li, Y. Biologically Inspired Synthetic Enzymes Made from DNA. Chem. Biol. 2009, 16, 311–322. [Google Scholar] [CrossRef]
  122. Lam, C.H.; Hipolito, C.J.; Hollenstein, M.; Perrin, D.M. A divalent metal-dependent self-cleaving DNAzyme with a tyrosine side chain. Org. Biomol. Chem. 2011, 9, 6949–6954. [Google Scholar] [CrossRef]
  123. Wiegand, T.W.; Janssen, R.C.; Eaton, B.E. Selection of RNA amide synthase. Chem. Biol. 1997, 4, 675–683. [Google Scholar] [CrossRef]
  124. Tarasow, T.M.; Tarasow, S.L.; Eaton, B.E. RNA-catalysed carbon-carbon bond formation. Nature 1997, 389, 54–57. [Google Scholar]
  125. Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009, 109, 1948–1998. [Google Scholar] [CrossRef]
  126. Shamah, S.M.; Healy, J.M.; Cload, S.T. Complex Target SELEX. Acc. Chem. Res. 2008, 41, 130–138. [Google Scholar] [CrossRef]
  127. Stoltenburg, R.; Reinemann, C.; Strehlitz, B. SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 2007, 24, 381–403. [Google Scholar] [CrossRef]
  128. Famulok, M.; Hartig, J.S.; Mayer, G. Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy. Chem. Rev. 2007, 107, 3715–3743. [Google Scholar] [CrossRef]
  129. Battersby, T.R.; Ang, D.N.; Burgstaller, P.; Jurczyk, S.C.; Bowser, M.T.; Buchanan, D.D.; Kennedy, R.T.; Benner, S.A. Quantitative Analysis of Receptors for Adenosine Nucleotides Obtained via In Vitro Selection from a Library Incorporating a Cationic Nucleotide Analog. J. Am. Chem. Soc. 1999, 121, 9781–9789. [Google Scholar]
  130. Huizenga, D.E.; Szostak, J.W. A DNA Aptamer That Binds Adenosine and ATP. Biochemistry 1995, 34, 656–665. [Google Scholar] [CrossRef]
  131. Lin, C.H.; Patel, D.J. Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: Distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP. Chem. Biol. 1997, 4, 817–832. [Google Scholar] [CrossRef]
  132. Hollenstein, M.; Hipolito, C.; Lam, C.; Dietrich, D.; Perrin, D.M. A highly selective DNAzyme sensor for mercuric ions. Angew. Chem. Int. Ed. 2008, 47, 4346–4350. [Google Scholar] [CrossRef]
  133. Sawai, H.; Ozaki, A.N.; Satoh, F.; Ohbayashi, T.; Masud, M.M.; Ozaki, H. Expansion of structural and functional diversities of DNA using new 5-substituted deoxyuridine derivatives by PCR with superthermophilic KOD Dash DNA polymerase. Chem. Commun. 2001, 2604–2605. [Google Scholar]
  134. Shoji, A.; Kuwahara, M.; Ozaki, H.; Sawai, H. Modified DNA aptamer that binds the (R)-Isomer of a thalidomide derivative with high enantioselectivity. J. Am. Chem. Soc. 2007, 129, 1456–1464. [Google Scholar] [CrossRef]
  135. Latham, J.A.; Johnson, R.; Toole, J.J. The application of a modified nucleotide in aptamer selection: Novel thrombin aptamers containing -(1-pentynyl)-2'-deoxyuridine. Nucleic Acids Res. 1994, 22, 2817–2822. [Google Scholar] [CrossRef]
  136. Bock, L.C.; Griffin, L.C.; Latham, J.A.; Vermaas, E.H.; Toole, J.J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 1992, 355, 564–566. [Google Scholar] [CrossRef]
  137. Schubert, S.; Gül, D.C.; Grunert, H.-P.; Zeichhardt, H.; Erdmann, V.A.; Kurreck, J. RNA cleaving “10-23” DNAzymes with enhanced stability and activity. Nucleic Acids Res. 2003, 31, 5982–5992. [Google Scholar] [CrossRef]
  138. Fahmy, R.G.; Khachigian, L.M. Locked nucleic acid modified DNA enzymes targeting early growth response-1 inhibit human vascular smooth muscle cell growth. Nucleic Acids Res. 2004, 32, 2281–2285. [Google Scholar] [CrossRef]
  139. Doessing, H.; Vester, B. Locked and Unlocked Nucleosides in Functional Nucleic Acids. Molecules 2011, 16, 4511–4526. [Google Scholar] [CrossRef] [Green Version]

Share and Cite

MDPI and ACS Style

Hollenstein, M. Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids. Molecules 2012, 17, 13569-13591. https://doi.org/10.3390/molecules171113569

AMA Style

Hollenstein M. Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids. Molecules. 2012; 17(11):13569-13591. https://doi.org/10.3390/molecules171113569

Chicago/Turabian Style

Hollenstein, Marcel. 2012. "Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids" Molecules 17, no. 11: 13569-13591. https://doi.org/10.3390/molecules171113569

Article Metrics

Back to TopTop