A nucleoside triphosphate for site-specific labelling of DNA by the Staudinger ligation

Abstract

A novel nucleotide building block for enzymatic synthesis of azide modified DNA and subsequent conjugation via Staudinger ligation was developed.Many studies of complex biological systems are only feasible through conjugation of biopolymers with other functional molecules like dyes or affinity tags. Linkage of a complex biomolecule with another molecular entity is often a challenging task. Albeit several bioconjugation methods are available, truly chemoselective methods are rarely represented. 1,2 Recently, two reactions based on the specific reactivity of the azide moiety were introduced. [3][4][5] One method is based on the [3 + 2] cyclo addition reaction of an azide and a terminal alkyne 6 promoted by copper catalysis. 7,8 This reaction has been employed extensively and recently been extended for conjugation of DNA. 9,10 On the other side, the so-called Staudinger ligation developed by Bertozzi [11][12][13] readily occurs between an azide and a phosphine to form an azaylide that can be trapped by an acyl group to form a stable amide bond. This reaction has been employed in numerous applications e.g. for the conjugation of carbohydrates, protein 14,15 and phage particles 16 as well as peptide ligation and immobilization. [17][18][19][20][21] Recently, methods were devised to employ the Staudinger ligation for conjugation of DNA. It was shown that conjugation of an azide functionality via a suitable linker system enables conjugation of dyes to the 59-end of single stranded DNA. 22 Rajski et al. employed the Staudinger ligation to conjugate DNA with phenanthroline moieties for subsequent Cu(I) induced strand scission. 23 Here we report on the development of a novel azide modified nucleoside triphosphate building block, that is readily incorporated into DNA enzymatically by a DNA polymerase. The resulting double stranded azide modified DNA can be smoothly conjugated with modified phosphines via the Staudinger ligation.Our strategy to modify DNA site-specifically with azide functions by use of DNA polymerases for subsequent Staudinger ligation is depicted in Scheme 1B. The first step consists of a DNA polymerase reaction in which one of the natural nucleoside triphosphates (dNTPs) is substituted by a modified analogue that contains an azide functionality. Obviously the success of this step is contingent on the ability of a DNA polymerase to accept the modified nucleotide. The azide-modified double stranded DNA in turn should serve as substrate for Staudinger ligation with a suitably functionalized phosphine.Thus, first we developed the synthesis of the nucleoside triphosphate 1, a 29-deoxyadenosine analogue (Scheme 1A). Since it has been shown that 7-modified 7-deaza-29-deoxyadenosine derivatives are accepted by DNA polymerases 24-28 we focused our synthesis on these kinds of analogues. Following a published procedure 24 we synthesised a 7-deaza-29-deoxyadenosine 2, which was readily converted into the corresponding triphosphate 3. After saponification 24 the azide moiety was introduced via EDC promoted amide bond...

Direct Polymerase Synthesis of Reactive Aldehyde‐Functionalized DNA and Its Conjugation and Staining with Hydrazines

“…The introduction of alkyne, azide, or diene groups either by chemical phosphoramidite synthesis or by enzymatic polymerase synthesis has been achieved and the modified DNA was used for click-chemistry, [2,3] Staudinger ligation, [4] and Diels-Alder reactions.[5] An aldehyde functional group is a very attractive group because of its high and specific reactivity with diverse reagents. However, it was considered too reactive and fragile to be incorporated directly (chemically or enzymatically) [6] and the few successful examples were prepared indirectly by a click reaction with azide derivatives of reducing sugars, [3] or by introduction of 2,3-dihydroxypropyl or 3,4-dihydroxypyrrolidine moieties [7,8] and subsequent oxidative cleavage of the vicinal diols to (di)aldehydes.…”

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…These include next-generation sequencing approaches (1)(2)(3), single molecule sequencing (4), labeling of DNA and PCR amplificates, e.g., for microarray analysis (5)(6)(7)(8), DNA conjugation (9), or the in vitro selection of ligands such as aptamers by SELEX (systematic enrichment of ligands by exponential amplification) (10). Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities provides an entry to new classes of nucleic acids-based hybrid materials (9). In most cases the dNTP modifications were introduced to the nucleobase.…”

2‐Substituted dATP Derivatives as Building Blocks for Polymerase‐Catalyzed Synthesis of DNA Modified in the Minor Groove

“…[1] Themodification is mostly attached to position 5of pyrimidines or position 7of7-deazapurines,not only because it then points out into the major groove of DNAand thus does not destabilize the duplex, but because in most cases,t he corresponding substituted 2'-deoxyribonucleoside triphosphates (dNTPs) are good substrates for DNAp olymerases and can be used in the polymerase-catalyzed synthesis of modified DNA. [2,3] Diverse modifications,i ncluding fluorophores, [4] redox [5] or spin labels, [6] reactive groups for conjugations, [7] and biomolecules (e.g., oligonucleotides [8] or proteins [9] ), have been introduced into the major groove through the enzymatic incorporation of modified nucleotides and applied in different fields.Modification or labelling of the minor groove has mostly been reported with 2'-and 4'-sugarmodified derivatives. [10][11][12][13] 2-Chloroadenine [14] and 2,6-diaminopurine [15] dNTPs are the only minor-groove base-modified nucleotides that have been reported as substrates for DNA polymerases,w hereas 2-arylamino-dATP derivatives were found to act as polymerase inhibitors.…”