Generierung einer thermostabilen Reverse‐Transkriptase‐Aktivität aus einer DNA‐abhängigen DNA‐Polymerase

Sauter, Katharina B. M.; Marx, Andreas

doi:10.1002/ange.200602772

Cited by 14 publications

(9 citation statements)

References 26 publications

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“…[42]; KlenTaq DNA polymerase (Klenow Fragment of Thermus aquaticus DNA polymerase): see ref. [43]; Taq DNA polymerase (Thermus aquaticus DNA polymerase): see ref.…”

Section: Methodsmentioning

confidence: 99%

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

Maiti¹,

Siegmund²,

Abramov³

et al. 2011

Chemistry A European J

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Orthogonal nucleic acids are chemically modified nucle ic acid polyme rs that are un able to transfer informa tion with natural nucl eic acids a nd thus can be used in synthetic biology to store and tra nsfer ge netic inform ation independe ntly. Recently, it was proposed that xylose-DNA (dXNA) can be considered to be a potential ca ndidate for an orthogo nal syste m. Herein, we present the structure in solution a nd conformational analysis of two self-complementary, fully modified dXNA oligonucleotides, as de te rmin ed by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dXNAs. In aqueous solu tion , dXNA duplexes predominantly form a linear ladderlike (type-l) structure. This is the first example of a furanose nucleic acid that adopts a ladde rlike structure.Keywords: DNA structures • helical structures· NMR spectroscopy· nucleic acids • orthogonal nucleic acids· synthetic biologyIn the presence of salt, an equilibrium exists be tween two types of duplex form. The corresponding nucleoside triphosphates (dXNTPs) were synthesized and evaluated for the ir ability to be incorporated into a growing DNA chain by using several n a tural a nd mutant DNA polyme rases. D espite the structural orthogonality of dXNA, DNA polyme rase ~ mutant is able to incorporate th e dXNTPs, showing DNA-dependent dXNA polymerase activity.

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“…[42]; KlenTaq DNA polymerase (Klenow Fragment of Thermus aquaticus DNA polymerase): see ref. [43]; Taq DNA polymerase (Thermus aquaticus DNA polymerase): see ref.…”

Section: Methodsmentioning

confidence: 99%

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

Maiti¹,

Siegmund²,

Abramov³

et al. 2011

Chemistry A European J

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“…For the generation of an improved enzyme variant the mutation sites of two KlenTaq (KTq) variants M1 (L322M, L459M, S515R, I638F, S739G, E773G)6 and M747K7 were recombined by DNA shuffling. Both variants, M1 and M747K, were reported to possess either some reverse transcriptase activity or an expanded substrate spectrum.…”

Section: Specific Activities Of Ktq Variants On Dna or Rna Templatementioning

confidence: 99%

“…Proteins were expressed in 96‐well plates and, after lysis and heat denaturation of the host proteins, used in a real‐time PCR activity screen with DNA as the template. As approximately 80 % of the expressed enzymes were found to be PCR active, we subsequently screened for reverse transcriptase activity by using MS2 RNA as the template, as described earlier 6. In this screening, the two variants RT‐KTq 1 and RT‐KTq 2 were identified and selected for further characterization as they showed significantly higher reverse transcriptase activity compared to the parental enzymes as well as a minimal number of mutations.…”

Section: Specific Activities Of Ktq Variants On Dna or Rna Templatementioning

confidence: 99%

“…Thus, thermostable DNA polymerases capable of reverse transcription and PCR would supersede the use of two enzymes and reduce the time and work. First, the purified enzymes were used in real‐time RT‐PCR experiments using MS2 bacteriophage RNA as the template 6. Under the conditions studied, RT‐KTq 2 leads to product formation approximately 10 cycles earlier than the parental enzyme KTq M1, whereas the wild‐type enzyme has no detectable activity (Figure 3 a).…”

Section: Specific Activities Of Ktq Variants On Dna or Rna Templatementioning

confidence: 99%

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Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Blatter

Bergen

Nolte³

et al. 2013

Angew Chem Int Ed

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The high substrate specificity of DNA-dependent DNA polymerases is essential for the stability of the genome as well as many biotechnological applications. [1] The discrimination between ribo-and deoxyribonucleotides and between RNA and DNA, particularly in cells, is fundamental since the concentration of ribo moieties by far exceeds that of deoxyribo analogues. Although the selection mechanisms for the incorporation of nucleotides have been investigated intensively for DNA and RNA polymerases, [2] much less is known on how DNA-dependent DNA polymerases discriminate between the different nucleic acid templates (DNA versus RNA). Some viral DNA polymerases (e.g. reverse transcriptases) can utilize both DNA and RNA as a template for nucleic acid synthesis. Crystal-structure analysis of these enzymes, complexed either to an RNA or DNA template, have contributed significantly to our understanding of this process. [3] However, similar structural data of DNA-dependent DNA polymerases that have a poor propensity to process RNA templates are lacking, presumably because the crystallization is hampered by the formation of unstable complexes that leads to structural heterogeneity.Structure analysis of KlenTaq DNA polymerase, a shortened form of Thermus aquaticus DNA polymerase, has added significant contributions to the understanding of how DNA polymerases recognize the cognate substrate, [4] process abasic sites, [5] and non-natural nucleotides. [4d-g] Since our attempts to obtain suitable crystals of KlenTaq complexed to RNA failed, we set out to engineer the enzyme in such a way that it is capable of processing an RNA template more efficiently, which might result in improved crystallization properties. Indeed, we were able to obtain a significantly improved KlenTaq variant from which we obtained structural insights into a DNA-dependent DNA polymerase while processing RNA as a template for the first time. Furthermore, the generated KlenTaq variant turned out to be a thermostable DNA polymerase with significant reverse transcriptase activity, thus resulting in it being a valuable tool for crucial applications in clinical diagnostics and molecular biology, such as transcriptome analysis as well as the detection of pathogens and disease-specific markers.For the generation of an improved enzyme variant the mutation sites of two KlenTaq (KTq) variants M1 (L322M, L459M, S515R, I638F, S739G, E773G) [6] and M747K [7] were recombined by DNA shuffling. Both variants, M1 and M747K, were reported to possess either some reverse transcriptase activity or an expanded substrate spectrum. DNA shuffling was employed since the M1 variant, previously evolved in error-prone PCR, comprises six mutations distributed over the enzyme scaffold, but the individual contributions of the mutations are unknown. We generated a library of 1570 clones to obtain high coverage of all mutation combinations (as described in the Supporting Information). Proteins were expressed in 96-well plates and, after lysis and heat denaturation of the host proteins, use...

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“…[ [6] und M747K [7] über DNA- Eine Quantifizierung der jeweiligen Aktivitäten auf einem DNA-und RNA-Templat [9] bestätigte die Überlegen-heit der identifizierten Varianten M1/M747K, RT-KTq 1 und 2 gegenüber den parentalen Enzymen (Tabelle 1). Die Rekombination zweier KTq-Mutanten führte zu einer neuen Generation von KTq-Varianten, die gegenüber der parentalen Variante KTq M1 eine bis zu 20-fach und gegenüber KTq M747K eine bis zu 100-fach erhçhte Reverse-TranskriptaseAktivität aufweist (Tabelle 1).…”

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Struktur und Funktion einer RNA‐lesenden thermostabilen DNA‐Polymerase

Blatter

Bergen

Nolte³

et al. 2013

Angewandte Chemie

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Die hohe Substratspezifität von DNA-abhängigen DNA-Polymerasen ist einerseits für die Stabilität des Genoms, andererseits auch für viele biotechnologische Anwendungen essentiell.[1] Speziell in vivo ist die Diskriminierung zwischen Ribo-und Desoxyribonukleotiden sowie zwischen RNA und DNA wichtig, da die Anzahl der Ribosebausteine die der Desoxyribosebausteine bei weitem übersteigt. Während der Selektionsmechanismus beim Einbau von Nukleotiden durch DNA-und RNA-Polymerasen [2] schon intensiv studiert wurde, ist darüber hinaus wenig bekannt, wie DNA-abhän-gige DNA-Polymerasen zwischen den verschiedenen Nukleinsäuretemplaten (DNA zu RNA) unterscheiden. Einige virale DNA-Polymerasen (z. B. Reverse Transkriptasen) sind in der Lage, DNA und RNA als Templat für die Nukleinsäuresynthese zu verwenden. Die Analyse der Kristallstrukturen dieser Enzyme, im Komplex mit RNA oder DNA als Templat, hat maßgeblich zum Verständnis dieses Prozesses [3] beigetragen. Für DNA-abhängige DNA-Polymerasen, die RNA-Template nur schlecht prozessieren kçnnen, fehlt es allerdings an vergleichbaren strukturellen Daten, was vermutlich durch die Entstehung instabiler Komplexe und einer daraus resultierenden strukturellen Heterogenität, die die Kristallisation erschweren, begründet werden kann.Die Kristallstrukturanalyse der KlenTaq-DNA-Polymerase, einer verkürzten Form der DNA-Polymerase aus Thermus aquaticus, hat erheblich zum Verständnis beigetragen, wie DNA-Polymerasen das natürliche Substrat erkennen [4] sowie abasische Stellen [5] und nicht-natürliche Nukleotide prozessieren.[ [6] In diesem Screening wurden zwei Varianten, RT-KTq 1 und RT-KTq 2, identifiziert und aufgrund ihrer signifikant erhçhten Reverse-Transkriptase-Aktivität gegenüber den parentalen Enzymen und einer minimalen Anzahl an Mutationen weiter charakterisiert. RT-KTq 1 besitzt drei Mutationen (S515R, I638F und M747K), wohingegen RT-KTq 2 zusätzlich zu den Mutationen von RT-KTq 1 eine weitere Mutation aufweist (L459M, Abbildung 1 a). Für Vergleichsstudien wurde über zielgerichtete Mutagenese eine Variante generiert, die alle sieben Mutationen aufweist (KTq M1/M747K). Der KTq-Wildtyp, die parentalen Enzyme M1 und M747K, KTq M1/M747K und RT-KTq 1 und 2 wurden exprimiert, gereinigt, auf die gleiche Proteinkonzentration eingestellt (Abbildung S1) und weiter charakterisiert.

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Generierung einer thermostabilen Reverse‐Transkriptase‐Aktivität aus einer DNA‐abhängigen DNA‐Polymerase

Cited by 14 publications

References 26 publications

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Struktur und Funktion einer RNA‐lesenden thermostabilen DNA‐Polymerase

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