Terminal Phosphate Labeled Nucleotides: Synthesis, Applications, and Linker Effect on Incorporation by Dna Polymerases

Kumar, Shiv; Sood, Anup; Wegener, Jeffery; Finn, Patrick J.; Nampalli, Satyam; Nelson, John; Sekher, Anuradha; Mitsis, Paul G.; Macklin, John W.; Fuller, Carl W.

doi:10.1081/ncn-200059823

Cited by 38 publications

(41 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The tags are attached to the terminal phosphate of 2′-deoxynucleoside-5′-hexaphosphates using Huisgen cycloaddition azide/alkyne coupling chemistry (21). Tagged nucleoside hexaphosphates were chosen because they generally have better activity with DNA polymerases than tagged nucleoside triphosphates (22,23). With these tagged nucleotides, we demonstrate continuous single-molecule electronic DNA sequencing with single-base resolution by nanopore SBS.…”

Section: Significancementioning

confidence: 99%

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Fuller

Kumar

Porel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

126

122

View full text Add to dashboard Cite

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotidebased polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time singlemolecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...

show abstract

Section: Significancementioning

confidence: 99%

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Fuller

Kumar

Porel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

126

122

View full text Add to dashboard Cite

show abstract

“…A third approach also takes advantage of the release of inorganic pyrophosphate, but links a fluorescent dye to the γ-phosphate of the B substrate (Kumar, Sood, Wegener, Finn, Nampalli, Nelson et al , 2005) and incorporates a fluorescence quencher within that substrate. Fluorescence is quenched for the unreacted substrate, but not for the dye-labeled pyrophosphate that is released (Figure 4b).…”

Section: Real-time Florescence Assaysmentioning

confidence: 99%

Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes

Olea

Joyce

2015

Methods in Enzymology

View full text Add to dashboard Cite

A general analytical method for the detection of target ligands has been developed, based on a special class of self-replicating aptazymes. These “autocatalytic aptazymes” are generated by linking an aptamer domain to the catalytic domain of a self-replicating RNA enzyme. Ligand-dependent self-replication of RNA proceeds in a self-sustained manner, undergoing exponential amplification at a constant temperature without the assistance of any proteins or other biological materials. The rate of exponential amplification is dependent on the concentration of the ligand, thus enabling quantitative ligand detection. This system has the potential to detect any ligand that can be recognized by an aptamer, including small molecules and proteins. The instability of RNA in biological samples due to the presence of ribonucleases can be overcome by employing the enantiomeric L-RNA form of the self-replicating enzyme. Methods for real-time fluorescence monitoring over the course of exponential amplification are currently being developed.

show abstract

“…The structures of these nucleotides are illustrated in Figure 2 . More in-depth information regarding these nucleotides can be found in the following articles (Metzker et al, 1996; Lee et al, 1997; Kumar et al, 2005; Metzker, 2010; Chen et al, 2013a). From classical Sanger sequencing to modern NGS technologies, the nucleotide substrates used for sequencing have changed over time.…”

Section: Nucleotide Substrates For the Generations Of Dna Polymerase-mentioning

confidence: 99%

“…These molecular remnants may perturb the protein–DNA interaction and eventually impact the sequencing performance of the DNA polymerase (Metzker, 2010; Chen et al, 2013a). To circumvent this concern, terminal γ-phosphate, fluorescently labeled nucleoside polyphosphates ( Figure 2F ) were developed for the more advanced, third-generation DNA sequencing technique (Kumar et al, 2005; Korlach et al, 2010). There are two major advantages of performing DNA sequencing with γ-phosphate-labeled nucleotides over conventional chain terminators.…”

Section: Nucleotide Substrates For the Generations Of Dna Polymerase-mentioning

confidence: 99%

“…Alternatively stated, the extra phosphate spacer, linked to the terminal γ-phosphate of dNTP, makes the modified nucleotide better tolerated by the enzyme. Indeed, when nucleotide incorporation rates were evaluated with fluorescent, terminal phosphate-labeled nucleoside polyphosphates containing 3, or more, phosphates at the 5′-position of the nucleoside, the nucleotides possessing greater than three phosphates were more effective substrates for A and B-family DNA polymerases (Kumar et al, 2005). Later studies proved both dye-labeled nucleoside penta/hexaphosphates (dN5Ps and dN6P) alone can be used by enterobacterial phage ϕ29 DNA polymerase for incorporating thousands of bases in length, approaching natural dNTP rates (Korlach et al, 2008, 2010).…”

Section: Challenges Of Rapidly Evolving Nucleotide Substrates On Dna mentioning

confidence: 99%

See 1 more Smart Citation

DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

Chen

2014

Front. Microbiol.

View full text Add to dashboard Cite

Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage φ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphatelabeled nucleoside polyphosphates. Furthermore, φ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistorbased sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

show abstract

Terminal Phosphate Labeled Nucleotides: Synthesis, Applications, and Linker Effect on Incorporation by Dna Polymerases

Cited by 38 publications

References 7 publications

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes

DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

Contact Info

Product

Resources

About