Artificial Genetic Systems: Self‐Avoiding DNA in PCR and Multiplexed PCR

Hoshika, Shuichi; Chen, Fei; Leal, Nicole A.; Benner, Steven A.

doi:10.1002/anie.201001977

Cited by 43 publications

(24 citation statements)

References 20 publications

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“…First, to support high levels of multiplexing with minimal need to engineer complex primer sets, the recently described “self avoiding molecular recognition systems”, SAMRS (Hoshika et al, 2010) was employed (Figure 1B). SAMRS nucleotides look like DNA, but are built from four special nucleotide analogs (A*, T*, G*, and C*).…”

Section: Introductionmentioning

confidence: 99%

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Glushakova

Bradley

et al. 2015

Journal of Virological Methods

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Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito.

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Section: Introductionmentioning

confidence: 99%

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Glushakova

Bradley

et al. 2015

Journal of Virological Methods

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“…The concept of pc nucleobases goes back to Gamper and colleagues (30) and has meanwhile found application even in polymerase chain reaction (31). Thereby, on one hand, a thymine (T) is substituted for a 2-thiouridine (S), whereas on the other hand, an adenine (A) is changed for a 2,6-diaminopurine (D) residue (Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

Pseudo-complementary PNA actuators as reversible switches in dynamic DNA nanotechnology

Ackermann

Famulok

2013

Nucleic Acids Research

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The structural reorganization of nanoscale DNA architectures is a fundamental aspect in dynamic DNA nanotechnology. Commonly, DNA nanoarchitectures are reorganized by means of toehold-expanded DNA sequences in a strand exchange process. Here we describe an unprecedented, toehold-free switching process that relies on pseudo-complementary peptide nucleic acid (pcPNA) by using a mechanism that involves double-strand invasion. The usefulness of this approach is demonstrated by application of these peptide nucleic acids (PNAs) as switches in a DNA rotaxane architecture. The monomers required for generating the pcPNA were obtained by an improved synthesis strategy and were incorporated into a PNA actuator sequence as well as into a short DNA strand that subsequently was integrated into the rotaxane architecture. Alternate addition of a DNA and PNA actuator sequence allowed the multiple reversible switching between a mobile rotaxane macrocycle and a stationary pseudorotaxane state. The switching occurs in an isothermal process at room temperature and is nearly quantitative in each switching step. pcPNAs can potentially be combined with light- and toehold-based switches, thus broadening the toolbox of orthogonal switching approaches for DNA architectures that open up new avenues in dynamic DNA nanotechnology.

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“…Oligonucleotides containing SAMRS nucleotides were synthesized using published methods. [21] Human genomic DNA male was from Promega (G1471, WI, USA). The plasmid for RpoB in Topo 2.1 was a gift from the Ellington laboratory (University of Texas; Austin, TX).…”

Section: Methodsmentioning

confidence: 99%

“…[21] In SAMRS DNA (Scheme 1), the A, T, G and C in natural DNA are replaced by A*, T*, G*, and C*, each with special pairing properties within a Watson-Crick geometry. [22] SAMRS A* pairs with natural T, T* pairs with natural A, G* pairs with natural C, and C* pairs with natural G (Scheme 1, middle), each with two hydrogen bonds.…”

Section: Introductionmentioning

confidence: 99%

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Helicase‐Dependent Isothermal Amplification of DNA and RNA by Using Self‐Avoiding Molecular Recognition Systems

et al. 2015

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Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3′-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research.

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Artificial Genetic Systems: Self‐Avoiding DNA in PCR and Multiplexed PCR

Cited by 43 publications

References 20 publications

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Pseudo-complementary PNA actuators as reversible switches in dynamic DNA nanotechnology

Helicase‐Dependent Isothermal Amplification of DNA and RNA by Using Self‐Avoiding Molecular Recognition Systems

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