Next-generation DNA in pathogen detection, surveillance, and CLIA-waivable diagnostics

Benner, Steven A.; Kim, Hyo‐Joong; Merritt, Kristen B.; Yang, Zunyi; McLendon, D. Chris; Hoshika, Shuichi; Hutter, Daniel

doi:10.1117/12.2183481

Cited by 4 publications

(3 citation statements)

References 14 publications

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“…For those interested in alternative Darwinian systems with extra nucleobase pairs, Watson–Crick size-complementary pairs having rearranged hydrogen-bonding patterns are already well-advanced, with many applications in biotechnology, materials science, and medicine . This is undoubtedly due to the fact that, in addition to retaining hydrogen-bonding complementarity, they retain the size complementarity familiar to enzymes that are recruited to replicate them, including DNA polymerases.…”

Section: Discussionmentioning

confidence: 99%

“Skinny” and “Fat” DNA: Two New Double Helices

et al. 2018

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According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

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Section: Discussionmentioning

confidence: 99%

“Skinny” and “Fat” DNA: Two New Double Helices

et al. 2018

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“…Not surprisingly, many DNA polymerases replicate DNA containing P:Z pairs. This has allowed the pair to contribute to many applications, including some in diagnostics and therapy ( 22 , 23 ). Further, it allows GACT ZP 6-letter DNA to evolve under selective pressure to create functional molecules ( 19–21 , 24 ).…”

Section: Introductionmentioning

confidence: 99%

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

Singh

Laos

Hoshika

et al. 2018

Nucleic Acids Research

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The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2′- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2′-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

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“…During the past 10 years, synthetic biology has advanced to a second generation of AEGIS nucleotides that mitigates these problems [7] . To this has been added a new class of DNA known as self-avoiding molecular recognition systems (SAMRSs).…”

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confidence: 99%

Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA

Glushakova

Sharma

Hoshika

et al. 2015

Analytical Biochemistry

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Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by “transliteration” technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample.

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Next-generation DNA in pathogen detection, surveillance, and CLIA-waivable diagnostics

Cited by 4 publications

References 14 publications

“Skinny” and “Fat” DNA: Two New Double Helices

“Skinny” and “Fat” DNA: Two New Double Helices

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA

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