Self-avoiding Molecular Recognition Systems Research Articles

Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems.

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how “self-avoiding molecular recognition systems” (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer–primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination

Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3′-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.

A label-free technique for accurate detection of nucleic acid–based self-avoiding molecular recognition systems supplemented multiple cross-displacement amplification and nanoparticles based biosensor

Here, we devised a novel isothermal technique on the basis of standard multiple cross-displacement amplification (MCDA), which is assisted with self-avoiding molecular recognition system (SAMRS) components and antarctic thermal-sensitive uracil-DNA-glycosylase enzyme (AUDG), termed AUDG–SAMRS–MCDA. To enable product detection on the dipsticks, we firstly developed an analysis strategy, which did not require the labelled primers or probes, and thus, the analysis system avoids the false-positive results arising from undesired hybridization (between two labelled primers, or the labelled probe and primer). The SAMRS components are incorporated into MCDA primers for improve the assay’s specificity, which can prevent the false-positive results yielding from off-target hybrids, undesired interactions between (hetero-dimer) or within (self-dimerization) primers. Two additional components (AUDG enzyme and dUTP) were added into the reaction mixtures, which were used for removing the false-positive results generating from carryover contamination, and thus, the genuine positives results were produced from the amplification of target templates. For the demonstration, the label-free AUDG–SAMRS–MCDA technique was successfully applied to detect Pseudomonas aeruginosa from pure culture and blood samples. As a proof-of-concept technique, the label-free AUDG–SAMRS–MCDA method can be reconfigured to detect different target sequences by redesigning the specific primers.

Nanoparticles-based lateral flow biosensor coupled with multiple cross displacement amplification Plus for simultaneous detection of nucleic acid and prevention of carryover contamination

Abstract Isothermal amplification techniques, which avoid thermal cycling, are often confounded by non-specific amplification arising from off-target hybrids, interactions between (hetero-dimer) or within (self-dimerization) primers and carryover contaminants. Here, we developed a new method, termed multiple cross displacement amplification Plus (MCDA Plus), which uses self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme to improve the specificity of MCDA and prevent carryover contamination, respectively. In the presence of SAMRS components, the false positive results arising from off-target hybrids and undesired interactions between or within primers are completely eliminated, which increase the reproducibility of the assay. In the presence of AUDG, the false positive results generating from carryover contaminants are removed, thus the genuine positive results are obtained from the amplification of target sequences. Moreover, we also devised a new analysis strategy, which was intended for the detection of amplified products using lateral flow biosensor (LFB). The analysis system only required a single labeled primer, thus eliminated the false positive result yielding from hybridization (the labeled primer and probe, or between two labeled primers). Therefore, the SAMRS components, AUDG and LFB convert standard MCDA from an assay suited only for the research laboratory into one that has applicable value at points-of-care testing, in the field environments and in poor-resource settings. For demonstration purpose, Mycobacterium tuberculosis complex (MTC), which is the causes of human Tuberculosis (TB), are detected by the novel MCDA Plus technique to demonstrate the capability of target analysis. The performance of MCDA Plus assay in identifying MTC from pure culture and clinical samples is successfully evaluated. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA Plus primers.

Detection of chikungunya viral RNA in mosquito bodies on cationic (Q) paper based on innovations in synthetic biology

Chikungunya virus (CHIKV) represents a growing and global concern for public health that needs inexpensive and convenient methods to collect mosquitoes as potential carriers so that they can be preserved, stored and transported for later and/or remote analysis. Reported here is a cellulose-based paper, derivatized with quaternary ammonium groups (“Q-paper”) that meets these needs. In a series of tests, infected mosquito bodies were squashed directly on Q-paper. Aqueous ammonia was then added on the mosquito bodies to release viral RNA that adsorbed on the cationic surface via electrostatic interactions. The samples were then stored (frozen) or transported. For analysis, the CHIKV nucleic acids were eluted from the Q-paper and PCR amplified in a workflow, previously developed, that also exploited two nucleic acid innovations, (“artificially expanded genetic information systems”, AEGIS, and “self-avoiding molecular recognition systems”, SAMRS). The amplicons were then analyzed by a Luminex hybridization assay. This procedure detected CHIKV RNA, if present, in each infected mosquito sample, but not in non-infected counterparts or ddH2O samples washes, with testing one week or ten months after sample collection.

Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus

This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of “synthetic biology”. Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing “signal amplification”. Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.

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

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.

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

Assays that detect 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, and requires equipment and power that might not be available in these environments. Isothermal procedures, which 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 and gives 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, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.

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

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.

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single‐stranded primers into the duplex DNA product; these are then extended using a strand‐displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base‐pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self‐avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS‐RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

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

Discerning in their choice of partner: The DNA of a self-avoiding molecular-recognition system (SAMRS) binds to natural DNA but not to members of the same SAMRS. Multiplexed PCR with an SAMRS based on 2-aminopurine (A*), 2-thiothymine (T*), 2′-hypoxanthine (G*), and N4-ethylcytosine (C*; see examples) showed the benefits of such systems for clinical analysis, which requires many DNA molecules to interact with target DNA but not with each other.

Self-Avoiding molecular Recognition Systems (SAMRS)

Reported here is a "Self-Avoiding Molecular Recognition Systems" (SAMRS), a species of DNA that can bind via simple rules to natural DNA but cannot bind to other members of the same SAMRS species. A system having these properties has been achieved with 2-aminopurine-2'-deoxyriboside (A*), 2'-deoxy-2-thiothymidine (T*), 2'-deoxyinosine (G*) and N4-ethyl-2'-deoxycytidine. These were designed to form more stable base pairs with natural complements than with SAMRS complements, based on the number of hydrogen bonds. Thermal melting studies were performed using duplexes containing SAMRS components. All SAMRS species, A*, T*, G* and C*, formed more stable base pairs with natural complements, T, A, C and G than with SAMRS complements, T*, A*, C* and G* respectively. This property of SAMRS would be useful for avoiding to be produced undesired products derived from intra- and intermolecular interaction between primers in multiplexed polymerase chain reactions.