Crystal structure of a B-form DNA duplex containing (L)-alpha-threofuranosyl (3'-->2') nucleosides: a four-carbon sugar is easily accommodated into the backbone of DNA

(L)-alpha-Threofuranosyl-(3'-->2')-oligonucleotides (TNA) containing vicinally connected phosphodiester linkages undergo informational base pairing in an antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. TNA is derived from a sugar containing only four carbon atoms and is one of the simplest potentially natural nucleic acid alternatives investigated thus far in the context of a chemical etiology of nucleic acid structure. Compared to DNA and RNA that contain six covalent bonds per repeating nucleotide unit, TNA contains only five. We have determined the atomic-resolution crystal structure of the B-form DNA duplex [d(CGCGAA)Td(TCGCG)](2) containing a single (L)-alpha-threofuranosyl thymine (T) per strand. In the modified duplex base stacking interactions are practically unchanged relative to the reference DNA structure. The orientations of the backbone at the TNA incorporation sites are slightly altered in order to accommodate fewer atoms and covalent bonds. The conformation of the threose is C4'-exo with the 2'- and 3'-substituents assuming quasi-diaxial orientation.     

An in vitro selection system for TNA

(3'-2')-alpha-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.     

Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current/time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3'-5' exonuclease activity of wild-type phi29 DNAP to excise a 3'-H terminal residue, yielding a primer strand 3'-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real-time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at ?ngstrom-scale precision as the template strand was drawn through the nanopore lumen during replication.     

Recognition of chromosomal DNA by PNAs

The recognition of cellular nucleic acids by synthetic oligonucleotides is a versatile strategy for regulating biological processes. The vast majority of published studies have focused on antisense oligonucleotides that target mRNA, but it is also possible to design antigene oligonucleotides that are complementary to chromosomal DNA. Antigene oligomers could be used to inhibit the expression of any gene or analyze promoter structure and the mechanisms governing gene regulation. Other potential applications of antigene oligomers include activation of expression of chosen genes or the introduction of mutations to correct genetic disease. Peptide nucleic acid (PNA) is a nonionic DNA/RNA mimic that possesses outstanding potential for recognition of duplex DNA. Here we describe properties of PNAs and the challenges for their development as robust antigene agents.     

Fluorescence Polarization Based Nucleic Acid Testing for Rapid and Cost‐Effective Diagnosis of Infectious Disease

A new nucleic acid detection method was developed for a rapid and cost‐effective diagnosis of infectious disease. This approach relies on the three unique elements: 1) detection probes that regulate DNA polymerase activity in response to the complementary target DNA; 2) universal reporters conjugated with a single fluorophore; and 3) fluorescence polarization (FP) detection. As a proof‐of‐concept, the assay was used to detect and sub‐type Salmonella bacteria with sensitivities down to a single bacterium in less than three hours.     

A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences

Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode's surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four‐way junction (4J)‐forming (UE4J) sensor consists of a universal DNA stem‐loop (USL) probe attached to the electrode's surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re‐useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets.     

An RNA‐Guided Cas9 Nickase‐Based Method for Universal Isothermal DNA Amplification

We have developed an ingenious method, termed Cas9 nickase‐based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 °C. Cas9nAR employs a sgRNA:Cas9n complex with a single‐strand nicking property, a strand‐displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 μL of reaction system) within 60 min and a single‐base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple‐to‐implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.     

Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.     

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids

Hybridization probes are often inefficient in the analysis of single‐stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real‐time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T_m>80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure‐folded analytes, whereas a MB‐based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.     

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide‐terminated template strand and a phosphorothiolate‐modified deoxynucleotide‐terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2‐bromoisobutyryl group and demonstrated that it could be well recognized by ?29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo⁽), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single‐stranded DNA at high‐density using ?29 DNA polymerase by rolling circle amplification.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Miniaturized isothermal nucleic acid amplification, a review

Micro-Total Analysis Systems (μTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.