Direct sequence detection of structured h5 influenza viral RNA

Abstract

We describe the development of sequence-specific molecular beacons (dual-labeled DNA probes) for identification of the H5 influenza subtype, cleavage motif, and receptor specificity when hybridized directly with in vitro transcribed viral RNA (vRNA). The cloned hemagglutinin segment from a highly pathogenic H5N1 strain, A/Hanoi/30408/2005(H5N1), isolated from humans was used as template for in vitro transcription of sense-strand vRNA. The hybridization behavior of vRNA and a conserved subtype probe was characterized experimentally by varying conditions of time, temperature, and Mg2+ to optimize detection. Comparison of the hybridization rates of probe to DNA and RNA targets indicates that conformational switching of influenza RNA structure is a rate-limiting step and that the secondary structure of vRNA dominates the binding kinetics. The sensitivity and specificity of probe recognition of other H5 strains was calculated from sequence matches to the National Center for Biotechnology Information influenza database. The hybridization specificity of the subtype probes was experimentally verified with point mutations within the probe loop at five locations corresponding to the other human H5 strains. The abundance frequencies of the hemagglutinin cleavage motif and sialic acid recognition sequences were experimentally tested for H5 in all host viral species. Although the detection assay must be coupled with isothermal amplification on the chip, the new probes form the basis of a portable point-of-care diagnostic device for influenza subtyping.

Figure 1

Cleavage motif frequency of the HA segment of H5 influenza isolated from all hosts (A) and humans (B). The cleavage sequence for H5 in humans is dominated by two motifs, RRRKKR (58%) and SRRKKR (33%). The polybasic amino acid sequence of lysine (K) and arginine (R) is a hallmark of high pathogenic H5 strains. The all-host high pathogenic population consists of RRRKKR (46%) and RRRKRG (27%) with only minor contribution from SRRKKR (3%). The sialic acid recognition motif distribution at amino acids 225–230 in H5 HA sequences isolated from all hosts (C) and humans (D). As of January 2007, no H5 sequences in the NCBI database contain the Q226 and G228S mutations, which confer human sialic receptor binding and are present in currently circulating human influenza subtypes.

Figure 2

A: Thermodynamic response of H5G1112 with and without complementary target DNA at a fixed 6 mmol/L [Mg²⁺]. The fluorescence is normalized to the probe maximum when hybridized with its DNA complement. Temperature is fixed at 25°C. B: The probe, H5G1112, hybridized with complementary target DNA as a function of magnesium concentrations of 6, 16, and 25 mmol/L. The upper trace is 6 mmol/L and the bottom trace is 25 mmol/L [Mg²⁺]. Temperature is fixed at 25°C. C: The maximum fluorescence (squares) of probe H5G1112 and probe hybridization time (circles) as a function of [Mg²⁺] at a fixed 25°C temperature when mixed with 45 nmol/L cDNA.

Figure 3

H5G1112 subtype probe with vRNA. A: The fluorescence response in time of probe H5G1112 mixed with H5 vRNA at increasing concentrations of Mg²⁺. Temperature was fixed at 25°C. B: The maximum fluorescence (squares) of probe H5G1112 and probe hybridization time (circles) as a function of [Mg²⁺] at a fixed 25°C temperature when mixed with H5 vRNA. C: The H5G1112 probe mixed with H5 influenza RNA at multiple temperatures at a fixed 20 mmol/L [Mg²⁺]. At 800s, the fluorescence is ranked in the following order: 45°C, 60°C, 55°C, 37°C, 25°C, 65°C. D: The maximum fluorescence (squares) of probe H5G1112 and probe hybridization time (circles) as a function of temperature at a fixed 25 mmol/L [Mg²⁺] when mixed with H5 vRNA. E: The detection limit of vRNA is 0.61 nmol/L or 4 × 10⁸ molecules/μl in 30 nmol/L of H5G1112 and 25 mmol/L [Mg²⁺]. The fluorescence value at 300 seconds was used in the calculations.

Figure 4

H5G1192 subtype probe. A: The fluorescence response in time of probe H5G1192 mixed with H5 vRNA at increasing concentrations of Mg²⁺. Temperature was fixed at 45°C. B: The maximum fluorescence (squares) of probe H5G1192 and probe hybridization time (circles) as a function of [Mg²⁺] at a fixed 25°C temperature when mixed with 45 nmol/L H5 vRNA.

Figure 5

Fluorescence response in time of H5C1045 (upper trace), H5C1046 (lower trace) cleavage probes and H5R734 (middle trace at 1000 seconds) receptor probe when mixed with 45 nmol/L H5 vRNA at 25 mmol/L [Mg²⁺]. Temperature was fixed at 25°C.

Figure 6

The effect of point mutations on the fluorescence of probe H5G1112 as a function of temperature at a fixed 25 mmol/L [Mg²⁺]. The fluorescence values at 300 seconds were used.