Cheminformatics methods for novel nanopore analysis of HIV DNA termini

Abstract

Background: Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis.

Hidden Markov Model Variants and their Application

“…The results clearly demonstrate the superior performance of the HMM-with-duration over its simpler, HMM without Duration, formulation. With use of the EVA-projection method described in the Background (and in [13]) this affords a robust means to obtain kinetic feature extraction. The emission broadening introduced in the HMM w/wo duration comparison in Figure 8a and 8b, is precisely what occurs with EVA-projection, with similar susceptibility to failure due to over-representation of brief blockade lifetimes in the HMM without Duration parse.…”

“…Emission broadening, via amplification of the emission state variances, is a filtering heuristic that leads to level-projection that strongly preserves transition times between major levels (see [13] for further details). Results from the emission variance amplification (EVA) emission broadening method are described in [13] (with varying amounts of variance amplification). This approach does not require the user to define the number of levels (classes).…”

Solid state nanopores for gene expression profiling

“…On the other hand, a non complementary oligonucleotide is rapidly drawn by the voltage bias into the constriction of the transmembrane region, producing a marked current reduction that signals molecular translocation without duplex formation. In this way, engineered biological nanopore devices have been used to sequence a codon in a single molecule of DNA [4], or to detect both single-base pair and single-nucleotide differences between molecules [5].…”

Nanopore Detector based analysis of single-molecule conformational kinetics and binding interactions

“…Brief snapshots of binding events, often with significant time-averaging of events, are possible with molecular beacon approaches, but these approaches come no where near matching the potential of a nanopore detector to observe single molecules for extensive periods, unmodified by chromophore attachment, etc. The nanopore detector also presents the possibility of observing conformational change within a molecule (see [26] for latest results), something not easily addressed by other methods.…”

“…The work on antibody conformations is not complete, so is not described here, (but preliminary results on the antibody-antigen binding experiments have been obtained, and are described in that section). The objective of the DNA HIV-hairpin conformational study was to systematically test how DNA dinucleotide flexibility (and reactivity) could be discerned using channel current blockade information (see [26], in this same journal issue, for a complete description of the results pertaining to this study). The structural and physical properties of DNA depend upon nucleotide sequence, as is manifest in differences in three dimensional structure and anisotropic flexibility.…”