RESUMO
We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.
Assuntos
Nanopartículas/química , Ácidos Nucleicos/química , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , Citocinas/metabolismo , DNA/química , DNA/genética , DNA/imunologia , Humanos , Imageamento Tridimensional , Leucócitos Mononucleares/metabolismo , Microscopia de Força Atômica , Modelos Moleculares , Nanotecnologia , Conformação de Ácido Nucleico , Ácidos Nucleicos/genética , Ácidos Nucleicos/imunologia , Oligonucleotídeos/química , Oligonucleotídeos/imunologia , RNA/química , RNA/genética , RNA/imunologia , Interferência de RNA , Termodinâmica , Transcrição Gênica , TransfecçãoRESUMO
A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.
