Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network.

Department of Chemical Engineering The University of Melbourne Parkville Victoria 3010 Australia

Department of Infectious Diseases Alfred Hospital and Monash University Melbourne Victoria 3004 Australia

Department of Infectious Diseases The University of Melbourne at the Peter Doherty Institute for Infection and Immunity Melbourne Victoria 3000 Australia

Dipartimento di Scienze e Tecnologie Chimiche Università degli Studi di Roma Tor Vergata Via della Ricerca Scientifica 1 00133 Rome Italy

International Clinical Research Center St Anne's University Hospital CZ 65691 Brno Czech Republic

Kirby Institute University of New South Wales New South Wales 2052 Australia

School of Science RMIT University Victoria 3000 Australia

Victorian Infectious Diseases Service Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity Melbourne Victoria 3000 Australia

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